Products and methods for full-length smchd1 expression using split inteins

Abstract

Nucleic acids, vectors, compositions, systems, and methods for expressing a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide to epigenetically silence double homeobox 4 (DUX4) for the treatment of a disease or disorder associated with DUX4 are provided. DUX4 regulates gene expression and plays a role in development, muscular dystrophy (including, but not limited to, facioscapulohumeral dystrophy (FSHD)), a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). The disclosure describes a split intein-mediated protein trans-splicing approach that was utilized to express SMCHD1 to downregulate or inhibit DUX4 expression.

Description

2835 / 708182024-079-02 PRODUCTS AND METHODS FOR FULL-LENGTH SMCHD1 EXPRESSION USING SPLIT INTEINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 721 ,139, filed on November 15, 2024, the entire contents of each of which are fully incorporated herein by reference.INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (Filename: 70818_SeqListing. xml; Size: 121,520 bytes; Created: November 13, 2025) which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0003] The disclosure provides nucleic acids, vectors, compositions, systems, and methods for expressing a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1 ) polypeptide to silence double homeobox 4 (DUX4) for the treatment of a disease or disorder associated with DUX4. DUX4 regulates gene expression and plays a role in development, muscular dystrophy (including, but not limited to, facioscapulohumeral dystrophy (FSHD)), a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). The disclosure describes a split intein-mediated protein trans-splicing approach that was utilized to express SMCHD1 to downregulate or inhibit DUX4.BACKGROUND

[0004] Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent forms of muscular dystrophy, affecting approximately 870,000 people globally. FSHD is associated with inappropriate expression of the double homeobox 4 (DUX4) gene, caused by contraction of the D4Z4 repeats on chromosome 4q or insufficient epigenetic silencing of the normal D4Z4 repeat arrays. Mutations in the chromatin modifier, structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) gene have been linked to hypomethylation of D4Z4 repeats and re-expression of DUX4 in skeletal muscle. Thus, overexpression of SMCHD1 could be beneficial in promoting hypermethylation and repressing the DUX4 locus in muscles. However, gene replacement using an adeno-associated virus (AAV) vector comprising the very large 6,018 base pair (bp) coding sequence of SMCHD1 is not possible to date because of the AAV packaging capacity of 4,700 bps.

[0005] The disclosure provides new products, systems, and methods for addressing this problem in the art in the field of gene therapy.2835 / 708182024-079-02 SUMMARY

[0006] As described herein, the disclosure provides nucleic acids, vectors, compositions, systems, and methods for expressing a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide to silence double homeobox4 (DUX4) for the treatment of a disease or disorder associated with DUX4. DUX4 regulates gene expression and plays a role in development, muscular dystrophy (including, but not limited to, facioscapulohumeral dystrophy (FSHD)), a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). The disclosure describes a split intein-mediated protein trans-splicing approach that was utilized to express SMCHD1 to downregulate or inhibit DUX4.

[0007] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide.

[0008] The disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

[0009] In some aspects, the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide2835 / 708182024-079-02 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

[0010] In some aspects, the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

[0011] In some aspects, the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

[0012] In some aspects, the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17.

[0013] In some aspects, the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

[0014] In some aspects, the nucleotide sequence further comprises an enhancer. In some aspects, the enhancer is a CMV enhancer. In some aspects, the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0015] In some aspects, the nucleotide sequence further comprises a promoter. In some aspects, the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter. In some aspects, the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0016] In some aspects, the nucleotide sequence further comprises an SV40 poly(A) signal. In some aspects, the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.2835 / 708182024-079-02

[0017] In some aspects, the nucleotide sequence further comprises an AAV 5’ ITR sequence. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22. In some aspects, the nucleotide sequence further comprises an AAV 3’ ITR sequence. In some aspects, the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23. In some aspects, the nucleotide sequence further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR sequence. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

[0018] In some aspects, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.

[0019] In some aspects, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

[0020] The disclosure provides a vector comprising any one or more of the nucleic acids disclosed herein. In some aspects, the vector is a recombinant adeno-associated virus (rAAV) vector. In some aspects, the virus lacks rep and / or cap genes. In some aspects, the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV). In some aspects, the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11, AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof. In some aspects, the vector comprises the capsid of MYOAAV3A or a derivative thereof.

[0021] The disclosure provides a composition comprising any of the nucleic acids disclosed herein, or any of the vectors disclosed herein, and a carrier, diluent, excipient, buffer, and / or adjuvant.

[0022] The disclosure provides a method of decreasing and / or inhibiting the expression of a DUX4 gene in a cell comprising introducing into the cell2835 / 708182024-079-02 (a) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and(ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide; and(b) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and(ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

[0023] In some aspects, the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

[0024] In some aspects, the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.2835 / 708182024-079-02

[0025] In some aspects, the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

[0026] In some aspects, the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17.

[0027] In some aspects, the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

[0028] In some aspects, the nucleotide sequence further comprises an enhancer. In some aspects, the enhancer is a CMV enhancer. In some aspects, the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0029] In some aspects, the nucleotide sequence further comprises a promoter. In some aspects, the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter. In some aspects, the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0030] In some aspects, the nucleotide sequence further comprises an SV40 poly(A) signal. In some aspects, the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

[0031] In some aspects, the nucleotide sequence further comprises an AAV 5’ ITR sequence. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22. In some aspects, the nucleotide sequence further comprises an AAV 3’ ITR sequence. In some aspects, the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23. In some aspects, the nucleotide sequence further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR2835 / 708182024-079-02 sequence. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

[0032] In some aspects, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.

[0033] In some aspects, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

[0034] In some aspects, each of the nucleic acids is present in a vector. In some aspects of the methods of the disclosure, each of the nucleic acids of (a) and (b) are in a vector. In some aspects, the vector is a recombinant adeno-associated virus (rAAV) vector. In some aspects, the virus lacks rep and / or cap genes. In some aspects, the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV). In some aspects, the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11, AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof. In some aspects, the vector comprises the capsid of MYOAAV3A or a derivative thereof.

[0035] In some aspects of the methods of the disclosure, introducing into the cell is carried out by injection or by electroporation. In some aspects, the cell is a human cell. In some aspects, the cell is in a human subject.

[0036] The disclosure provides a system comprising (a) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) an N-intein domain coding sequence positioned at the 3’2835 / 708182024-079-02 end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide; and (b) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

[0037] In some aspects, the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

[0038] In some aspects, the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

[0039] In some aspects, the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

[0040] In some aspects, the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17.

[0041] In some aspects, the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

[0042] In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises an enhancer. In some aspects, the enhancer is a CMV enhancer. In some2835 / 708182024-079-02 aspects, the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0043] In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises a promoter. In some aspects, the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiacspecific promoter. In some aspects, the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0044] In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises an SV40 poly(A) signal. In some aspects, the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

[0045] In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 5’ ITR sequence. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22. In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 3’ ITR sequence. In some aspects, the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23. In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR sequence. In some aspects, the nucleotide sequence of at least one of the nucleic acids further comprises wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

[0046] In some aspects, the nucleic acid comprising the nucleotide sequence encoding a fusion protein comprising the N-terminal fragment of the SMCHD1 polypeptide and the N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.2835 / 708182024-079-02

[0047] In some aspects, the nucleic acid comprising the nucleotide sequence encoding a fusion protein comprising the C-terminal SMCHD1 polypeptide fragment and the C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

[0048] In some aspects, each of the nucleic acids is present in a vector. In some aspects, the vector is a recombinant adeno-associated virus (rAAV) vector. In some aspects, the virus lacks rep and / or cap genes. In some aspects, the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV). In some aspects, the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11, AAV12, AAV13, AAV-anc80, AAV-B1, AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof. In some aspects, the vector comprises the capsid of MYOAAV3A or a derivative thereof.

[0049] The disclosure includes a method of inhibiting and / or interfering with expression of a double homeobox 4 (DUX4) gene in a cell comprising introducing into the cell a system as disclosed herein. In some aspects, the cell is in a human subject. In some aspects, introducing the system into the cell is carried out by injection or by electroporation. In some aspects, introducing the system comprising two vectors into the cell is carried out by injection or by electroporation. In some aspects, the two vectors are introduced into the cell or administered to the subject in a single composition.

[0050] The disclosure includes a method of treating a subject suffering from a disease or disorder associated with expression of a double homeobox 4 (DUX4) gene comprising administering to the subject an effective amount of a system as disclosed herein. In some aspects, the disease or disorder associated with expression of a double homeobox 4 (DUX4) gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). In some aspects, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some aspects, the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus. In some aspects, the system comprising two vectors is administered to the subject in a single composition. In some aspects, an effective amount or effective dose is a dosage of the vector or vectors comprising the nucleic acid of about 1.0x1010vg / kg to about 1.0x1015vg / kg.2835 / 708182024-079-02

[0051] The disclosure includes uses of any of the nucleic acids, vectors, compositions, systems, and / or methods disclosed herein for the preparation of a medicament for inhibiting expression of a double homeobox 4 (DLIX4) gene in a cell.

[0052] The disclosure includes uses of any of the nucleic acids, vectors, compositions, systems, and / or methods disclosed herein for treating or ameliorating a disease or disorder associated with expression of a double homeobox 4 (DUX4) gene, or for the preparation of a medicament for treating or ameliorating a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). In some aspects, the disease or disorder associated with expression of a double homeobox 4 (DUX4) gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). In some aspects, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some aspects, the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus.

[0053] The disclosure includes any of the nucleic acids, vectors, compositions, methods, systems, and / or uses disclosed herein, wherein the nucleic acid, vector, composition, or medicament is formulated for intramuscular administration or injection, oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, transdermal transport, injection into the blood stream, or aerosol administration.

[0054] The disclosure includes a composition for treating a disease or disorder associated with expression of a double homeobox 4 (DLIX4) gene in a subject in need thereof, wherein the composition comprises any of the nucleic acids, vectors, compositions, or systems disclosed herein. In some aspects, the disease or disorder associated with expression of the DUX4 gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). In some aspects, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some aspects, the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus. In some aspects, the composition is formulated for intramuscular administration or injection, oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, transdermal transport, injection into the blood stream, or aerosol administration.2835 / 708182024-079-02

[0055] Further aspects and advantages of the disclosure will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. It should be understood, however, that the detailed description (including the drawings and the specific examples), while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0057] Fig. 1 provides a schematic representation of split intein-mediated protein trans-splicing. The large target protein is split at sites compatible with intein-mediated splicing and the corresponding coding sequences (CDS) are packaged into two AAV vectors. The first vector contains the CDS of the N-terminal half of the target protein followed by the CDS of the N-terminal half of the intein. The second vector includes the C-terminal half of the intein attached to the C-terminal half of the target protein. Both AAV vectors contain the same regulatory elements, including inverted terminal repeats (ITR), a promoter, and a polyadenylation signal (pA). The polypeptides are expressed from each expression cassette individually. Upon association, inteins catalyze protein trans-splicing, excising themselves out and ligating the flanking polypeptide sequence.

[0058] Fig. 2A-B shows strategy and amino acid requirements for intein insertion and functional domains in SMCHD1 protein. Fig. 2A provides a table showing amino acid sequence specificity and sites amenable for intein insertion. The native amino acid residues flanking the inteins that promote splicing are highlighted in bold. Additional proximal amino acids that promote intein-mediated splicing are indicated in subsequent rows in the order of preference. Amino acids preceding the N-terminus of the intein are depicted as -1 , -2, and -3, while those flanking the C-terminus of intein are labelled as +1 , +2 and +3. Fig. 2B provides a schematic showing the position of functional domains in SMCHD1 protein, and the region of interest screened for intein insertion. The expanded region of interest shows the amino acid residues, secondary structure, and the selected split sites (SS) in SMCHD1 protein. Amino acids forming the p-sheet are highlighted in yellow, and those forming the a-helix are in pink. The putative amino acids flanking the N-terminus of the intein are boxed in brown and the amino acids flanking the C-terminus of intein are boxed in blue.2835 / 708182024-079-02

[0059] Fig. 3A-D shows the design strategies for the various dual AAV split-intein vectors for SMCHD1 of the disclosure. Fig. 3A-B are schematic representations of dual AAV-vectors for split sites 2 (SS2) and SS3 containing split intein derived from Nostoc punctiforme (Npu). Fig. 3C-D are schematic representations of dual AAV-vectors for split sites 2 (SS2) and SS3 containing split intein derived from Rhodothermus marinus (Rma). In each vector pair, the first vector, represented as AAV.SMCHD1-N, contains the CDS of the N-terminal half of SMCHD1 followed by the CDS of the N-terminal half of the intein. The second vector, depicted as AAV.SMCHD1-C has the C-terminal half of the intein fused to the C-terminal half of SMCHD1 and a terminal V5 epitope tag. Both AAV vectors possess the same regulatory elements, including ITR, CAG promoter (CMV enhancer (C), chicken p actin promoter (A) and an intron from globin (G)) and SV40 pA.

[0060] Fig. 4A-F shows data obtained in the validation of AAV split intein constructs in vitro in HEK293 cells, i.e., Western blot analysis of lysates from HEK 293 cells transfected with either single or dual split intein AAV vectors encoding either the N-terminal half or C-terminal half of SMCHD1. Fig. 4A shows a Western blot image showing the reconstituted full-length SMCHD1 for SS2 and SS3-Npu constructs. Fig 4B shows a Western blot image showing the reconstituted full-length SMCHD1 for SS2 and SS3-Rma constructs. Fig. 4C is a Western blot image displaying effective full-length SMCHD1 protein reconstitution in SMCHD1-SS2-Rma constructs. Fig. 4E is a Western blot image displaying effective full-length SMCHD1 protein reconstitution in SMCHD1-SS3-Rma constructs. Figs. 4D and 4 F are graphs of the densitometry quantification of the experiments shown in Figs. 4C and 4E, respectively, showing the relative expression of split intein-mediated full-length SMCHD1 protein compared to full-length SMCHD1 construct. Actin served as a loading control. Neg, control AAV vector-transfected cells; N, AAV.SMCHD1-N; C, AAV.SMCHD1-C, N+C, AAV.SMCHD1-N + AAV.SMCHD1-C transfected cells. Differences were assessed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. *P<0.05, **P<0.01.

[0061] Fig. 5A-B provide a schematic and results of an animal study involving dual Split intein vector mediated SMCHD1 full-length protein reconstitution in mouse tibialis anterior (TA) muscle. Fig. 5A is a schematic diagram of the animal study. Wild-type mice were intramuscularly injected with single or dual AAV split intein vectors into the TA muscle at three different doses (1.0E+10 vg / vector, 5.0E+10 vg / vector, 1.0E+11 vg / vector; n = 10 TA / dose). TA muscles were harvested 5 weeks post-injection. Fig. 5B shows Western blot analysis of protein expression in harvested TA muscles using an anti-V5 antibody, revealing that full-length SMCHD1 protein reconstitution was achieved at all three doses. Alpha tubulin was used as a loading control.2835 / 708182024-079-02

[0062] Fig. 6A-C provides the experimental plans for functional analysis of the reconstituted full-length SMCHD1. Fig. 6A is a schematic diagram of an in vitro experimental plan using human myotubes that are differentiated for four to seven days. Single and dual Myo-AAV split intein vectors are transduced into the human myotubes. The myotubes are harvested 7 days post-transduction for protein function analysis. Fig. 6B is a schematic diagram of the human facioscapulohumeral muscular dystrophy (FSHD) xenograft model. TA muscles are harvested two to four weeks after grafting human myoblasts and quantification of protein expression in the TA muscles is then carried out. Fig. 6C is a schematic diagram showing retro-orbital (RO) injections of AAV vectors into D4Z42.5 mice that have the FSHD-permissive DUX4 locus. TA and gastrocnemius (Gas) muscles are harvested 4-5 weeks post-injection. The harvested cells and muscles are evaluated for protein, downstream DUX4 target genes, and epigenetic outcome measures.

[0063] Fig. 7A-C provides a schematic of the 5-SMCHD1-SS2-Npu expression cassette (Fig. 7A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 7B), and the expression cassette nucleotide sequence (SEQ ID NO: 24) where various components are highlighted (Fig. 7C).

[0064] Fig. 8A-C provides a schematic of the 3-SMCHD1-SS2-Npu expression cassette (Fig. 8A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 8B), and the expression cassette nucleotide sequence (SEQ ID NO: 25) where various components are highlighted (Fig. 8C).

[0065] Fig. 9A-C provides a schematic of the 5-SMCHD1-SS3-Npu expression cassette (Fig. 9A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 9B), and the expression cassette nucleotide sequence (SEQ ID NO: 26) where various components are highlighted (Fig. 9C).

[0066] Fig. 10A-C provides a schematic of the 3-SMCHD1 -SS3-Npu expression cassette (Fig. 10A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 10B), and the expression cassette nucleotide sequence (SEQ ID NO: 27) where various components are highlighted (Fig. 10C).

[0067] Fig. 11A-C provides a schematic of the 5-SMCHD1-SS2-Rma expression cassette (Fig. 11 A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 11 B), and the expression cassette nucleotide sequence (SEQ ID NO: 28) where various components are highlighted (Fig. 11C).

[0068] Fig. 12A-C provides a schematic of the 3-SMCHD1 -SS2-Rma expression cassette (Fig. 12A), a detailed map of the components of the expression cassette and their sequence2835 / 708182024-079-02 positions in the cassette (Fig. 12B), and the expression cassette nucleotide sequence (SEQ ID NO: 29) where various components are highlighted (Fig. 12C).

[0069] Fig. 13A-C provides a schematic of the 5-SMCHD1 -SS3-Rma expression cassette (Fig. 13A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 13B), and the expression cassette nucleotide sequence (SEQ ID NO: 30) where various components are highlighted (Fig. 13C).

[0070] Fig. 14A-C provides a schematic of the 3-SMCHD1 -SS3-Rma expression cassette (Fig. 14A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 14B), and the expression cassette nucleotide sequence (SEQ ID NO: 31) where various components are highlighted (Fig. 14C).

[0071] Fig. 15 shows Western blot analysis using an anti-V5 antibody to compare the reconstitution efficiency of dual vector split intein constructs (Npu: Lanes 3-5; Rma: Lanes 6-8) with those of homologous recombination (HR: Lanes 9, 10) and RNA trans-splicing (RNA-TS: Lanes 11, 12) constructs. The blot demonstrates that both the Npu and Rma split intein constructs achieved efficient full-length SMCHD1 protein reconstitution, significantly outperforming the RNA-TS and HR constructs. Beta actin was used as a loading control.

[0072] Fig. 16A-F provides immunofluorescence images of reconstituted SMCHD1 localizing to the nuclear speckles in HEK293T cells. These immunofluorescence images show the localization of SMCHD1 protein 72 hours post transfection with single or dual split intein vectors in 293T cells. In Fig. 16A-C, i.e., the figures in the top panels, the localization of individual halves and full length SMCHD1 for Npu split intein constructs is shown. In Fig.16D-F, the figures in the bottom panels, the location of SMCHD1 for Rma split intein constructs is shown. In Fig. 16C and Fig. 16F, the full-length reconstituted SMCHD1 localizes to nuclear speckles. In Fig. 16B and Fig. 16E, the C-terminal half of SMCHD1 display diffused nuclear localization. SMCHD1 was detected using anti-V5 antibody. The nucleus was stained with DAPI. Scale bar, 50pm.

[0073] Fig. 17 provides an assessment of cytotoxicity of SMCHD1 split intein fragments in 293T cells. Apo-ONE homogenous caspase 3 / 7 assay was performed 48 hrs after transfection with single or dual split intein vectors. Transfection of the known apoptosis inducer DUX4 served as a positive control, resulting in an increase in caspase 3 / 7 activity. However, the SMCHD1 N-terminal half or the C-terminal half and their combined expression (via the dual vector), did not induce apoptosis, suggesting that the split-intein fragments are non-toxic to the 293T cells.

[0074] Fig. 18A-D shows the stability of Rma split intein mediated reconstituted full-length SMCHD1 assessed by cycloheximide (CHX) chase assay. HEK 293 cells were transfected2835 / 708182024-079-02 with single or dual SMCHD1 split intein vectors. After 48 hrs, cells were treated with CHX (50pg / mL) and samples were collected at 0,4,8,12,16 and 24h time points. Western blot analysis using anti-FLAG antibody revealed the stability of full-length SMCHD1 (Fig. 18A) and N-terminal SMCHD1 half (Fig. 18C). Western blot analysis using anti-V5 antibody revealed the stability of dual split intein mediated reconstituted SMCHD1 (Fig. 18B) and C-terminal SMCHD1 half (Fig. 18D). The SMCHD1 protein level at zero hours was set to 100%. The graph shows the mean densities of full-length and split SMCHD1 halves, relative to DMSO control at 0 hours and normalized to alpha tubulin. Data presented as mean ± S.D (n=3).

[0075] Fig. 19A-I shows the histological analysis of AAV Npu split intein vector dose response in TA muscle following intramuscular injection. Hematoxylin and eosin (H&E) staining demonstrates a dose-dependent accumulation of cellular infiltration at low dose (Fig.19A-19C; 1.0E+10 vg / vector / TA), mid dose (Fig. 19D-19F;5.0E+10 vg / vector / TA), and high dose (Fig. 19G-19I;1.0E+11 vg / vector / TA). The nuclei of the infiltrating cells appear dark blue due to staining with hematoxylin. This dose-dependent cellular infiltration indicates an immune response to the viral capsid. Scale bar, 50pm.

[0076] Fig. 20 A-C shows the dual AAV Rma split intein vector dose optimization for intramuscular injection. Fig. 20A provides a schematic of the intramuscular injection of single or dual AAV Rma split intein vectors into the TA muscle at two doses: 5.0E+9 vg / vector, 3.0E+9 vg / vector (n = 4). TA muscles were harvested four weeks post-injection. Fig. 20B shows Western blot analysis using an anti-V5 antibody confirms full-length SMCHD1 protein reconstitution at both tested doses. Alpha tubulin is used as a loading control. Fig. 20C shows that H&E staining showed no signs of toxicity at 3.0E+9 vg / vector dose (top panel). Minor infiltration (arrows) was observed at 5.0E+9 vg / vector dose (bottom panel). Scale bar, 50pm.

[0077] Fig. 21 A-F provide experimental results resulting from the transduction of human FSHD myotubes with single or dual myo-AAV split intein vectors. Fig. 21 A provides an in vitro experimental plan illustrating the transduction of human FSHD myotubes with single or dual myo-AAV split intein vectors. The myotubes were harvested 7 days post-transduction for protein function analysis. Fig. 21 B shows a western blot analysis using an anti-V5 antibody shows full-length SMCHD1 protein reconstitution in human myotubes. Beta actin is used as a loading control. (Fig. 21C-F) ddPCR analysis shows the mRNA levels of the DLIX4 target genes ZSCAN4 (Fig. 21 C, Fig. 21 E) and SLC34A2 (Fig. 21 D, Fig. 21 F) in two different FSHD2 patient myotubes PMB-13 (Fig. 21C and Fig. 21 D) and PMB-14 (Fig. 21 E and Fig.21 F) following split intein vector transduction. Data presented as mean ± S.E.M. Differences2835 / 708182024-079-02 were assessed using two-way ANOVA followed by Tukey post hoc test. **P<0.01 , ****P<0.0001.DETAILED DESCRIPTION

[0078] The disclosure provides novel products and methods in the treatment of a disease or disorder associated with DUX4 expression by expressing or overexpressing structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) using a split intein approach to epigenetically silence DUX4. DUX4, or double homeobox 4, is a protein that regulates gene expression and plays a role in development, a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS). The disclosure describes a split intein-mediated protein trans-splicing approach that was utilized to express SMCHD1.

[0079] Mutations in the chromatin modifier, SMCHD1 gene have been linked to hypomethylation of D4Z4 repeats and re-expression of DUX4 in skeletal muscle. Thus, overexpression of SMCHD1 is beneficial in promoting hypermethylation and repressing the DUX4 locus in muscles. However, inserting the 6,018 base pair (bp) coding sequence of SMCHD1 is limited by the AAV packaging capacity of 4,700 bps. This disclosure provides products and methods for a split intein-mediated protein trans-splicing approach to overexpress large full-length SMCHD1 protein successfully into cells and / or into a subject in order to downregulate and / or inhibit DUX4 expression in an effort to treat diseases associated with DUX4 expression or overexpression, such as a muscular dystrophy, e.g., facioscapulohumeral dystrophy (FSHD), a cancer, or Bosma arhinia microphthalmia syndrome (BAMS).

[0080] FSHD is an autosomal dominant disease affecting approximately 870,000 people worldwide. FSHD weakens the muscles of the face, shoulder girdle, and arms, leading to progressive muscle wasting. The root cause of FSHD is the inappropriate expression of DUX4 gene in adults, which is usually silenced post-embryogenesis. Specifically, the DUX4 coding sequence (CDS) resides within the tandem 3.3 kb D4Z4 repeats located in the subtelomeric region of the human chr. 4q (short arm). Expression of DUX4 is triggered when there is a reduction in the number of D4Z4 repeats to less than 10 (95% cases; FSHD1) or due to inadequate epigenetic silencing (less methylation) of the normal D4Z4 repeat arrays (5%; FSHD2).

[0081] Notably, mutations in the chromatin modifier, structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) gene, are strongly linked to hypomethylation of D4Z4 repeats and re-expression of DUX4 in skeletal muscle. The SMCHD1 gene provides instructions for making the SMCHD1 protein that is involved in regulating gene activity by altering the structure of DNA. Specifically, the SMCHD1 protein is2835 / 708182024-079-02 associated with DNA methylation, which is the addition of methyl groups (consisting of one carbon atom and three hydrogen atoms) to DNA molecules. The addition of methyl groups is associated with the turning off (silencing) of genes, so regions of DNA with many methyl groups (hypermethylated regions) tend to have fewer genes that are turned on (active).

[0082] The SMCHD1 protein is involved in the hypermethylation of a region near the end of chromosome 4 called D4Z4. This region consists of 11 to more than 100 repeated segments, each of which is about 3,300 DNA building blocks (3.3 kb) long. The segment closest to the end of chromosome 4 contains a gene called DUX4. Because the D4Z4 region is hypermethylated, the DUX4 gene is silenced in most adult cells and tissues. Little is known about the function of the protein produced from the DUX4 gene; it appears to help control the activity of other genes. Accordingly, the disclosure provides a gene replacement strategy targeting SMCHD1 to epigenetically silence DUX4 at the DNA locus.

[0083] One of the challenges in transducing and expressing or overexpressing SMCHD1 is that the full-length coding sequence of SMCHD1 (6018 base pair (bp) or nucleotides; see, for example, CCDS45822.1 , Homo sapiens SMCHD1 gene and protein, NCBI; also see SEQ ID NOs: 1 and 2) cannot be packaged into a single adeno-associated virus (AAV) vector. To overcome this challenge, a split intein-mediated protein trans-splicing approach was utilized to express SMCHD1. Inteins are naturally existing intervening protein domains that are expressed along with the host protein, following translation, they spontaneously excise themselves out and ligate the flanking polypeptide sequences (exteins) via a normal peptide bond. Some inteins exist naturally in a split form where the N-terminal and C-terminal halves are embedded in two separately expressed genes. Upon expression, the two halves of the split inteins associate, fold, catalyze splicing in trans and subsequently ligate the flanking peptides (Fig. 1).

[0084] Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This selfexcision process is called "protein splicing," by analogy to the splicing of RNA introns from pre-mRNA. The segments are called "intein" for internal protein sequence, and "extein" for external protein sequence, with upstream exteins termed "N-exteins" and downstream exteins called "C- exteins." The products of the protein splicing process are two stable proteins: the mature protein and the intein.

[0085] Thus, the disclosure provides new products and methods for SMCHD1 gene replacement, i.e., expressing SMCHD1 by utilizing the protein trans-splicing mechanism mediated by split inteins. More specifically, the disclosure provides four pairs of adeno-2835 / 708182024-079-02 associated virus (AAV) constructs using two different split intein pairs that efficiently join the 5’-terminus comprising partial coding sequence of the SMCHD1 gene to the rest of the coding sequence and 3’-terminus of the SMCHD1 gene to generate expression of the full-length SMCHD1 protein. Thus, for each vector pair, the first vector contains the coding sequence for the N-terminal half of SMCHD1 followed by the N-intein coding sequence, and the second vector contains the coding sequence of a C-intein in frame with the C-terminal half of SMCHD1 and a terminal V5 epitope tag.

[0086] In this approach, and as described herein in this disclosure, the coding sequence of SMCHD1 is split into two fragments at sites compatible for intein insertion and protein trans-splicing. A schematic representation of how split intein-mediated protein trans-splicing is and was carried out is shown in Fig. 1. SMCHD1 protein was split at sites compatible with intein-mediated splicing and the corresponding SMCHD1 coding sequences (CDS) were packaged into two AAV vectors. The first vector contains the CDS of the N-terminal half of SMCHD1 protein followed by the CDS of the N-terminal half of the intein. The second vector includes the C-terminal half of the intein attached to the C-terminal half of the SMCHD1 protein. Both AAV vectors contain the same regulatory elements, including inverted terminal repeats (ITR), a promoter, and a polyadenylation signal (pA). The polypeptides are expressed from each expression cassette individually. Upon association, inteins catalyze protein trans-splicing, excising themselves out and ligating the flanking polypeptide sequence.

[0087] Successful intein-mediated splicing relies on specific amino acid residues in the flanking sites to promote proper folding, splicing, and ligation. The native splicing preferred sequence (Iwai et al., FEBS Lett. 580: 1853-58, 2006), N-extein (polypeptide region proximal to intein) residues A(-3) E(-2) Y(-1), split site, C-extein (polypeptide proximal to intein) residues C(+1) F(+2) N(+3) and the non-native residues ( Cheriyan et al., J. Biol. Chem. 288: 6202-11 , 2013; Cheriyan et al., J. Mol. Biol. 426: 4018-29, 2014; Zhu et al., Sci. China Life Sci. 56: 262-67, 2013) are listed in Fig. 2A-B. Among these six residues, the first and second residues in C-extein (C>S>T(+1) and large hydrophobic amino acids F>W>M(+2)) are critical for effective splicing. Thus, the split sites were selected considering factors such as i) the amino acid residue preference surrounding the intein (Fig. 2A-B), ii) secondary structure, iii) avoidance of protein functional domains, and iv) exclusion of residues that are known to be associated with polymorphisms.

[0088] The strategy and amino acid requirements for intein insertion and functional domains in SMCHD1 protein are provided in Fig. 2A-B. In this approach, the coding sequence of SMCHD1 was split into two fragments at sites compatible with intein insertion and protein trans-splicing. Successful intein-mediated splicing relies on specific amino acid2835 / 708182024-079-02 residues in the flanking sites to promote proper folding, splicing, and ligation. The native splicing preferred sequence (Iwai et al., FEBS Lett. 580: 1853-58, 2006), N-extein (polypeptide region proximal to intein) residues A(-3) E(-2) Y(-1), split site, C-extein (polypeptide proximal to intein) residues C(+1) F(+2) N(+3) and the non-native residues (Cheriyan et al., J. Biol. Chem. 288: 6202-11, 2013; Cheriyan et al., J. Mol. Biol. 426: 4018-29, 2014; Zhu et al., Sci. China Life Sci. 56: 262-67, 2013) are listed in Fig. 2A-B. Among these six residues, the first and second residues in C-extein (C>S>T(+1) and large hydrophobic amino acids F>W>M(+2)) are critical for effective splicing. As a result, the split sites were selected considering factors such as i) the amino acid residue preference surrounding the intein (Fig. 2A-B), ii) secondary structure, iii) avoidance of protein functional domains, and iv) exclusion of residues that are known to be associated with polymorphisms.

[0089] The SMCHD1 protein was screened for split sites in the non-functional region, between amino acids 771 to 1190. Five split sites were selected, SS1 at amino acid 882, SS2 at amino acid 956, SS3 at amino acid 9692, SS4 at amino acid 1017, and SS5 at amino acid 1042 (Fig. 2A-B). Two naturally-occurring, fast-acting split inteins derived from Nostoc punctiforme (Npu) and Rhodothermus marinus (Rma) were used for vector designing. An AAV vector harboring AAV2-ITRs, CAG promoter (CMV enhancer (C), chicken 0 actin promoter (A), an intron from 0 globin (G)), and a SV40 polyadenylation signal was used for vector construction (Fig. 3A-D). After specifying the split sites in SMCHD1 , two vectors were constructed in silico using SnapGene software (Dotmatics; snapgene.com). The first vector constructed, i.e.,SMCHD1 -N, contains the coding sequence (CDS) for the N-terminal half of the SMCHD1 protein, along with the N-intein, while the second vector constructed, i.e., SMCHD1-C, contains the C-intein CDS linked to the C-terminal half of SMCHD1, followed by a V5 tag nucleotide sequence. In total, ten constructs were designed using SnapGene software. Considering the ITR-to-ITR packaging capacity, SS2 and SS3 were selected for vector synthesis. Four pairs of AAV constructs, i.e., a total of 8 AAV vector constructs, were created using two distinct split sites (SSs) in combination with two different split inteins, i.e., a split intein derived from Nostoc punctiforme (Npu) and a split intein derived from Rhodothermus marinus (Rma) (see Fig. 3A-D). In each pair, the first vector contains the first half of the SMCHD1 coding sequence fused to the N-intein coding sequence. The second vector contains the C-intein coding sequence, the second half of the SMCHD1 coding sequence, and a V5 tag nucleotide sequence. The insert sequences were synthesized and cloned into AAV vector under CAG promoter (CMV enhancer, chicken 0 actin promoter, and an intron from 0 globin). The relevant split sites and insert sequences for all vector pairs, i.e., 8 individual vectors and 4 vector pairs, are shown in Figs. 7A-D through Figs. 14A-D.2835 / 708182024-079-02

[0090] Fig. 2A provides a table showing amino acid sequence specificity and sites amenable for intein insertion. The native amino acid residues flanking the inteins that promote splicing are highlighted in bold. Additional proximal amino acids that promote intein-mediated splicing are indicated in subsequent rows in the order of preference. Amino acids preceding the N-terminus of the intein are depicted as -1 , -2, and -3, while those flanking the C-terminus of intein are labelled as +1 , +2 and +3. Fig. 2B provides a schematic showing the position of functional domains in SMCHD1 protein, and the region of interest screened for intein insertion. The expanded region of interest shows the amino acid residues, secondary structure, and the selected split sites (SS) in SMCHD1 protein. Amino acids forming the beta-sheet are highlighted in yellow, and those forming the alpha-helix are in pink. The putative amino acids flanking the N-terminus of the intein are boxed in brown and the amino acids flanking the C-terminus of intein are boxed in blue.

[0091] The SMCHD1 protein was screened for split sites in the non-functional region, between amino acids 771 to 1190. Five split sites were selected, SS1 at amino acid 882, SS2 at amino acid 956, SS3 at amino acid 9692, SS4 at amino acid 1017, and SS5 at amino acid 1042 (Fig. 2B). Two naturally occurring fast-acting split inteins derived from Nostoc punctiforme (Npu) and Rhodothermus marinus (Rma) were used for vector designing. AAV vector harboring AAV2-ITR, CAG promoter (CMV enhancer (C), chicken beta actin promoter (A), and an intron from beta globin (G)) and a SV40 polyadenylation signal was used for vector construction (Fig. 3A-D). After specifying the split sites in SMCHD1 , we constructed two vectors in silico using SnapGene: the first vector contains the coding sequence (CDS) for the N-terminal half of the SMCHD1 protein, along with the N-intein, while the second vector consists of the C-intein CDS linked to the C-terminal half of SMCHD1 , followed by a V5 tag nucleotide sequence. The first vector is labeled as SMCHD1-N and the second vector as SMCHD1-C along with the respective intein used. In total, ten constructs were designed using Snapgene. Considering the ITR-to-ITR packaging capacity, SS2 and SS3 were selected for vector synthesis. Four pairs of AAV constructs were created using two distinct SSs in combination with two different split inteins (Fig. 3A-D).

[0092] Fig. 3A-D shows the design strategies for the various dual AAV split-intein vectors for SMCHD1 of the disclosure. Fig. 3A-B are schematic representations of dual AAV-vectors for split sites 2 (SS2) and SS3 containing split intein derived from Nostoc punctiforme (Npu). Fig. 3C-D are schematic representations of dual AAV-vectors for split sites 2 (SS2) and SS3 containing split intein derived from Rhodothermus marinus (Rma). In each vector pair, the first vector, represented as AAV.SMCHD1-N, contains the CDS of the N-terminal half of SMCHD1 followed by the CDS of the N-terminal half of the intein. The second vector, depicted as AAV.SMCHD1-C has the C-terminal half of the intein fused to the C-terminal half2835 / 708182024-079-02 of SMCHD1 and a terminal V5 epitope tag. Both AAV vectors possess the same regulatory elements, including ITR, CAG promoter (CMV enhancer (C), chicken beta actin promoter (A) and an intron from beta globin (G)) and SV40 pA.

[0093] Four pairs of adeno-associated virus (AAV) constructs using two different split inteins were designed. For each vector pair, the first vector contains the coding sequence for the N-terminal half of SMCHD1 followed by the N-intein coding sequence, and the second vector contains the coding sequence of a C-intein in frame with the C-terminal half of SMCHD1 and a terminal V5 epitope tag. More specifically, the AAV split intein constructs were designed as shown in Fig. 3A-D.

[0094] The SMCHD1 protein was screened for splitting sites (SS) suitable for intein insertion and protein trans-splicing. The splitting sites (SS) were selected based on several factors, including the amino acid residue preference surrounding the intein (Fig. 2A-B), secondary structure, the integrity of protein functional domains, and avoidance of the residues that are known to be associated with polymorphisms. Five SS were chosen, SS1 at amino acid 882, SS2 at amino acid 956, SS3 at amino acid 9692, SS4 at amino acid 1017 and SS5 at amino acid 1042. Two different split inteins, one derived from Nostoc punctiforme (Npu) and the other from Rhodothermus marinus (Rma) were used for vector construction. An AAV plasmid (Addgene: 37825) containing AAV2-ITR, CAG promoter (CMV enhancer (C), chicken beta-actin promoter (A), and an intron from the beta-globin (G)) and a SV40 polyadenylation signal was used for vector construction. After splitting the SMCHD1 at a specific site, the corresponding coding sequence (CDS) of the N-terminal half of the SMCHD1 protein and the CDS of N-intein were cloned and represented as SMCHD1-N along with the intein used. The C-intein coding sequence was attached to the second half of the SMCHD1 coding sequence followed by a V5 tag nucleotide sequence. In total, ten constructs were designed using SnapGene software. Taking into account the ITR-to-ITR packaging capacity, SS2 and SS3 were selected for vector generation. Four pairs of AAV constructs were then created using two different SSs in combination with two different split inteins. The relevant split sites and insert sequences / cassettes for all 8 vectors, i.e., 4 vector pairs, are shown in Fig. 7A-C through Fig. 14A-C.

[0095] Fig. 7A-C provides a schematic of the 5-SMCHD1-SS2-Npu expression cassette and its components and sequences, and Fig. 8A-C provides a schematic of the 3-SMCHD1-SS2-Npu expression cassette and its components and sequences. These two expression cassettes were designed to be used together to so that the SMCHD1 polypeptide is expressed or overexpressed in a cell or in a subject.2835 / 708182024-079-02

[0096] Fig. 9A-C provides a schematic of the 5-SMCHD1-SS3-Npu expression cassette and its components and sequences, and Fig. 10A-C provides a schematic of the 3-SMCHD1-SS3-Npu expression cassette and its components and sequences. These two expression cassettes were designed to be used together.

[0097] Fig. 11A-C provides a schematic of the 5-SMCHD1-SS2-Rma expression cassette and its components and sequences, and Fig. 12A-C provides a schematic of the 3-SMCHD1-SS2-Rma expression cassette and its components and sequences. These two expression cassettes were designed to be used together.

[0098] Fig. 13A-C provides a schematic of the 5-SMCHD1 -SS3-Rma expression cassette (Fig. 13A), a detailed map of the components of the expression cassette and its components and sequences, and Fig. 14A-C provides a schematic of the 3-SMCHD1-SS3-Rma expression cassette and its components and sequences. These two expression cassettes were designed to be used together.

[0099] The disclosure thus provides novel nucleic acids designed for use with this strategy, i.e., delivering two expression cassettes, each comprising a 5’ or a 3’ fragment of the SMCHD1 gene, to express SMCHD1 in cells in vitro, and / or in cells of a subject to downregulate and / or inhibit DUX4 expression. The nucleic acids are designed so that they can be delivered together so that the 5’ end of the SMCHD1 gene and the 3’ end of the SMCHD1 gene will be recombined for the expression of a functional SMCHD1 protein once they are delivered to the cell or subject.

[0100] The disclosure thus provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide.

[0101] The disclosure also provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide,2835 / 708182024-079-02 wherein the nucleotide sequence comprises at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

[0102] Thus, the SMCHD1 gene / SMCHD1 polypeptide is divided according to the strategy described herein, including as depicted in Figs. 1-4A-F, so there is an N-terminal (5’-terminal) half and a C-terminal (3’-terminal half). In some aspects, each nucleic acid, either the 5’ half or the 3’ half, is inserted in a vector, e.g., an AAV vector. In each vector pair, the first vector, represented as AAV.SMCHD1-N, contains the CDS of the N-terminal half of SMCHD1 followed by the CDS of the N-terminal half of the intein. The second vector, depicted as AAV.SMCHD1-C has the C-terminal half of the intein fused to the C-terminal half of SMCHD1. As disclosed herein, the so-called “half” of the SMCHD1 gene does not need to be a true “half” but instead is designed so that it is conducive to the split intein design to accomplish protein trans-splicing.

[0103] These two nucleic acids, i.e., (1) the nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1 ) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide; and (2) the nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide are designed so that they can be transduced separately into a cell, into a cell of a subject, or into a subject so that the large SMCHD1 gene can be expressed in the cell, int the cell of the subject, or in the subject in order to downregulate and / or inhibit the expression of DUX4.

[0104] In some aspects, the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 1. In some aspects, the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at2835 / 708182024-079-02 least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0105] The disclosure thus provides various nucleic acids comprising, consisting essentially of, or consisting of the various nucleotide sequences described herein. In some aspects, the nucleic acid comprises the nucleotide sequence. In some aspects, the nucleic acid consists essentially of the nucleotide sequence. In some aspects, the nucleic acid consists of the nucleotide sequence.

[0106] The terms “nucleotide sequence” and “polynucleotide sequence” are used interchangeably herein. In some aspects, a nucleic acid of the disclosure comprises a nucleotide sequence comprising the sequence of any one of SEQ ID NOs: 1 and 3-31 , or a variant thereof comprising at least or about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of any one of SEQ ID NOs: 1 and 3-31. In some aspects, a polypeptide of the disclosure comprises an amino acid sequence comprising the sequence of SEQ ID NO: 2, or a variant thereof comprising at least or about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 2.

[0107] The disclosure includes a nucleotide sequence encoding an N-terminal fragment of the SMCHD1 polypeptide comprising the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

[0108] The disclosure includes a nucleotide sequence encoding a C-terminal fragment of the SMCHD1 polypeptide comprising the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

[0109] The disclosure includes a nucleotide sequence encoding the N-intein domain comprising the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11 , 13, 15, or 17.

[0110] The disclosure includes a nucleotide sequence encoding the C-intein domain comprising the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.2835 / 708182024-079-02

[0111] In some aspects, a nucleic acid of the disclosure comprises an enhancer. Different types of enhancers for gene expression include, but are not limited to, cell type-specific enhancers (activated only in certain cell types), tissue-specific enhancers (active in specific tissues), developmental enhancers (regulating gene expression during development), superenhancers (highly active enhancers controlling key regulatory genes), and long-range enhancers (located far away from the gene they regulate), all of which work by binding transcription factors to influence gene expression levels depending on the cellular context. In some aspects, the enhancer is a CMV enhancer. In some aspects, the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19 or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0112] In some aspects, a nucleic acid of the disclosure comprises a promoter. In some aspects, the promoter is a polymerase II promoter or a polymerase III promoter. In some aspects, the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a U6 promoter, a U7 promoter, a tRNA promoter, an H1 promoter, a T7 promoter, an, EF1 -alpha promoter, a minimal EF1 -alpha promoter, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter. In some aspects, the musclespecific promoter is unc45b, tMCK, minimal MCK, CK6, CK7, CK8, MHCK7, or CK1 . In some exemplary aspects, a beta-actin is used. In some aspects, the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising a nucleotide sequence comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0113] In some aspects, a nucleic acid of the disclosure comprises an SV40 poly(A) signal. In some aspects, the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising a nucleotide sequence comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

[0114] In various aspects, a nucleic acid of the disclosure comprises a 5’ ITR sequence and a 3’ ITR sequence flanking the sequence encoding the fusion protein comprising the SMCHD1 5’ or 3’ fragment and the intein 5’ or 3’ fragment. In some aspects, the single or pair of AAV ITR sequences are AAV2, AAV6, AAV8, AAVrh.74, or AAV9 ITR sequences. In some aspects, the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising a nucleotide sequence comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22. In some aspects, the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.2835 / 708182024-079-02

[0115] The disclosure includes a nucleic acid construct designated 5-SMCHD1 -SS2-Npu comprising the nucleotide sequence of SEQ ID NO: 24, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24.

[0116] The disclosure includes a nucleic acid construct designated 3-SMCHD1 -SS2-Npu comprising the nucleotide sequence of SEQ ID NO: 25, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25.

[0117] The nucleic acid construct designated 5-SMCHD1-SS2-Npu and the nucleic acid construct designated 3-SMCHD1-SS2-Npu are designed to work together to express SMCHD1 protein.

[0118] The disclosure includes a nucleic acid construct designated 5-SMCHD1-SS3-Npu comprising the nucleotide sequence of SEQ ID NO: 26, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 26.

[0119] The disclosure includes a nucleic acid construct designated 3-SMCHD1-SS3-Npu comprising the nucleotide sequence of SEQ ID NO: 27, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 27.

[0120] The nucleic acid construct designated 5-SMCHD1-SS3-Npu and the nucleic acid construct designated 3-SMCHD1-SS3-Npu are designed to work together to express SMCHD1 protein.

[0121] The disclosure includes a nucleic acid construct designated 5-SMCHD1-SS2-Rma comprising the nucleotide sequence of SEQ ID NO: 28, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 28.

[0122] The disclosure includes a nucleic acid construct designated 3-SMCHD1-SS2-Rma comprising the nucleotide sequence of SEQ ID NO: 29, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 29.

[0123] The nucleic acid construct designated 5-SMCHD1-SS2-Rma and the nucleic acid construct designated 3-SMCHD1 -SS2-Rma are designed to work together to express SMCHD1 protein.

[0124] The disclosure includes a nucleic acid construct designated 5-SMCHD1-SS3-Rma comprising the nucleotide sequence of SEQ ID NO: 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 30.

[0125] The disclosure includes a nucleic acid construct designated 3-SMCHD1-SS3-Rma comprising the nucleotide sequence of SEQ ID NO: 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 31.2835 / 708182024-079-02

[0126] The nucleic acid construct designated 5-SMCHD1-SS3-Rma and the nucleic acid construct designated 3-SMCHD1 -SS3-Rma are designed to work together to express SMCHD1 protein.

[0127] In some aspects, the sequence identity is over the full-length sequence. In some aspects, the sequence identity is not limited to the full-length sequence. In some aspects, the nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more substitutions. In some aspects, when the sequence is smaller, the nucleotide sequence comprises 1 , 2, 3, 4, or 5 substitutions. In some aspects, the substitutions are conservative substitutions.

[0128] The terms “polypeptide sequence” and “amino acid sequence” are used interchangeably herein. In some aspects, a polypeptide of the disclosure comprises an amino acid sequence comprising at least or about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 2. In some aspects, the sequence identity is over the full-length sequence. In some aspects, the sequence identity is not limited to the full-length sequence. In some aspects, the amino acid sequence comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more substitutions. In some aspects, the sequence comprises 1, 2, 3, 4, or 5 substitutions. In some aspects, the substitutions are conservative substitutions. Nucleotide and polypeptide sequences of the disclosure are also provided herein in Table 1 below.

[0129] Table 1. Nucleotide and Polypeptide Sequences of the Disclosure.2835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-022835 / 708182024-079-02

[0130] In some embodiments, the disclosure includes a vector comprising any of the nucleic acids described herein. Thus, embodiments of the disclosure utilize vectors (for example, viral vectors, such as adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, herpes simplex virus, polio virus, sindbis virus, vaccinia virus or a synthetic virus, e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and / or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule) to deliver the nucleic acids disclosed herein.

[0131] In some aspects, the disclosure provides a viral vector to deliver a nucleic acid disclosed herein. In some aspects, the viral vector is an AAV vector. AAV is a replicationdeficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV and the nucleotide sequences of the genomes of the AAV serotypes are known in the art. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1 , VP2, and VP3. Alternative splicing2835 / 708182024-079-02 and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka (Current Topics in Microbiology and Immunology, 158: 97-129 (1992)).

[0132] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and nondividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. In some aspects, the rep and cap proteins are provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56Qto 65QC for several hours), making cold preservation of AAV less critical. AAV may be lyophilized and AAV-infected cells are not resistant to superinfection.

[0133] In some aspects, the AAV vector is recombinant AAV (rAAV) vector. In some aspects, the vector is a single-stranded AAV vector (ss-AAV or ss-rAAV). In some aspects, the vector is a self-complementary AAV vector (sc-AAV or sc-rAAV). In some aspects, the rAAV lack rep and cap genes.

[0134] Thus, in some aspects, the viral vector is an adeno-associated virus (AAV), such as an AAV1 (i.e., an AAV containing AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 capsid proteins), AAV9 (i.e., an AAV containing AAV9 capsid proteins), AAVrh74 (i.e., an AAV containing AAVrh74 capsid proteins), AAVrh.8 (i.e., an AAV containing AAVrh.8 capsid proteins), AAVrh.10 (i.e., an AAV containing AAVrh.10 capsid proteins), AAV11 (i.e., an AAV containing AAV11 capsid proteins), AAV12 (i.e., an AAV containing AAV12 capsid proteins), AAV13 (i.e., an AAV containing AAV13 capsid proteins), AAV-anc80, AAV-B1, AAV-BR1, AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1 A,2835 / 708182024-079-02 MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9) (e.g., SEQ ID NO: 13 of WO 2021 / 072197), or AAV-SLB101 (e.g., SEQ ID NO: 14 of WO 2021 / 072197), or any derivative thereof. In some exemplary aspects, the vector comprises a capsid of MYOAAV3A or a derivative thereof.

[0135] In some embodiments, DNA plasmids of the disclosure are provided which comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1 -deleted adenovirus or herpes virus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art.

[0136] In certain embodiments, the rAAV is produced based on the helper-virus-free transient transfection method, with all cis and trans components (vector plasmid and packaging plasmids, along with helper genes isolated from adenovirus) in suitable host cells such as 293 cells. The transient-transfection method is simple in vector plasmid construction and generates high-titer AAV vectors that are free of adenovirus. The modified VP1 capsid proteins can be encoded by one of the plasmids used in transient transfection of the producer cell line.

[0137] In certain embodiments, the rAAV is produced using a recombinant herpes simplex virus (rHSV)-based AAV production system, which utilizes rHSV vectors to bring the AAV vector and the Rep and Cap genes (i.e., the modified VP1 capsid gene of the invention) into the producer cells. The modified cap gene can be present in the rHSV vector that may also hosts the rAAV genome.

[0138] In certain embodiments, the rAAV is produced using a baculovirus system that requires simultaneous infection of insect cells with several baculovirus vectors to deliver the AAV vector cassette and the Rep and Cap genes (i.e., the modified VP1 capsid gene of the invention).

[0139] In certain embodiments, the rAAV is produced based on certain AAV producer cell lines derived from, e.g., HeLa or A549 or HEK293 cells, which stably harbored AAV Rep / cap genes (i.e., the modified VP1 capsid gene of the invention). The AAV vector cassette can either be stably integrated in the host genome or be introduced by an adenovirus that contained the cassette.

[0140] In certain embodiments, such producer cell line for rAAV production comprises an rAAV provirus that encodes the DLIX4 miRNA sequence of interest flanked by the AAV ITR sequences, wherein the rAAV provirus is integrated into the genome of the producer cell line for rAAV production.2835 / 708182024-079-02

[0141] In certain embodiments, production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhl 0, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof.

[0142] In some aspects, AAV DNA in the rAAV genomes is from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhl 0, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof. Other types of rAAV variants, for example rAAV with capsid mutations, are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900-1909 (2014). More specifically, rAAV variants, as disclosed in WO 2021 / 072197 are included in the disclosure. WO 2021 / 072197 is incorporated by reference herein in its entirety. As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692 which is incorporated by reference herein in its entirety.

[0143] Recombinant AAV genomes of the disclosure comprise one or more AAV ITRs flanking at least one nucleotide sequence encoding the fusion protein sequence comprising the SMCH1 D fragment and the intein fragment as described herein. In some aspects, it is contemplated that the nucleotide sequence encoding the fusion protein sequence comprising the SMCH1 D fragment and the intein fragment is administered with other polynucleotide constructs targeting DUX4.

[0144] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1 -deleted adenovirus or herpes virus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep2835 / 708182024-079-02 and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1 A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof

[0145] In some aspects, AAV DNA in the rAAV genomes is from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof. Other types of rAAV variants, for example rAAV with capsid mutations, are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900-1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692 which is incorporated by reference herein in its entirety.

[0146] In some embodiments, packaging cells are provided. Packaging cells are created in order to have a cell line that stably expresses all the necessary components for AAV particle production. Retroviral vectors are created by removal of the retroviral gag, pol, and env genes. These are replaced by the therapeutic gene. In order to produce vector particles, a packaging cell is essential. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. Thus, packaging cell lines are made so that they contain the gag, pol and env genes. Following insertion of the desired gene into in the retroviral DNA vector, and maintenance of the proper packaging cell line, it is now a simple matter to prepare retroviral vectors. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081], addition of synthetic linkers containing restriction endonuclease cleavage sites [Laughlin et al., 1983, Gene, 23:65-73] or by direct, blunt-end ligation [Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666]. The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are2835 / 708182024-079-02 that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0147] In some embodiments, therefore, a method of generating a packaging cell to create a cell line that stably expresses all the necessary components for AAV particle production is provided. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081], addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy et al., 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0148] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbiol, and Immunol. 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); T ratschin et al., Mo1. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No.5,658.776 ; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al., Vaccine, 13:1244-1250 (1995); Paul et al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy, 3:1124-1132 (1996); U.S. Patent. No. 5,786,211 ; U.S. Patent No. 5,871,982; U.S. Patent. No. 6,258,595; and McCarty, Mol. Then, 16(10): 1648-1656 (2008). The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production. The production and use of various types of rAAV are specifically contemplated and exemplified.Recombinant AAV ( / .e., infectious encapsidated rAAV particles) are thus provided herein. In some aspects, genomes of the rAAV lack AAV rep and cap genes; that is, there is no AAV rep or cap DNA between the ITRs of the genomes of the rAAV. In some embodiments, the2835 / 708182024-079-02 AAV is a recombinant linear AAV (rAAV), a single-stranded AAV (ssAAV), or a recombinant self-complementary AAV (scAAV).

[0149] The disclosure thus provides in some embodiments packaging cells that produce infectious rAAV. In one embodiment, packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0150] The rAAV, in some aspects, are purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Then, 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0151] In some embodiments, the disclosure provides a composition or compositions comprising a nucleic acid or a vector, as described herein with other ingredients, such as a carrier, diluent, excipient, buffer, or adjuvant. Acceptable carriers, diluents, excipients, buffers, and / or adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG). Thus, compositions comprising delivery vehicles (such as rAAV) described herein are provided. In various aspects, such compositions also comprise a pharmaceutically acceptable carrier. In general, as used herein, "pharmaceutically acceptable carrier" means all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, e.g. phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media and coatings, which are compatible with pharmaceutical administration, in particular with parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and the compositions comprising such carriers can be formulated by well-known conventional methods.2835 / 708182024-079-02

[0152] In some aspects, the disclosure provides AAV transducing cells for the delivery of nucleic acids and / or vectors as described herein. Methods of transducing a target cell with rAAV, in vivo or in vitro, are included in the disclosure. The methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the disclosure to a subject, including an animal (such as a human being) in need thereof. If the dose is administered prior to development of the disease, e.g., a muscular dystrophy, including but not limited to FSHD, a cancer, or BAMS, the administration is prophylactic. If the dose is administered after the development of the disease, the administration is therapeutic. In embodiments of the disclosure, an effective dose or a therapeutically effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disease being treated, that slows or prevents progression of the disease, that diminishes the extent of disease, that results in remission (partial or total) of the disease, and / or that prolongs survival.

[0153] In some other aspects, a nucleic acid of the disclosure is introduced into the cell via non-vectorized delivery. Thus, in an embodiment, the disclosure includes non-vectorized delivery of a nucleic acid of the disclosure. In some aspects, in this context, synthetic carriers able to form complexes with nucleic acids, and protect them from extra- and intracellular nucleases, are an alternative to viral vectors. In some aspects, such non-vectorized delivery includes the use of nanoparticles, extracellular vesicles, or exosomes comprising the nucleic acids of the disclosure. The disclosure also includes compositions comprising any of the constructs described herein alone or in combination.

[0154] Methods of transducing a target cell with a nucleic acid, vector, composition, or system, in vivo or in vitro, are contemplated by the disclosure. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a nucleic acid, vector, including but not limited to an rAAV, composition, or system of the disclosure to an animal or mammal (including a human being) in need thereof. If the dose is administered prior to development of a disorder / disease, the administration is prophylactic. If the dose is administered after the development of a disorder / disease, the administration is therapeutic. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, that slows or prevents progression to a disorder / disease state, that slows or prevents progression of a disorder / disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and / or that prolongs survival. In some aspects, a therapeutically effective dose is defined as a dose that yields a therapeutic benefit to a subject.2835 / 708182024-079-02

[0155] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-f iltered solution thereof.

[0156] Titers of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1x106, about 1x107, about 1x108, about 1x109, about 1x101°, about 1x1011, about 1x1012, about 1x1013, about 1x1014, about 1x1015, about 1x1016, to about 1x1017or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (e.g., 1x107vg, 1x108vg, 1x109vg, 1x101° vg, 1x1011vg, 1x1012vg, 1x1013vg, 1x1014vg, 1x1015vg, 1x1016vg, and 1x1017vg, respectively). In some aspects, dosages are expressed in units of viral genomes (vg) per kilogram (kg) body weight (e.g., 1x107vg / kg, 1x108vg / kg, 1x109vg / kg, 1x101° vg / kg, 1x1011vg / kg, 1x1012vg / kg, 1x1013vg / kg, 1x1014vg / kg, 1x1015vg / kg, 1x1016vg / kg, and 1x1017vg / kg, respectively). Additional information regarding an effective dose, or a therapeutically effective dose, as used herein, is provided herein below.

[0157] In embodiments of the disclosure, an effective dose or a therapeutically effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disease being treated, that slows or prevents progression of the disease, that diminishes the extent of disease, that results in remission (partial or total) of the disease, and / or that prolongs survival.

[0158] In some aspects, the disclosure provides a method of delivering to a cell or to a subject any one or more nucleic acids of the disclosure. In some aspects, the disclosure provides a method of expressing in a cell or in a subject any one or more nucleic acids of the disclosure. In some aspects, the disclosure provides a method of inhibiting and / or interfering with expression of a DLIX4 gene in a cell comprising contacting the cell with or administering to the subject at least two nucleic acids of the disclosure, each nucleic acid comprising a 5’ or 3’ fragment of the large coding sequence of SMCHD1 with a split intein, so that the full-length SMCHD1 protein, or a variant thereof comprising SMCHD1 biological activity, is expressed in the cell or subject to interfere or inhibit the expression of DUX4.2835 / 708182024-079-02

[0159] Thus, the disclosure provides a method of decreasing and / or inhibiting the expression of a DUX4 gene in a cell comprising introducing into the cell (a) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide; and (b) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and (ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

[0160] In some embodiments, the disclosure provides a method of delivering to a cell any one of more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma expression cassette and the 3-SMCHD1-SS3-Rma expression cassette, wherein the method comprises introducing into the cell any one or more of the four combinations of expression cassettes as set out above.

[0161] In some embodiments, the disclosure provides a method of expressing in a cell any one of more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma2835 / 708182024-079-02 expression cassette and the 3-SMCHD1-SS3-Rma expression cassette, wherein the method comprises introducing into the cell any one or more of the four combinations of expression cassettes as set out above.

[0162] In some embodiments, the disclosure provides a method of decreasing and / or inhibiting the expression of a DLIX4 gene in a cell comprising introducing into the cell any one of more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma expression cassette and the 3-SMCHD1-SS3-Rma expression cassette.

[0163] In some embodiments, the disclosure provides a method of treating a subject suffering from or at risk of suffering from a disease or disorder associated with DLIX4 expression comprising administering to the subject an effective amount of any one or more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma expression cassette and the 3-SMCHD1-SS3-Rma expression cassette.

[0164] In some aspects, the nucleic acid, or pair of nucleic acids comprising the nucleotide sequence encoding the two fusion proteins, is administered to the subject in a vector as described herein. In some aspects, the nucleic acid or vector is provided in a composition which is administered to the subject. In some aspects, the subject is a human subject.

[0165] In some aspects, the disease or disorder associated with DUX4 is muscular dystrophy, cancer, or Bosma arhinia microphthalmia syndrome (BAMS), a rare genetic disorder that causes abnormalities in the nose, eyes and puberty, e.g., absence of nose, eye defects, and absence of sexual maturation. In some aspects, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some aspects, the cancer is a cancer associated with DUX4 overexpression. In some aspects, the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus. In some aspects, the disease or

[0166] In some aspects, the disclosure provides a method of treating muscular dystrophy (such as FSHD), a cancer, or BAMS associated with DLIX4 overexpression in a subject in2835 / 708182024-079-02 need thereof, the method comprising administering to the subject an effective amount, or a therapeutically effective amount, of a recombinant AAV (rAAV) vector comprising any one or more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma expression cassette and the 3-SMCHD1-SS3-Rma expression cassette.

[0167] In some aspects, expression of DLIX4 or the expression of functional DLIX4 is decreased in a cell or in a subject by the methods provided herein by at least or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100 percent.

[0168] Combination therapies are also contemplated by the disclosure. Combination as used herein includes simultaneous treatment or sequential treatments. Combinations of methods of the disclosure with standard medical treatments (e.g., corticosteroids and / or immunosuppressive drugs) or with other inhibitory RNA constructs are specifically contemplated, as are combinations with other therapies such as those disclosed in International Publication No. WO 2013 / 016352, which is incorporated by reference herein in its entirety.

[0169] Administration of an effective dose of the nucleic acids, compositions, or vectors, including AAV, nanoparticles, extracellular vesicles, and exosomes comprising the compositions and nucleic acids of the disclosure, may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intracerebroventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and / or matched by those skilled in the art taking into account the disease state being treated and the target cells / tissue(s), such as cells that express DUX4. In some embodiments, the composition or medicament is formulated for intramuscular injection, oral administration, subcutaneous, intradermal, intraventricular, or transdermal transport, injection into the blood stream, or for aerosol administration. In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is intravenous.

[0170] In some aspects, actual administration of rAAV of the present disclosure may be accomplished by using any physical method that will transport the rAAV recombinant vector2835 / 708182024-079-02 into the target tissue of an animal. Administration according to the disclosure includes, but is not limited to, injection into muscle, the bloodstream, the central nervous system, and / or directly into the brain or other organ. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated by reference herein.Pharmaceutical compositions can be prepared for oral administration, as injectable formulations, or as topical formulations to be delivered to the muscles by subcutaneous, intradermal, intraventricular, and / or transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the disclosure. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0171] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0172] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. In some aspects, proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by2835 / 708182024-079-02 the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0173] In some aspects, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient which protects the formulation from adverse conditions, such as those which occur during heating or freezing, and / or prolongs the stability or shelflife of the formulation in a stable state. Examples of stabilizers include, but are not limited to, sugars, such as sucrose, lactose and mannose; sugar alcohols, such as mannitol; amino acids, such as glycine or glutamic acid; and proteins, such as human serum albumin or gelatin.

[0174] In some aspects, the formulation comprises an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance which is added to the composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container being used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0175] The term "transduction" is used to refer to the administration / delivery of any one or more of (1) the 5-SMCHD1-SS2-Npu expression cassette and the 3-SMCHD1-SS2-Npu expression cassette; (2) the 5-SMCHD1-SS3-Npu expression cassette and the 3-SMCHD1-SS3-Npu expression cassette; (3) the 5-SMCHD1-SS2-Rma expression cassette and the 3-SMCHD1-SS2-Rma expression cassette; and / or (4) the 5-SMCHD1-SS3-Rma expression cassette and the 3-SMCHD1-SS3-Rma expression cassette via a replication-deficient rAAV of the disclosure resulting in expression of SMCHD1 and resulting in decreased expression of DUX4 by the recipient cell or the recipient subject.

[0176] Methods of transducing a target cell with a delivery vehicle (such as rAAV), in vivo or in vitro, are contemplated. Transduction of cells with an rAAV of the disclosure results in sustained expression the sequences of the expression cassettes resulting in sustained expression of SMCHD1. The disclosure thus provides rAAV and methods of administering / delivering rAAV which express SMCHD1 in the cell(s) in vitro or in a subject in vivo. In some aspects, the subject is a mammal. In some aspects, the mammal is a human. These methods include transducing cells and tissues (including, but not limited to, tissues such as muscle) with one or more rAAV described herein. Transduction may be carried out2835 / 708182024-079-02 with gene cassettes comprising cell-specific control elements, as disclosed herein. The term “transduction” is used to refer to, as an example, the administration / delivery of the gene cassettes designed to deliver SMCHD1 , to a target cell either in vivo or in vitro, via a replication-deficient rAAV described herein resulting in the decreased expression or inhibition of expression of DLIX4 by the target cell.

[0177] In one aspect, transduction with rAAV is carried out in vitro. In one embodiment, desired target cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.

[0178] Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells are transduced in vitro by combining rAAV with cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and / or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter.

[0179] The disclosure provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV that comprise DNA that encodes SMCHD1 which in turn is designed to downregulate or inhibit the expression of DUX4 to a cell or to a subject in need thereof. In some aspects, the effective dose is therefore a therapeutically effective dose.

[0180] In some embodiments, the dose or effective dose of rAAV administered is about 1.0x1010vg / kg to about 1.0x1016vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 1.0x1011vg / kg to about 1.0x1015vg / kg. In some embodiments, the dose or effective dose of rAAV administered is from about 9.0x1012vg / kg to about 6x1014vg / kg. In some embodiments, the dose or effective dose of rAAV administered is from about 1.0x1013vg / kg to about 1.0x1014vg / kg. In some embodiments, the dose or effective dose of rAAV administered is from about 3.0x1013vg / kg to about 6.0x1013vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 1 .0x1014vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 6.0x1012vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 9.0x1012vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 3.0x1013vg / kg. In some embodiments, the dose or effective dose of rAAV administered is about 6.0x1013vg / kg. In some aspects the dose or effective dose of rAAV is2835 / 708182024-079-02 about 1.0x1010vg / kg, about 2.0x1010vg / kg, about 3.0x1010vg / kg, about 4.0x1010vg / kg, about 5.0x1010vg / kg, about 6.0x1010vg / kg, about 7.0x1010vg / kg, about 8.0x1010vg / kg, about 9.0x1010about 1.0x1011vg / kg, about 2.0x1011vg / kg, about 3.0x1011vg / kg, about 4.0x1011vg / kg, about 5.0x1011vg / kg, about 6.0x1011vg / kg, about 7.0x1011vg / kg, about 8.0x1011vg / kg, about 9.0x1011vg / kg, about 1.0x1012vg / kg, about 2.0x1012vg / kg, about 3.0x1012vg / kg, about 4.0x1012vg / kg, about 5.0x1012vg / kg, about 6.0x1012vg / kg, about 7.0x1012vg / kg, about 8.0x1012vg / kg, about 9.0x1012vg / kg, about 1.0x1013vg / kg, about 2.0x1013vg / kg, about 3.0x1013vg / kg, about 4.0x1013vg / kg, about 5.0x1013vg / kg, about 6.0x1013vg / kg, about 7.0x1013vg / kg, about 8.0x1013vg / kg, about 9.0x1013vg / kg, about 1.0x1014vg / kg, about 2.0x1014vg / kg, about 3.0x1014vg / kg, about 4.0x1014vg / kg, about 5.0x1014vg / kg, about 6.0x1014vg / kg, about 7.0x1014vg / kg, about 8.0x1014vg / kg, about 9.0x1014vg / kg, about 1.0x1015vg / kg, about 2.0x1015vg / kg, about 3.0x1015vg / kg, about 4.0x1015vg / kg, about 5.0x1015vg / kg, about 6.0x1015vg / kg, about 7.0x1015vg / kg, about 8.0x1015vg / kg, about 9.0x1015vg / kg, or about 1 .0x1016vg / kg.

[0181] In some aspects, 1 .0x1010vg / kg is also designated 1 .0 E10 vg / kg, which is simply an alternative way of indicating the scientific notation. Likewise, 1011is equivalent to E11 , 1012is equivalent to E12, 1013is equivalent to E13, and the like. In some aspects, the dose or effective dose of rAAV administered is about 1 .0x1011vg / kg to about 1.0x1015vg / kg. In some aspects, the dose or effective dose of rAAV administered is about 1 .0x1010vg / kg to about 1.0x1015vg / kg. In some aspects, the dose is about 1 .0x1011vg / kg to about 5.0x1014vg / kg.

[0182] In some aspects, an initial dose is followed by a second greater dose. In some aspects, an initial dose is followed by a second same dose. In some aspects, an initial dose is followed by one or more lesser doses. In some aspects, an initial dose is followed by multiple doses which are the same or greater doses.

[0183] The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a delivery vehicle (such as rAAV) to a subject (including a human subject) in need thereof. Thus, methods are provided of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV described herein to a subject in need thereof. If the dose or doses is administered prior to development of a disorder / disease, the administration is prophylactic. If the dose or doses is administered after the development of a disorder / disease, the administration is therapeutic. An effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, that slows or prevents progression to a disorder / disease state, that2835 / 708182024-079-02 slows or prevents progression of a disorder / disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and / or that prolongs survival.

[0184] In some embodiments, nucleic acids, vectors, compositions, systems, and methods of the disclosure are used in treating, ameliorating, or preventing a disease or disorder associated with DUX4 expression.

[0185] In some aspects, such disease or disorder is a muscular dystrophy (MD), a cancer, or BAMS. In various aspects, such MD is FSHD. FSHD is among the most commonly inherited muscular dystrophies, estimated to affect as many as 870,000 individuals.Classical descriptions of FSHD presentation include progressive muscle weakness in the face, shoulder-girdle and arms, but disease can manifest more broadly, including in muscles of the trunk and lower extremities. Variability is also commonly seen within individuals, as asymmetrical weakness is common. Age-at-onset can range from early childhood to adulthood, and is usually related to disease severity, where earlier onset is often associated with more severe muscle weakness. Although most patients with FSHD have a normal life span, respiratory insufficiency can occur, and the disease can be debilitating, as approximately 25% of affected individuals may become wheelchair dependent by their fifties, and even earlier in more severe forms of the disease, while others maintain lifelong ambulation. FSHD is caused by aberrant expression of the double homeobox 4 gene (DUX4), which produces a transcription factor that is toxic to skeletal muscle. DUX4 is normally functional during the two-cell stage of human development but repressed thereafter in essentially all other tissues, except perhaps the testes. In skeletal muscles of people with FSHD, specific genetic and epigenetic factors conspire to permit DUX4 de-repression, where it then initiates several aberrant gene expression cascades, including those involved in differentiation abnormalities, oxidative stress, inflammatory infiltration, cell death and muscle atrophy. In families known to carry pathological FSHD, the methods of the disclosure, in various aspects, are methods of preventing disease and they are carried out before the onset of disease. In other various aspects, the methods of the disclosure are carried out after diagnosis and, therefore, are methods of treating or ameliorating disease.

[0186] In some embodiments, nucleic acids, vectors, compositions, systems, and methods of the disclosure are used in treating, ameliorating, or preventing a disease, such as a cancer. DUX4 has been shown to be activated in some cancer types, where it functions to mask tumor cells from the immune system [Chew et al., Dev. Cell 50(5):658-71 (2019)]. For example, DUX4 protein fusions are known to cause cancer, such as rhabdomyosarcoma and Ewing's sarcoma. A CIC-DUX4 gene fusion induces sarcomas and drives sarcoma metastasis [Yoshimoto et al., Cancer Res. 2017 Jun 1 ; 77(11): 2927-2937; Okimoto et al., J Clin Invest. 2019; 129(8):3401-3406)]. Other cancer tissues, such2835 / 708182024-079-02 as those tissues from the adrenal, B-cell lymphoma, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain (e.g., lower grade glioma), lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, and thymus, also were shown to express DLIX4 [Chew et al., Dev. Cell 50(5):658-71 (2019)]. Thus, the nucleic acids, vectors, systems, and compositions described herein are used in inhibiting DLIX4 expression in the treatment, amelioration, or prevention of cancer.

[0187] In some embodiments, nucleic acids, vectors, compositions, systems, and methods of the disclosure are used in treating, ameliorating, or preventing a disease, such as Bosma arhinia microphthalmia syndrome (BAMS). BAMS or BAM syndrome is an extremely rare genetic disorder that is defined by three major features: 1) complete absence of the nose 2) eye defects and 3) absent sexual maturation. Thus, the nucleic acids, vectors, systems, and compositions described herein are used in inhibiting DLIX4 expression in the treatment, amelioration, or prevention of BAMS.

[0188] Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the nucleic acids, vectors, compositions, systems, and methods disclosed herein for decreasing the expression of the DUX4 gene and protein and treating muscular dystrophies, such as FSHD. Outcome measures are described, for example, in Chapters 32, 35 and 43 of Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4thEdition, Volume 1 (2005) and in Burgess et al., Methods Mol. Biol., 602: 347-393 (2010). Outcome measures include, but are not limited to, reduction or elimination of DLIX4 mRNA or protein in affected tissues. The lack of expression of DLIX4 and / or the downregulation of expression of DUX4 in the cell is detected by measuring the level of DUX4 protein by methods known in the art including, but not limited to, RT-PCR, QRT-PCR, RNAscope, Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of the rAAV to determine the improvement.

[0189] In some embodiments, the level of DLIX4 gene expression or protein expression in a cell of the subject is decreased after administration of the nucleic acids encoding the two SMCHD1 fragments so that there is SMCHD1 recombination or the vector, e.g., rAAV, comprising the nucleic acids encoding the two SMCHD1 fragments so that there is SMCHD1 recombination as compared to the level of DUX4 gene expression or protein expression before administration of the nucleic acids encoding the two SMCHD1 fragments so that there is SMCHD1 recombination or the vector, e.g. rAAV. In some aspects, expression of DLIX4 is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100% percent, or at least about greater than 100%. In various aspects, improved muscle2835 / 708182024-079-02 strength, improved muscle function, and / or improved mobility and stamina show an improvement by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100% percent, or at least about greater than 100%.

[0190] Other outcome measures include measuring the level of serum creatinine kinase (CK) in the subject before and after treatment. Increased CK levels are a hallmark of muscle damage. In muscular dystrophy patients, CK levels are significantly increased above the normal range (10 to 100 times the normal level since birth). When elevated CK levels are found in a blood sample, it usually means muscle is being disintegrated by some abnormal process, such as a muscular dystrophy or inflammation. Thus, a positive therapeutic outcome for treatment with the methods of the disclosure is a reduction in the level of serum creatinine kinase after administration of the rAAV as compared to the level of serum creatinine kinase before administration of the rAAV.

[0191] Other outcome measures include, but are not limited to, measuring to determine if there is improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject after treatment. Such outcome measures are important in determining muscular dystrophy progression in the subject and are measured by various tests known in the art. Some of these tests include, but are not limited to, the six minute walk test, time to rise test, ascend 4 steps test, ascend and descend 4 steps test, North Star Ambulatory Assessment (NSAA) test, 10 meter timed test, 100 meter timed test, hand held dynamometry (HHD) test, Timed Up and Go test, Gross Motor Subtest Scaled (Bayley-Ill) score, maximum isometric voluntary contraction test (MVICT), or a combination of two or more thereof.

[0192] Combination therapies are also contemplated by the disclosure. A combination therapy, as used herein, includes both simultaneous treatment(s) and sequential treatment(s). Combinations of methods described herein with standard medical treatments and supportive care are specifically contemplated, as are combinations with therapies, such as glucocorticoids. All types of glucocorticoids are included for use in the combination therapies disclosed herein. Such glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.

[0193] Other combination therapies included in the disclosure are the administration of DUX4 miRNAs in combination with other miRNAs, or in combination with U7-snRNA-based gene therapy, a small molecule inhibitor of DUX4 expression, oligonucleotides to inhibit2835 / 708182024-079-02 DUX4 through RNAi or RNAse H or exon skipping mechanisms, U7-snRNA plus a theoretical CRISPR-based gene therapy approach.

[0194] "Treating" includes ameliorating or inhibiting one or more symptoms of a disease or disorder associated with DUX4 expression. In some aspects, treating ameliorates a symptom of a muscular dystrophy including, but not limited to, muscle wasting, muscle weakness, myotonia, skeletal muscle problems, abnormalities of the retina, hip weakness, facial weakness, abdominal muscle weakness, joint and spinal abnormalities, lower leg weakness, shoulder weakness, hearing loss, muscle inflammation, and nonsymmetrical weakness. In some aspects, treating ameliorates a symptom of a cancer, i including, but not limited to, tumor size or growth, weight loss, fatigue, weakness, pain, skin problems, eating problems, bowel or bladder problems, coughing, difficulty breathing, fever, lumps, night sweats, bleeding or bruising, and dizziness. In some aspects, treating ameliorates a symptom of BAMS, including, but not limited to, nose defects, eye defects, or problems with sexual maturation.

[0195] Administration of an effective dose of a nucleic acid, vector, composition, or system of the disclosure may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intracerebroventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal. In some aspects, an effective dose is delivered by a systemic route of administration, i.e., systemic administration. Systemic administration is a route of administration into the circulatory system so that the entire body is affected. Such systemic administration, in various aspects, takes place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally via injection, infusion, or implantation). In various aspects, an effective dose is delivered by a combination of routes. For example, in various aspects, an effective dose is delivered intravenously and / or intramuscularly, or intravenously and intracerebroventricularly, and the like. In some aspects, an effective dose is delivered in sequence or sequentially. In some aspects, an effective dose is delivered simultaneously. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure, in various aspects, are chosen and / or matched by those skilled in the art taking into account the condition or state of the disease or disorder being treated, the condition, state, or age of the subject, and the target cells / tissue(s) that are to express the nucleic acid or protein.

[0196] In particular, actual administration of delivery vehicle (such as rAAV) may be accomplished by using any physical method that will transport the delivery vehicle (such as rAAV) into a target cell of an animal. Administration includes, but is not limited to, injection2835 / 708182024-079-02 into muscle, the bloodstream and / or directly into the nervous system or liver. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV).Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as neurons. See, for example, WO 02 / 053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the disclosure. The delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0197] A dispersion of delivery vehicle (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques known to those skilled in the art.

[0198] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, sorbitol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0199] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as2835 / 708182024-079-02 required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-f iltered solution thereof.

[0200] The disclosure also provides a kit comprising a nucleic acid, vector, or composition of the disclosure or produced according to a process of the disclosure. In the context of the disclosure, the term "kit" means two or more components, one of which corresponds to a nucleic acid, vector, or composition of the disclosure, and the other which corresponds to a container, recipient, instructions, or otherwise. A kit, therefore, in various aspects, is a set of products that are sufficient to achieve a certain goal, which can be marketed as a single unit.

[0201] The kit may comprise one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material containing the nucleic acid, vector, or composition of the disclosure in an appropriate dosage for administration (see above). The kit may additionally contain directions or instructions for use (e.g. in the form of a leaflet or instruction manual), means for administering the nucleic acid, vector, or composition, such as a syringe, pump, infuser or the like, means for reconstituting the nucleic acid, vector, or composition and / or means for diluting the nucleic acid, vector, or composition.

[0202] In some aspects, the kit comprises a label and / or instructions that describes use of the reagents provided in the kit. The kits also optionally comprise catheters, syringes, or other delivering devices for the delivery of one or more of the compositions used in the methods described herein.

[0203] The disclosure also provides kits for a single dose of administration unit or for multiple doses. In some embodiments, the disclosure provides kits containing singlechambered and multi-chambered pre-filled syringes.

[0204] This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure2835 / 708182024-079-02 described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning.

[0205] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. 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 disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.

[0206] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term." The terms "and / or" and "any combination thereof" and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" can mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C."

[0207] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, up to 2.5%, or up to 1% of a given value or range. It also includes the concrete number; for example, about 10 includes 10. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0208] Throughout this disclosure and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having." When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim, but instead refers to those elements required for a given embodiment or aspect. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the2835 / 708182024-079-02 disclosure. When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0209] Throughout the disclosure, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, it is contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[0210] Reference in the specification to "some embodiments / aspects”, "an embodiment / aspect”, "one embodiment / aspect”, or "other embodiments / aspects" means that a particular feature, structure, or characteristic described in connection with the embodiments / aspects is included in at least some embodiments / aspects, but not necessarily all embodiments / aspects, of the disclosure. It is contemplated that any embodiment or aspect discussed in this disclosure can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0211] It should be understood that this disclosure is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the subject matter of the disclosure, which is defined solely by the claims.

[0212] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and / or with the aid of automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.

[0213] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications,2835 / 708182024-079-02 instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0214] A better understanding of the disclosure and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLES

[0215] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.Example 1Design and Construction of Split Intein SMCHD1 Constructs

[0216] A schematic representation of how split intein-mediated protein trans-splicing is and was carried out is shown in Fig. 1. SMCHD1 protein was split at sites compatible with intein-mediated splicing and the corresponding SMCHD1 coding sequences (CDS) were packaged into two AAV vectors. The first vector contains the CDS of the N-terminal half of SMCHD1 protein followed by the CDS of the N-terminal half of the intein. The second vector includes the C-terminal half of the intein attached to the C-terminal half of the SMCHD1 protein. Both AAV vectors contain the same regulatory elements, including inverted terminal repeats (ITR), a promoter, and a polyadenylation signal (pA). The polypeptides are expressed from each expression cassette individually. Upon association, inteins catalyze protein trans-splicing, excising themselves out and ligating the flanking polypeptide sequence.

[0217] The strategy and amino acid requirements for intein insertion and functional domains in SMCHD1 protein are provided in Fig. 2A-B. In this approach, the coding sequence of SMCHD1 was split into two fragments at sites compatible with intein insertion and protein trans-splicing. Successful intein-mediated splicing relies on specific amino acid residues in the flanking sites to promote proper folding, splicing, and ligation. The native splicing preferred sequence ( Iwai et al., FEBS Lett. 580: 1853-58, 2006), N-extein (polypeptide region proximal to intein) residues A(-3) E(-2) Y(-1), split site, C-extein (polypeptide proximal to intein) residues C(+1) F(+2) N(+3) and the non-native residues ( Cheriyan et al., J. Biol. Chem. 288: 6202-11 , 2013; Cheriyan et al., J. Mol. Biol. 426: 4018-2835 / 708182024-079-02 29, 2014; Zhu et al., Sci. China Life Sci. 56: 262-67, 2013) are listed in Fig. 2A-B. Among these six residues, the first and second residues in C-extein (C>S>T(+1) and large hydrophobic amino acids F>W>M(+2)) are critical for effective splicing. As a result, the split sites were selected considering factors such as i) the amino acid residue preference surrounding the intein (Fig. 2A-B), ii) secondary structure, iii) avoidance of protein functional domains, and iv) exclusion of residues that are known to be associated with polymorphisms.

[0218] Fig. 2A provides a table showing amino acid sequence specificity and sites amenable for intein insertion. The native amino acid residues flanking the inteins that promote splicing are highlighted in bold. Additional proximal amino acids that promote intein-mediated splicing are indicated in subsequent rows in the order of preference. Amino acids preceding the N-terminus of the intein are depicted as -1 , -2, and -3, while those flanking the C-terminus of intein are labelled as +1 , +2 and +3. Fig. 2B provides a schematic showing the position of functional domains in SMCHD1 protein, and the region of interest screened for intein insertion. The expanded region of interest shows the amino acid residues, secondary structure, and the selected split sites (SS) in SMCHD1 protein. Amino acids forming the beta-sheet are highlighted in yellow, and those forming the alpha-helix are in pink. The putative amino acids flanking the N-terminus of the intein are boxed in brown and the amino acids flanking the C-terminus of intein are boxed in blue.

[0219] The SMCHD1 protein was screened for split sites in the non-functional region, between amino acids 771 to 1190. Five split sites were selected, SS1 at amino acid 882, SS2 at amino acid 956, SS3 at amino acid 9692, SS4 at amino acid 1017, and SS5 at amino acid 1042 (Fig. 2B). Two naturally occurring fast-acting split inteins derived from Nostoc punctiforme (Npu) and Rhodothermus marinus (Rma) were used for vector designing. AAV vector harboring AAV2-ITR, CAG promoter (CMV enhancer (C), chicken beta actin promoter (A), and an intron from beta globin (G)) and a SV40 polyadenylation signal was used for vector construction (Fig. 3A-D). After specifying the split sites in SMCHD1 , we constructed two vectors in silico using SnapGene: the first vector contains the coding sequence (CDS) for the N-terminal half of the SMCHD1 protein, along with the N-intein, while the second vector consists of the C-intein CDS linked to the C-terminal half of SMCHD1 , followed by a V5 tag nucleotide sequence. The first vector is labeled as SMCHD1-N and the second vector as SMCHD1-C along with the respective intein used. In total, ten constructs were designed using Snapgene. Considering the ITR-to-ITR packaging capacity, SS2 and SS3 were selected for vector synthesis. Four pairs of AAV constructs were created using two distinct SSs in combination with two different split inteins (Fig. 3A-D).

[0220] Fig. 3A-D shows the design strategies for the various dual AAV split-intein vectors for SMCHD1 of the disclosure. Fig. 3A-B are schematic representations of dual AAV-vectors2835 / 708182024-079-02 for split sites 2 (SS2) and SS3 containing split intein derived from Nostoc punctiforme (Npu). Fig. 3C-D are schematic representations of dual AAV-vectors for split sites 2 (SS2) and SS3 containing split intein derived from Rhodothermus marinus (Rma). In each vector pair, the first vector, represented as AAV.SMCHD1-N, contains the CDS of the N-terminal half of SMCHD1 followed by the CDS of the N-terminal half of the intein. The second vector, depicted as AAV.SMCHD1-C has the C-terminal half of the intein fused to the C-terminal half of SMCHD1 and a terminal V5 epitope tag. Both AAV vectors possess the same regulatory elements, including ITR, CAG promoter (CMV enhancer (C), chicken beta actin promoter (A) and an intron from beta globin (G)) and SV40 pA.

[0221] Four pairs of adeno-associated virus (AAV) constructs using two different split inteins were designed. For each vector pair, the first vector contains the coding sequence for the N-terminal half of SMCHD1 followed by the N-intein coding sequence, and the second vector contains the coding sequence of a C-intein in frame with the C-terminal half of SMCHD1 and a terminal V5 epitope tag. More specifically, the AAV split intein constructs were designed as shown in Fig. 3A-D.

[0222] The SMCHD1 protein was screened for splitting sites (SS) suitable for intein insertion and protein trans-splicing. The splitting sites (SS) were selected based on several factors, including the amino acid residue preference surrounding the intein (Fig. 2A-B), secondary structure, the integrity of protein functional domains, and avoidance of the residues that are known to be associated with polymorphisms. Five SS were chosen, SS1 at amino acid 882, SS2 at amino acid 956, SS3 at amino acid 9692, SS4 at amino acid 1017 and SS5 at amino acid 1042. Two different split inteins, one derived from Nostoc punctiforme (Npu) and the other from Rhodothermus marinus (Rma) were used for vector construction. An AAV plasmid (Addgene: 37825) containing AAV2-ITR, CAG promoter (CMV enhancer (C), chicken beta-actin promoter (A), and an intron from the beta-globin (G)) and a SV40 polyadenylation signal was used for vector construction. After splitting the SMCHD1 at a specific site, the corresponding coding sequence (CDS) of the N-terminal half of the SMCHD1 protein and the CDS of N-intein were cloned and represented as SMCHD1-N along with the intein used. The C-intein coding sequence was attached to the second half of the SMCHD1 coding sequence followed by a V5 tag nucleotide sequence. In total, ten constructs were designed using SnapGene software. Taking into account the ITR-to-ITR packaging capacity, SS2 and SS3 were selected for vector generation. Four pairs of AAV constructs were then created using two different SSs in combination with two different split inteins. The relevant split sites and insert sequences / cassettes for all 8 vectors, i.e., 4 vector pairs, are shown in Fig. 7A-C through Fig. 14A-C.2835 / 708182024-079-02

[0223] Fig. 7A-C provides a schematic of the 5-SMCHD1-SS2-Npu expression cassette (Fig. 7A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 7B), and the expression cassette nucleotide sequence (SEQ ID NO: 24) where various components are highlighted (Fig. 7C). Fig. 8A-C provides a schematic of the 3-SMCHD1-SS2-Npu expression cassette (Fig. 8A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 8B), and the expression cassette nucleotide sequence (SEQ ID NO: 25) where various components are highlighted (Fig. 8C). These two expression cassettes were designed to be used together.

[0224] Fig. 9A-C provides a schematic of the 5-SMCHD1-SS3-Npu expression cassette (Fig. 9A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 9B), and the expression cassette nucleotide sequence (SEQ ID NO: 26) where various components are highlighted (Fig. 9C). Fig. 10A-C provides a schematic of the 3-SMCHD1 -SS3-Npu expression cassette (Fig. 10A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 10B), and the expression cassette nucleotide sequence (SEQ ID NO: 27) where various components are highlighted (Fig. 10C). These two expression cassettes were designed to be used together.

[0225] Fig. 11A-C provides a schematic of the 5-SMCHD1-SS2-Rma expression cassette (Fig. 11 A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 11 B), and the expression cassette nucleotide sequence (SEQ ID NO: 28) where various components are highlighted (Fig. 110). Fig. 12A-C provides a schematic of the 3-SMCHD1-SS2-Rma expression cassette (Fig. 12A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 12B), and the expression cassette nucleotide sequence (SEQ ID NO: 29) where various components are highlighted (Fig. 120). These two expression cassettes were designed to be used together.

[0226] Fig. 13A-C provides a schematic of the 5-SMCHD1 -SS3-Rma expression cassette (Fig. 13A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 13B), and the expression cassette nucleotide sequence (SEQ ID NO: 30) where various components are highlighted (Fig. 130). Fig. 14A-C provides a schematic of the 3-SMCHD1-SS3-Rma expression cassette (Fig. 14A), a detailed map of the components of the expression cassette and their sequence positions in the cassette (Fig. 14B), and the expression cassette nucleotide sequence (SEQ ID NO: 31) where various components are highlighted (Fig. 140). These two expression cassettes were designed to be used together.2835 / 708182024-079-02 Example 2Experimental Materials and Methods

[0227] In vitro validation of AAV split-intein vectors

[0228] To determine if the split-intein vector pairs could achieve efficient protein reconstitution in vitro validation experiments were carried out. Human embryonic kidney (HEK) 293 cells were used to validate the split intein-mediated protein reconstitution in vitro. The cells were plated in a 12-well plate at a density of 350,000 cells / well in growth media (DMEM (Gibco), 10%FBS (Corning), 1% antibiotics (Gibco)). The next day, single or dual AAV split intein plasmids (0.8pg of each plasmid / well) were transfected using Lipofectamine 2000 in opti-MEM media (Gibco). The total amount of DNA was kept equal across different conditions using a negative control plasmid. Cells were harvested 72 hours (hrs) posttransfection. Protein was extracted using ice-cold RIPA lysis buffer (Thermo Fisher Scientific) with protease inhibitor (Sigma). The split-intein vector pairs that displayed efficient protein reconstitution in vitro were selected for in vivo validation.

[0229] Western blotting

[0230] Protein quantification was carried out using a DC Protein Assay Kit (Bio-Rad) as per manufacturer’s protocol. Proteins (20 pg) were separated in a 4-20% Mini-PROTEAN® TGX™ stain-free gel (Bio-Rad), a precast gel designed for the separation of proteins, and transferred onto a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). The following antibodies were used for Western blotting, anti-V5-horse radish peroxidase (HRP) (Bethyl Lab), anti-V5-HRP (Invitrogen), anti-p actin (abeam), and anti-a-tubulin (abeam). The PVDF membrane was blocked with 5% skimmed milk for one hour at room temperature and incubated with anti-V5 antibody, followed by three washes with tris-buffered saline with 0.1% Tween® 20 detergent (TBST) (Sigma-Aldrich). Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for visualizing protein bands. The membrane was stripped using Restore™ Western Blot Stripping Buffer (Invitrogen), blocked again with 5% skimmed milk, and incubated with primary antibody for loading control. Goat anti-mouse IgG-HRP (Jackson Immunoresearch) secondary antibody was used for p-actin and a-tubulin. Densitometric analysis was performed using Imaged (NIH).

[0231] AA V vector production

[0232] The SMCHD1 -SS3-Npu plasmid pairs were used for vector production at Andelyn Bioscience. The expression cassette was packaged into AAV using the Myo-AAV3A serotype. SMCHD1-SS3-F?ma plasmid pairs are used if SMCHD1-SS3-A / pu vector constructs display toxicity.

[0233] Animals2835 / 708182024-079-02

[0234] Eight-week-old wild-type mice (Strain: C57BL / 6J; Stock number: 000664) were purchased from the Jackson Laboratory. D4Z4-2.5 mice were bred in-house at the Nationwide Children’s Hospital animal facility and were genotyped using tail samples. D4Z4-2.5 transgenic mice recapitulate important epigenetic and DUX4 expression attributes seen in human FSHD1 . D4Z4-2.5 mice, along with the control strain (D4Z4-12.5 transgenic mice carrying a normal sized, non-pathogenic allele) are also available at the Jackson Laboratory. All animal experiments were conducted in accordance with the Nationwide Children’s Hospital Institutional Animal Care and Use Committee.

[0235] Intramuscular injection

[0236] For dose-finding, wild-type mice were anesthetized using isofluorane. Three different doses of split intein vectors (1.0E+10 vg / vector, 5.0E+10 vg / vector, 1 .0E+11 vg / vector; n = 10) were intramuscularly injected in one tibialis anterior (TA) muscle. For each dose, either AAV-SMCHD1-N or AAV-SMCHD1-C, or a mix of both was injected unilaterally into one TA. Five animals were utilized for each dose. Mice were sacrificed 4-5 weeks after injection, and TA muscles were harvested for analysis. One-third of the TA was used for Western blotting and the remaining two-thirds of the TA muscle was embedded in Tissue-Tek® O.C.T. Compound (TissueTek), snap frozen in cold methyl butane and stored at -80°C for histology.

[0237] Systemic Delivery

[0238] Two doses (2.0E+13 vg / kg and 6.0E+13 vg / kg) of vector were injected into the vein via the retro-orbital route to assess a safe dosage for systemic delivery in adult mice. For each dose, either AAV-SMCHD1-N or AAV-SMCHD1-C, or a mix of both are injected. A minimum of two animals is used for each condition / dose. Mice are sacrificed 4-5 weeks postinjection, and the TA and gastrocnemius (gas) muscles are harvested for Western blotting and histology.

[0239] Protein extraction from muscles

[0240] The frozen TA muscles were weighed, and ice-cold RIPA buffer (Thermo Scientific) with protease inhibitor (Sigma) was added to the tissue at a volume of 20pl per mg of tissue. To each tissue, one 5mm stainless steel bead (Qiagen) was added and the tissue was lysed using TissueLyser II (Qiagen) at 30Hz frequency. The lysed tissue was centrifuged at 14,000rpm and the supernatant was collected. The supernatant was used for protein quantification and Western blotting.

[0241] Histological analysis2835 / 708182024-079-02

[0242] Frozen cryosections (10pm) were taken from the O.C.T. Compound-embedded muscles and stained for hematoxylin and eosin (H&E). Four representative images were taken from each muscle using cellSens Standard Software (Olympus LS). Muscle sections are evaluated for the presence of central nuclei, regenerated fibers, and infiltrating cells.

[0243] RNA extraction and Digital Droplet Polymerase Chain Reaction (ddPCR)

[0244] Mouse muscles were lysed with 5mm stainless steel beads (Qiagen) using TissueLyser II (Qiagen) at 30Hz frequency for 2 minutes. The RNA was extracted from mouse muscles or human myoblast cells using TRIzol (Invitrogen) according to manufacturer’s instruction. The extracted RNA (2pg) was treated with dsDNase (Thermo Fisher Scientific) for 30 minutes followed by DNase inactivation. Reverse transcription was carried out using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) with random primers. For ddPCR, ddPCR Supermix for Probes (No dllTP) (Bio-Rad) was used along with Taqman™ Probes for DLIX4, ZSCAN4, TRIM43 and Wfdc3 (Thermo Fisher Scientific). RpH 3 was used as a reference gene for relative expression analysis.

[0245] Methylation analysis

[0246] Methylation pattern changes mediated by the reconstituted SMCHD1 protein was evaluated using nanopore sequencing or AT AC sequencing (Azenta).

[0247] Immunofluorescence

[0248] For immunofluorescence experiments, HEK293 cells were plated in a 6-well plate containing coverslips at a density of 700,000 cells / well in growth media. The following day, cells were transfected with single or dual AAV split intein plasmids (1.6pg of each plasmid / well) using Lipofectamine 2000 in opti-MEM media (Gibco). Seventy-two hours posttransfection, the cells were fixed with 4% paraformaldehyde for 15 mins. The fixed cells were then washed three times with 1X PBS and permeabilized with PBS containing 0.25% Triton X-100 for 10 mins. After another three PBS washes, the coverslips were incubated overnight at 4°C with anti-V5 antibody (Abeam) diluted in 4% normal goat serum. Following three more washes, the cells were incubated with goat-anti-rabbit 594 diluted in 4% normal goat serum for 1 hr. Finally, the coverslips were washed three times and mounted with Prolong Gold antifade mounting reagent containing DAPI.

[0249] Statistical analysis

[0250] Data were presented as mean ± standard error of the mean (SEM). For experiments involving one independent variable, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (GraphPad Software) was carried out (Fig.4D and 4F). *P<0.05, **P<0.01.2835 / 708182024-079-02 Example 3In Vitro and In Vivo Reconstitution of Full-Length SMCHD1 Protein after Ligation of bland C-Termini

[0251] To assess the efficiency of full-length SMCHD1 protein reconstitution, the transfected the AAV split intein vector pairs were transfected into HEK293 cells, both as single and dual vectors. Cell lysates were harvested 72 hrs post-transfection. Western blotting and densitometric analyses were performed as described herein, including in the Materials and Methods of Example 2, to evaluate full-length protein reconstitution (Fig. 4A-F).

[0252] Fig. 4A-F shows data obtained in the validation of AAV split intein constructs in vitro in HEK293 cells, i.e., Western blot analysis of lysates from HEK 293 cells transfected with either single or dual split intein AAV vectors encoding either the N-terminal half or C-terminal half of SMCHD1. Fig. 4A shows a Western blot image showing the reconstituted full-length SMCHD1 for SS2 and SS3-Npu constructs. Fig 4B shows a Western blot image showing the reconstituted full-length SMCHD1 for SS2 and SS3-Rma constructs. Fig. 4C is a Western blot image displaying effective full-length SMCHD1 protein reconstitution in SMCHD1-SS2-Rma constructs. Fig. 4E is a Western blot image displaying effective full-length SMCHD1 protein reconstitution in SMCHD1-SS3-Rma constructs. Figs. 4D and 4 F are graphs of the densitometry quantification of the experiments shown in Figs. 4C and 4E, respectively, showing the relative expression of split intein-mediated full-length SMCHD1 protein compared to full-length SMCHD1 construct. Actin served as a loading control. Neg, control AAV vector-transfected cells; N, AAV.SMCHD1-N; C, AAV.SMCHD1-C, N+C, AAV.SMCHD1-N + AAV.SMCHD1-C transfected cells. Differences were assessed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. *P<0.05, **P<0.01.

[0253] All four split intein vector pairs demonstrated ligation of both halves of the SMCHD1 protein. Notably, both the Rma and Npu split intein vectors for split site three (SS3) exhibited the highest reconstitution levels of full-length SMCHD1 (Fig. 4E-F). Thus, the SMCHD1-SS3-Npu split intein vector pair was selected first for in vivo validation.

[0254] The SMCHD1 -SS3-Npu split intein vector pair expression cassette was packaged into MyoAAV3A vectors to determine in vivo full-length SMCHD1 protein reconstitution efficiency of SMCHD1 in skeletal muscles, e.g., mouse tibialis anterior (TA) muscle. In vivo testing was carried out using dual AAV split intein vectors in wild-type mice to determine the optimal dose for intramuscular delivery. Fig. 5A provides a schematic diagram of the animal study. Three different doses of split intein vectors (1.0E+10 vg / vector, 5.0E+10 vg / vector, 1.0E+11 vg / vector) were tested. Each dose was intramuscularly injected (IM) into one tibialis2835 / 708182024-079-02 anterior (TA) muscle, utilizing five animals per dose. Injections included either AAV-SMCHD1 -N-Npu (n=2 TAs) or AAV-SMCHD1 -C-Npu (n=2 TAs) or a mix of both (n=6 TAs).

[0255] Mice were sacrificed five weeks post-injection, and TA muscles were harvested for analysis. Fig. 5B shows Western blot analysis of protein expression in harvested TA muscles using an anti-V5 antibody, revealing that full-length SMCHD1 protein reconstitution was achieved at all three doses. Alpha tubulin was used as a loading control. All three doses displayed effective full-length SMCHD1 protein reconstitution in a dose-dependent manner in vivo (Fig. 5A-B).

[0256] The data shows that the Intramuscular injection of dual AAV split intein vectors in wild-type mice showed efficient reconstitution of the full-length SMCHD1 protein useful for the delivery and expression, including the overexpression of SMCHD1.Example 4Functional Analysis Testing of the Split Intein Gene Replacement Therapy

[0257] Fig. 6A-C provides the experimental plans for functional analysis of the reconstituted full-length SMCHD1. Fig. 6A is a schematic diagram of an In vitro experimental plan using human myotubes that are differentiated for four to seven days. Single and dual Myo-AAV3A split intein vectors are transduced into the human myotubes. The myotubes are harvested 7 days post-transduction for protein function analysis. Fig. 6B is a schematic diagram of the human facioscapulohumeral muscular dystrophy (FSHD) xenograft model. TA muscles are harvested two to four weeks after grafting human myoblasts and quantification of protein expression in the TA muscles is then carried out. Fig. 6C is a schematic diagram showing retro-orbital (RO) injections of AAV vectors into D4Z42.5 mice that have the FSHD-permissive DLIX4 locus. TA and gastrocnemius (Gas) muscles are harvested 4-5 weeks post-injection. The harvested cells and muscles are evaluated for protein, downstream DUX4 target genes, and epigenetic outcome measures.

[0258] Additionally, in vivo efficacy studies in human FSHD2 myoblasts xenografted in mice, and the D4Z4-2.5 mouse model expressing human, FSHD-permissive D4Z4 repeats (Fig. 6C) are being carried out. Functional outcome measures, such as measuring DUX4 downstream target gene expression and changes in methylation pattern, are being evaluated.Example 5Toxicity Testing of the Split Intein Gene Replacement Therapy

[0259] Experiments are carried out to assess toxicity of vector delivery in IM injected TA tissues using precise histological outcome measures.2835 / 708182024-079-02

[0260] Experiments are carried out to determine a safe dosage for retro-orbital vein injections. Systemic delivery of two doses (2.0E+13 vg / kg and 6.0E+13 vg / kg) of the split intein vectors are administer to mice via retro-orbital vein injections. Optimal dosing experiments are likewise being carried out to ensure safety.Example 6In Vitro Validation of Expression Efficiency, Subcellular Localization and Stability of Reconstituted Full-Length SMCHD1 protein

[0261] The efficiency of dual split intein AAV vectors in reconstituting the full-length SMCHD1 was compared with the RNA trans-splicing (RNA-TS) and homologous recombination (HR) dual vector constructs. Western blot analysis (Fig. 15) performed using an anti-V5 antibody, revealed that both the Npu and Rma split intein constructs achieved efficient full-length SMCHD1 protein reconstitution, significantly outperforming the RNA-TS and HR constructs. Beta actin was used as a loading control.

[0262] Additionally, immunostaining using an anti-V5 antibody was carried out to evaluate the localization of the reconstituted full-length SMCHD1 protein in HEK293 cells following transfection with single or dual split intein vectors.

[0263] For immunofluorescence experiments, HEK293 cells were plated in a 6-well plate containing coverslips at a density of 700,000 cells / well in growth media. The following day, cells were transfected with single or dual AAV split intein plasmids (1.6pg of each plasmid / well) using Lipofectamine 2000 in opti-MEM media (Gibco). Seventy-two hours posttransfection, the cells were fixed with 4% paraformaldehyde for 15 mins. The fixed cells were then washed three times with 1X PBS and permeabilized with PBS containing 0.25% Triton X-100 for 10 mins. After another three PBS washes, the coverslips were incubated overnight at 4°C with anti-V5 antibody (Abeam) diluted in 4% normal goat serum. Following three more washes, the cells were incubated with goat-anti-rabbit 594 diluted in 4% normal goat serum for 1 hr. Finally, the coverslips were washed three times and mounted with ProLong Gold antifade mounting reagent containing DAPI.

[0264] Fig. 16A-C display the localization of individual halves and full-length SMCHD1 for Npu split intein constructs. Fig.16D-F show the location of SMCHD1 for Rma split intein constructs. In cells transfected with the dual split intein vectors, the reconstituted full-length SMCHD1 localized to nuclear speckles for both Npu (Fig. 16C) and Rma (Fig. 16F) split intein constructs. The C-terminal half of SMCHD1 displayed diffused nuclear localization (Fig. 16B and Fig. 16E). The nucleus was counterstained with DAPI. Scale bar, 50pm.

[0265] The safety of the SMCHD1 split intein fragments was assessed in HEK293 cells using the Apo-ONE homogenous caspase 3 / 7 assay, 48 hrs post-transfection with single or2835 / 708182024-079-02 dual split intein vectors (Fig. 17). Transfection of the known apoptosis inducer DLIX4 served as a positive control, resulting in an increase in caspase 3 / 7 activity. However, the SMCHD1 N-terminal half or the C-terminal half and their combined expression (via the dual vector), did not induce apoptosis. This indicates that the split-intein fragments are non-toxic to the cells.

[0266] Fig. 18A-D shows the stability of Rma split intein mediated reconstituted full-length SMCHD1 assessed by cycloheximide (CHX) chase assay. HEK 293 cells were transfected with single or dual SMCHD1 split intein vectors. After 48 hrs, cells were treated with CHX (50pg / mL) and samples were collected at 0,4,8,12,16 and 24h time points. Western blot analysis using anti-FLAG antibody revealed the stability of full-length SMCHD1 (Fig. 18A) and N-terminal SMCHD1 half (Fig. 18C). Western blot analysis using anti-V5 antibody revealed the stability of dual split intein mediated reconstituted SMCHD1 (Fig. 18B) and C-terminal SMCHD1 half (Fig. 18D). The SMCHD1 protein level at zero hours was set to 100%. The graph shows the mean densities of full-length and split SMCHD1 halves, relative to DMSO control at 0 hours and normalized to alpha tubulin. Data presented as mean ± S.D (n=3).

[0267] Collectively, these data establish the high efficiency of split intein technology in expressing large proteins. Furthermore, the results confirm the appropriate localization, safety, and stability of the split intein-mediated, reconstituted, full-length SMCHD1 protein.Example 7Toxicity assessment of the Split Intein vectors In Vivo

[0268] To evaluate the safety of the Npu- split intein vector pair doses used for IM delivery, as shown in Fig. 5A-C, H&E staining was carried out on muscle cryosections (Fig.19A-I). Histological analysis revealed dose-dependent toxicity, characterized by the accumulation of infiltrating cells (inflammation) for all three tested doses. The highest dose (Fig. 19G-I) exhibited more inflammation, while the lowest dose (Fig. 19A-C) showed less inflammation. Scale bar, 50pm.

[0269] Both efficacy and optimal IM dose of the Rma split intein vector pairs (SMCHD1-SS3-Rma) were subsequently tested in vivo in wild-type mice. Fig. 20A provides a schematic diagram of the animal study. Two doses of Rma split intein vectors, packaged in the MyoAAV3A serotype (5.0E+9 vg / vector and 3.0E+9 vg / vector) were tested. Each dose was injected intramuscularly into one TA muscle, utilizing four animals per dose. Injections included either AAV-SMCHD1 -N-Rma (n=2 TAs) or AAV-SMCHD1 -C-Rma (n=2 TAs) or a mix of both (n=4 TAs).

[0270] Mice were sacrificed four weeks post-injection, and TA muscles were harvested for analysis. Western blot analysis using anti-V5 antibody confirmed full-length SMCHD1 protein2835 / 708182024-079-02 reconstitution at both tested doses (Fig. 20B). Alpha tubulin was used as a loading control. Histology analysis using H&E staining showed no toxicity (no cellular infiltration or central nuclei) at 3.0E+9 vg / vector dose (Fig. 20C; top panel), but some cellular infiltration was noted at 5.0E+10 vg / vector dose (Fig. 20C; bottom panel). Scale bar, 50pm.

[0271] The data shows that the Rma split intein vector pairs exhibited safe and efficient SMCHD1 reconstitution at an optimal dose of 3.0E+9 vg / vector.Example 8Functional Analysis of the SMCHD1 Split Intein Gene Replacement Therapy in FSHD Myotubes In Vitro

[0272] The functional effect of the SMCHD1 split intein replacement therapy was assessed in vitro using human FSHD myotubes and control myotubes. Fig. 21 A provides the experimental plan illustrating the transduction of human FSHD myotubes with single or dual myo-AAV split intein vectors. The myotubes were harvested 4-7 days post-transduction for protein function analysis. Western blot analysis using an anti-V5 antibody shows full-length SMCHD1 protein reconstitution in human myotubes (Fig. 21 B). Beta actin was used as a loading control. Functional outcome readout using ddPCR assessed the mRNA levels of the DUX4 target genes ZSCAN4 (Fig. 21 C, Fig. 21 E) and SLC34A2 (Fig. 21 D, Fig. 21 F) in two different FSHD2 patient myotubes PMB-13 (Fig. 21C, Fig. 21 D) and PMB-14 (Fig. 21 E, Fig.21 F) following split intein vector transduction. Data are presented as mean ± S.E.M.Differences were analyzed using two-way ANOVA followed by Tukey post hoc test.**P<0.01, ****P<0.0001.

[0273] These results highlight the protective effect of reconstituted full length SMCHD1 protein as well as the individual SMCHD1 fragments in FSHD myotubes, in reducing the expression of DUX4 target genes.

[0274] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

Claims

1. 2835 / 708182.2024-079-02 CLAIMS3.What is claimed is:

1. A nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises5.(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and6.(ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide.

2. A nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises8.(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and9.(ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

3. The nucleic acid of claim 1 or 2, wherein the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

4. The nucleic acid of claim 1 or 3, wherein the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of2835 / 7081812.2024-079-02 SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

5. The nucleic acid of claim 2 or 3, wherein the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

6. The nucleic acid of any one of claims 1 , 3, or 4, wherein the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17.

7. The nucleic acid of any one of claims 2, 3, or 5, wherein the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

8. The nucleic acid of any one of claims 1-7, wherein the nucleotide sequence further comprises an enhancer.

9. The nucleic acid of claim 8, wherein the enhancer is a CMV enhancer.

10. The nucleic acid of claim 9, wherein the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

11. The nucleic acid of any one of claims 1 -10, wherein the nucleotide sequence further comprises a promoter.

12. The nucleic acid of claim 11 , wherein the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter.

13. The nucleic acid of claim 12, wherein the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

14. The nucleic acid of any one of claims 1-13, wherein the nucleotide sequence further comprises an SV40 poly(A) signal.2835 / 7081823.2024-079-02 15. The nucleic acid of claim 14, wherein the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

16. The nucleic acid of any one of claims 1 -15, wherein the nucleotide sequence further comprises an AAV 5’ ITR sequence.

17. The nucleic acid of claim 16, wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22.

18. The nucleic acid of any one of claims 1 -17, wherein the nucleotide sequence further comprises an AAV 3’ ITR sequence.

19. The nucleic acid of claim 18, wherein the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

20. The nucleic acid of any one of claims 1 -19, wherein the nucleotide sequence further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR sequence.

21. The nucleic acid of claim 20, wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

22. The nucleic acid of claim 1 , wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.

23. The nucleic acid of claim 2, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

24. A vector comprising the nucleic acid of any one of claims 1 -23.

25. The vector of claim 24, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

26. The vector of claim 25, wherein the virus lacks rep and / or cap genes.

27. The vector of claim 25 or 26, wherein the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV).2835 / 7081836.2024-079-02 28. The vector of any one of claims 25-27, wherein the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rhW, AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1 A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof.

29. The vector of claim 28, wherein the vector comprises the capsid of MYOAAV3A or a derivative thereof.

30. A composition comprising39.(a) the nucleic acid of any one of claims 1-23; or40.(b) the vector of any one of claims 24-28; and41.a carrier, diluent, excipient, buffer, and / or adjuvant.42.31 . A method of decreasing and / or inhibiting the expression of a DLIX4 gene in a cell comprising introducing into the cell43.(a) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises44.(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and45.(ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide; and46.(b) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a C-terminal structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises 2835 / 7081847.2024-079-02 (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and48.(ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

32. The method of claim 31 , wherein the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

33. The method of claim 31 or 32, wherein the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

34. The method of any one of claims 31 -33, wherein the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.

35. The method of any one of claims 31 -34, wherein the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17.

36. The method of any one of claims 31 -35, wherein the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

37. The method of any one of claims 31 -36, wherein the nucleotide sequence further comprises an enhancer.

38. The method of claim 37, wherein the enhancer is a CMV enhancer.2835 / 7081856.2024-079-02 39. The method of claim 38, wherein the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

40. The method of any one of claims 31 -39, wherein the nucleotide sequence further comprises a promoter.

41. The method of claim 40, wherein the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter.59.42 The method of claim 41 , wherein the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

43. The method of any one of claims 31 -42, wherein the nucleotide sequence further comprises an SV40 poly(A) signal.

44. The method of any one of claims 43, wherein the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

45. The method of any one of claims 31 -44, wherein the nucleotide sequence further comprises an AAV 5’ ITR sequence.

46. The method of claim 45, wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22.

47. The method of any one of claims 31 -46, wherein the nucleotide sequence further comprises an AAV 3’ ITR sequence.

48. The method of claim 47, wherein the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

49. The method of any one of claims 31 -48, wherein the nucleotide sequence further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR sequence.

50. The method of claim 49, wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR2835 / 7081868.2024-079-02 sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

51. The method of claim 31 , wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.

52. The method of claim 31 , wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

53. The method of any one of claims 31 -52, wherein each of the nucleic acids of (a) and (b) are in a vector.

54. The method of claim 53, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

55. The method of claim 54, wherein the virus lacks rep and / or cap genes.

56. The method of claim 54 or 55, wherein the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV).

57. The method of any one of claims 54-56, wherein the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11, AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof.

58. The method of claim 57, wherein the vector comprises the capsid of MYOAAV3A or a derivative thereof.

59. The method of any one of claims 31 -58, wherein introducing into the cell is carried out by injection or by electroporation.

60. The method of any one of claims 31 -59, wherein the cell is in a human subject.

61. A system comprising:80.(a) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising an N-terminal fragment of a structural maintenance of chromosomes hinge domain containing 1 (SMCHD1) polypeptide and an N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises 2835 / 7081881.2024-079-02 (i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 5’ end of a nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 3’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and82.(ii) an N-intein domain coding sequence positioned at the 3’ end of the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide; and.83.(b) a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a SMCHD1 polypeptide fragment and a C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide, wherein the nucleotide sequence comprises84.(i) at least about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3200, about 3300, or about 3400 consecutive nucleotides of the 3’ end of a nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide, wherein the sequence is designed so that nucleotides at the 5’ end of the nucleotide sequence encode amino acids compatible to intein-mediated splicing; and85.(ii) a C-intein domain coding sequence positioned at the 5’ end of the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide.

62. The system of claim 61 , wherein the nucleotide sequence encoding SMCHD1 comprises the nucleotide sequence of SEQ ID NO: 1 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1 , or encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising at least 80% sequence identity to amino acid sequence of SEQ ID NO: 2.

63. The system of claim 61 or 62, wherein the nucleotide sequence encoding the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, 5, 7, or 9.

64. The system of any one of claims 61-63, wherein the nucleotide sequence encoding the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 4, 6, 8, or 10.2835 / 7081889.2024-079-02 65. The system of any one of claims 61-64, wherein the nucleotide sequence encoding the N-intein domain comprises the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 11, 13, 15, or 17.

66. The system of any one of claims 61-65, wherein the nucleotide sequence encoding the C-intein domain comprises the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 12, 14, 16, or 18.

67. The system of any one of claims 61-66, wherein the nucleotide sequence of at least one of the nucleic acids further comprises an enhancer.

68. The system of claim 67, wherein the enhancer is a CMV enhancer.

69. The system of claim 68, wherein the CMV enhancer comprises the nucleotide sequence of SEQ ID NO: 19, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

70. The system of any one of claims 61-69, wherein the nucleotide sequence of at least one of the nucleic acids further comprises a promoter.

71. The system of any one of claims 61 -70, wherein the promoter is any of a beta-actin promoter, a CMV promoter, a minimal CMV promoter, a T7 promoter, an EF1-alpha promoter, a minimal EF1 -alpha, a tissue-specific promoter, a muscle-specific promoter, or a cardiac-specific promoter.

72. The system of claim 71 , wherein the beta-actin promoter comprises the nucleotide sequence of SEQ ID NO: 20, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

73. The system of any one of claims 61-72, wherein the nucleotide sequence of at least one of the nucleic acids further comprises an SV40 poly(A) signal.

74. The system of claim 73, wherein the SV40 poly(A) signal comprises the nucleotide sequence of SEQ ID NO: 21 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 21.

75. The system of any one of claims 61-74, wherein the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 5’ ITR sequence.

76. The system of claim 75, wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22.2835 / 70818101.2024-079-02 77. The system of any one of claims 61-76, wherein the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 3’ ITR sequence.

78. The system of claim 77, wherein the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

79. The system of any one of claims 61-78, wherein the nucleotide sequence of at least one of the nucleic acids further comprises an AAV 5’ ITR sequence and an AAV 3’ ITR sequence.

80. The system of claim 79, wherein the nucleotide sequence of at least one of the nucleic acids further comprises wherein the AAV 5’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 22, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 22, and / or the AAV 3’ ITR sequence comprises the nucleotide sequence of SEQ ID NO: 23, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 23.

81. The system of claim 61 , wherein the nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising the N-terminal fragment of the SMCHD1 polypeptide and the N-intein domain positioned at the C-terminus of the N-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30, or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 24, 26, 28, or 30.

82. The system of claim 61 , wherein the nucleic acid comprising the nucleotide sequence encoding a fusion protein comprising the C-terminal SMCHD1 polypeptide fragment and the C-intein domain positioned at the N-terminus of the C-terminal fragment of the SMCHD1 polypeptide comprises the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31 , or a variant thereof comprising at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 25, 27, 29, or 31.

83. The system of any one of claims 61-82, wherein each of the nucleic acids is present in a vector.

84. The system of claim 83, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

85. The system of claim 84, wherein the virus lacks rep and / or cap genes.

86. The system of claim 84 or 85, wherein the vector is a self-complementary recombinant AAV (scAAV) or a single-stranded recombinant vector (ssAAV).2835 / 70818111.2024-079-02 87. The system of any one of claims 84-86, wherein the vector comprises a capsid of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh , AAV11 , AAV12, AAV13, AAV-anc80, AAV-B1 , AAV-BR1 , AAV.PHP.EB, AAVv66, AAV2 / 1 , AAV2 / 8, or AAV2 / 9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101 , or any derivative thereof.

88. The system of claim 87, wherein the vector comprises the capsid of MYOAAV3A or a derivative thereof.

89. A method of inhibiting and / or interfering with expression of a double homeobox 4 (DLIX4) gene in a cell comprising introducing into the cell the system of any one of claims 61-88.

90. The method of claim 89, wherein the cell is in a human subject.115.91 . The method of claim 89 or 90, wherein introducing into the cell is carried out by injection or by electroporation.

92. A method of treating a subject suffering from a disease or disorder associated with expression of a double homeobox 4 (DLIX4) gene comprising administering to the subject an effective amount of the system of any one of claims 61-88.

93. The method of claim 92, wherein the disease or disorder associated with expression of a double homeobox 4 (DUX4) gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS).

94. The method of claim 93, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

95. The method of claim 93, wherein the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus.

96. The method of any one of claims 92-95, wherein the effective amount is a dosage of the vector of about 1.0x1010vg / kg to about 1.0x1015vg / kg.

97. Use of122.(a) the nucleic acid of any one of claims 1 -23;123.(b) the vector of any one of claims 24-29;124.(c) the composition of claim 30; and / or125.(d) the system of any one of claims 61 -88 2835 / 70818126.2024-079-02 for the preparation of a medicament for inhibiting expression of a double homeobox 4 (DUX4) gene in a cell.

98. Use of128.(a) the nucleic acid of any one of claims 1 -23;129.(b) the vector of any one of claims 24-29;130.(c) the composition of claim 30; and / or131.(d) the system of any one of claims 61 -88132.for treating or ameliorating a disease or disorder associated with expression of a double homeobox 4 (DUX4) gene, or for the preparation of a medicament for treating or ameliorating a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS).

99. The use of claim 98, wherein the disease or disorder associated with expression of a double homeobox 4 (DUX4) gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS).

100. The use of claim 99, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

101. The use of claim 100, wherein the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus.

102. The137.(a) nucleic acid of any one of claims 1 -23;138.(b) vector of any one of claims 24-29;139.(c) composition of claim 30;140.(d) method of any one of claims 31-60 and 89-95;141.(e) system of any one of claims 61 -88; or142.(f) use of any one of claims 97-101 ,143.wherein the nucleic acid, vector, composition, or medicament is formulated for intramuscular administration or injection, oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, transdermal transport, injection into the blood stream, or aerosol administration. 2835 / 70818144.2024-079-02 103. A composition for treating a disease or disorder associated with expression of a double homeobox 4 (DLIX4) gene in a subject in need thereof, wherein the composition comprises145.(a) the nucleic acid of any one of claims 1 -23;146.(b) the vector of any one of claims 24-29;147.(c) the composition of claim 30; and / or148.(d) the system of any one of claims 61 -88.

104. The composition of claim 103, wherein the disease or disorder associated with expression of the DUX4 gene is a muscular dystrophy, a cancer, or Bosma arhinia microphthalmia syndrome (BAMS).

105. The composition of claim 104, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

106. The composition of claim 105, wherein the cancer is a sarcoma, a B-cell lymphoma, or a DUX4-expressing cancer of the adrenal, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head / neck, liver, brain, lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, or thymus.

107. The composition of any one of claims 106-106, wherein the composition is formulated for intramuscular administration or injection, oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, transdermal transport, injection into the blood stream, or aerosol administration.

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