Deficient CAS-transcriptional repressor fusions for regulation of gene expression

Abstract

Disclosed herein are polynucleotides including expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) allowing from the expression of a CRISPRi system from a single polynucleotide, vectors comprising the polynucleotides, as well as methods of treating FSHD using the polynucleotides and the vectors disclosed herein.

Description

DEFICIENT CAS -TRANSCRIPTIONAL REPRESSOR FUSIONS FOR REGULATION OF GENE EXPRESSIONRELATED INFORMATION

[0001] The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 683,727, filed August 16, 2024, the disclosure of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 14, 2025, is named 4140_076PC01_Sequencelisting_ST26.xml and is 110,432 bytes in size.FIELD OF DISCLOSURE

[0004] The present disclosure pertains to the medical field including gene therapy, for example, to the treatment of genetic diseases by gene therapy.BACKGROUND

[0005] Facioscapulohumeral muscular dystrophy (FSHD) (MIM: 158900 and 158901) is the third most common muscular dystrophy. FSHD is characterized by progressive and often asymmetric weakness and atrophy of specific muscle groups (facial (facio), shoulder girdle (scapulo), and upper arm muscles (humeral)). With progression, other muscles may become affected. Extramuscular symptoms, such as retinal vasculopathy, are rare, but can also occur.Although highly variable in disease presentation, FSHD can have rapid progression and lead to wheelchair dependency.

[0006] FSHD is caused by ectopic expression of the DUX4 (retro)gene in skeletal muscle cells, which disturbs muscle homeostasis and can eventually result in apoptosis. DUX4 is a cleavage stage and germline transcription factor that under normal conditions is silenced in somatic tissues, such as skeletal muscle. A copy of the DUX4 open reading frame is embedded within each 3.3 kb large and CpG-rich D4Z4 unit, which is organized in a polymorphic array of 8-100 units on chromosome 4 in non-affected individuals. Two forms of FSHD are known: FSHD1 and FSHD2, both forms are caused by epigenetic dysregulation of the D4Z4 macrosatellite repeat array at chromosome 4q35. FSHD1, the most common form of the disease, is linked to contractions at this array, resulting in relaxation of chromatin that is normally repressed. FSHD2 is caused by mutations in proteins that maintain epigenetic silencing of the D4Z4 array, leading to a similar chromatin relaxation. In both forms of the disease, loss of epigenetic repression leads to the aberrant expression of the DUX4 (retro)gene in skeletal muscle. The DUX4 protein, in turn, activates a host of genes normally expressed in early development, which cause pathology when misexpressed in adult skeletal muscle.

[0007] Although an intact DUX4 open reading frame resides in every D4Z4 repeat unit in the macrosatellite array, only the full-length DUX4 mRNA (DUX4-f) encoded by the distal-most repeat is stably expressed and translated due to a polyadenylation signal residing in an exon distal to the array in disease-permissive alleles. The D4Z4 repeat array maps to the subtelomere of the long arm of chromosome 4 of which two major variants exist: 4qA and 4qB, and only D4Z4 repeat arrays on 4qA (the permissive allele) can express DUX4 in skeletal muscle as this haplotype uniquely contains a somatic DUX4 polyadenylation signal immediately distal to the D4Z4 array.

[0008] In addition, other rare D4Z4 rearrangements, such as D4Z4 proximally extended deletions (DPED) and 4; 10 translocations, and duplications of the D4Z4 repeat array can be associated with FSHD.

[0009] FSHD is a toxic gain-of-function disease, and no cures or ameliorative treatments exist for the treatment of FSHD. Therefore, an effective therapy is urgently needed.BRIEF SUMMARY

[0010] In some aspects, provide herein are expression cassette comprising: (a) a first polynucleotide comprising a regulatory sequence operably linked to a first nucleotide sequenceencoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); (b) a second polynucleotide comprising a first promoter operably linked to a second nucleotide sequence encoding a single guide RNA; and wherein the cassettes further comprise a pair of inverted terminal repeats (ITRs) flanking (a) and (b). In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other. In some aspects, the first polynucleotide sequence is located 5' to the second polynucleotide. In some aspects, the second polynucleotide sequence is located 5' to the first polynucleotide. In some aspects, the nucleotide sequence encoding the fusion protein further comprises a polyadenylation signal. In some aspects, the polyadenylation signal is a SV40, or a bGH polyadenylation signal. In some aspects, the fusion protein further comprises at least one nuclear localization signal (NLS). In some aspects, the same or different NLS are located N-terminal and C-terminal to the dCas. In some aspects, the at least one NLS is a SV40, or a nucleoplasmin NLS. In some aspects, the regulatory sequence comprises a second promoter. In some aspects, the second promoter is a Muscle Creatine Kinase (Ckm) promoter. In some aspects, the regulatory sequence further comprises one or more enhancers. In some aspects, the one or more enhancers is a Ckm, enhancer. In some aspects, the regulatory sequence is a NH or a HLH regulatory sequence. In some aspects, the TRD is a SUV39H1 preSET, SET, and post-SET domain; a KRAB TRD; a MeCP2 TRD; a HPla TRD; or a HPly TRD. In some aspects, the first promoter is a U6 promoter, a Ml 1 hybrid promoter, a 7SK promoter, or a Hl promoter. In some aspects, the nucleotide sequence encoding the single guide RNA comprises a spacer portion and a scaffold portion. In some aspects, the scaffold portion of the single guide RNA is a scaffold portion capable of binding to a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY. In some aspects, the spacer portion of the single guide RNA comprises a sequence targeting & DUX4 gene promoter and / or & DUX4 gene exon 1. In some aspects, the dCas enzyme is a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY. In some aspects, the ITRs in the pair of ITRs are the same or different. In some aspects, the ITRs are derived from an AAV genome of serotype AAV2. In some aspects, the fusion protein the dCas is located N-terminal to the TRD.

[0011] In some aspects, the expression cassettes comprise: (a) first polynucleotide comprising: (i) a HLH regulatory cassette, (ii) a first portion of a nucleotide sequence encoding a bipartite SV40 NLS, (iii) a nucleotide sequence encoding a dSaCas9, (iv) a second portion of a nucleotide sequence encoding a bipartite SV40 NLS, (v) a nucleotide sequence encoding a MeCP2TRD, and (vi) an SV40 polyadenylation signal; and (b) a second polynucleotide comprising: (i) a nucleotide sequence encoding a single guide RNA targeting the DUX4 gene, and (ii) a U6 promoter.

[0012] In some aspects, the expression cassettes comprise a nucleotide sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.

[0013] In some aspects, provided herein are single-stranded expression cassettes comprising: (a) a first polynucleotide comprising a regulatory sequence operably linked to a first nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); (b) a second polynucleotide comprising the antisense of a second nucleotide sequence, wherein the second nucleotide sequence comprises a first promoter operably linked to a third nucleotide sequence encoding a single guide RNA; wherein the first polynucleotide is located 5' to the second polynucleotide; and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b). In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other.

[0014] In some aspects, provided herein are single-stranded expression cassettes comprising: (a) a first polynucleotide comprising a first promoter operably linked to a first nucleotide sequence encoding a single guide RNA; (b) a second polynucleotide comprising the antisense of a second nucleotide sequence, wherein the second nucleotide sequence comprises a regulatory sequence operably linked to a third nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); wherein the first polynucleotide is located 5' to the second polynucleotide; and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b). In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other.

[0015] In some aspects, in the single-stranded expression cassettes: (a) the nucleotide sequence encoding the fusion protein further comprises a SV40 or a bGH polyadenylation signal; (b) the fusion protein further comprises at least one SV40 or nucleoplasmin nuclear localization signal (NLS); (c) the regulatory sequence comprises a Muscle Creatine Kinase (Ckm) promoter; (d) the regulatory sequence comprises a Ckm enhancer; (e) the TRD is a SUV39H1 pre-SET, SET,and post-SET domain; a KRAB TRD; a MeCP2 TRD; a HPla TRD; or a HPly TRD; (f) the nucleotide sequence encoding the single guide RNA comprises a scaffold portion capable of binding to a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY, and a spacer portion comprising a sequence targeting &DUX4 gene promoter and / or a DUX4 gene exon 1; (g) the dCas enzyme is a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY; (h) the ITRs are derived from an AAV genome of serotype AAV2; and (i) in the fusion protein the dCas is located N- terminal to the TRD.

[0016] In some aspects, provided herein are self-complementary expression cassette comprising an expression cassette disclosed herein and its complementary sequence.

[0017] In some aspects, provided herein are AAV vectors comprising the expression cassettes, the single-stranded expression cassettes, or the self-complementary expression cassettes disclosed herein, or any combination thereof.

[0018] In some aspects, provided herein are lipid nanoparticles (LNP) comprising the expression cassettes, the single-stranded expression cassettes, the self-complementary expression cassettes disclosed herein, or any combination thereof.

[0019] In some aspects, provided herein are cells comprising the expression cassettes, the single-stranded expression cassettes, the self-complementary expression cassettes, the AAV vectors, the LNP disclosed herein, or any combination thereof.

[0020] In some aspects, provided herein are pharmaceutical compositions comprising the expression cassettes, the single-stranded expression cassettes, the self-complementary expression cassettes, the AAV vectors, the LNP, the cells disclosed herein, or any combination thereof, and a pharmaceutically acceptable carrier.

[0021] In some aspects, provided herein are methods of inducing transcriptional repression of a DUX4 gene in a population of cells comprising contacting the population of cells with the single-stranded expression cassettes, the self-complementary expression cassettes, the AAV vectors, the LNP, the pharmaceutical compositions disclosed herein, or any combination thereof.

[0022] In some aspects, in the methods disclosed herein the transcription level of the D UX4 gene in the population of cells is reduced compared to the transcription level of the DUX4 gene in the same population of cells before contacting the population of cells with the single-stranded expression cassettes, the self-complementary expression cassettes, the AAV vectors, the LNP, the pharmaceutical compositions disclosed herein, or any combination thereof.

[0023] In some aspects, in the methods disclosed herein the transcription level of the DUX4 gene in the population of cells is reduced compared to the transcription level of the DUX4 gene in a same population of cells not contacted with the single-stranded expression cassettes, the self- complementary expression cassettes, the AAV vectors, the LNP, the pharmaceutical compositions disclosed herein, or any combination thereof.

[0024] In some aspects, provided herein are methods of treating FSHD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the singlestranded expression cassettes, the self-complementary expression cassettes, the AAV vectors, the LNP, the pharmaceutical compositions, the cells disclosed herein, or any combination thereof. In some aspects, the FSHD is FSHD1 or FSHD2. In some aspects, the subject is a human.BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 shows schematics of the generated and tested single-vector expression cassettes for CRISPR inhibition (CRISPRi) (HLH reg: C w-based regulatory cassette; NLS: bipartite SV40 nuclear localization signal; dSaCas9: dead SaCas9; TRD: transcriptional repressor domain; HA: hemagglutinin tag; SV40 pA: SV40 polyadenylation signal; saCas9 Scaffold: scaffold portion of the sgRNA; sgRNA: spacer portion of the sgRNA; U6 Prom: U6 promoter).

[0026] FIGs. 2A-2B show charts representing the expression levels of DUX4-fl (FIG. 2A) and of the DUX4-FL target MBD3L2 (FIG. 2B) as assessed by qRT-PCR in FSHD1 myocytes transduced as indicated. Data are plotted as the mean + / - SEM from at least four independent experiments, with average expression from cells infected with each control vector set to 1. *p<0.05, ****p<0.0001 are from comparing to the equivalent control vector.

[0027] FIGs. 3A-3Q show charts representing the expression levels of DUX4-fl, DUX4- FL target genes MBD3L2 and TRIM43, and the dSaCas9 cargo (Cas9) as assessed by qRT-PCR in three lines of FSHD1 myocytes: MB073 (FIGs. 3A-3D), MB2306 (FIGs. 3E-3H), and RL-01 (FIG. 3I-3L), and one line of FSHD2 myocytes: MB2453 (FIGs. 3M, 3O-3Q), transduced as indicated. The expression levels of DUX4-fl from the 4AL FSHD-permissive allele in FSHD2 myocytes MB2453 are also shown (FIG. 3N). Data are plotted as the mean + / - SEM from five independent experiments, with average expression from cells infected with each control vector set to 1. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 are from comparing to NT.

[0028] FIGs. 4A-4P show charts and tables from RNA-seq analysis of cells from four FSHD lines (MB073, MB2306, RL-01, and MB2453) and two healthy lines (MB 135 and RL-06)transduced with TRD(AS DUX4) or TRD(AS NT). Heat maps (FIGs. 4A-4H) show the relative expression changes in cells treated with TRD(AS DUX4) compared to cells treated with TRD(AS NT) of differentially expressed genes (DEGs) that are DUX4 targets or members of DUX4 target families from FSHD lines: MB073 (FIGs. 4A-4B), MB2306 (FIGs. 4C-4D), RL-01 (FIGs. 4E- 4F), and MB2453 (FIGs. 4G-4H). Left and right panels list DUX4 targets whose expression is increased or decreased by TRD(AS DUX4) compared to TRD(AS NT), respectively. Relative expression levels are shown for each of 5 biological replicates and the composite (average). Gradient bars on the right indicate the fraction of relative expression compared to the average expression in control-treated cells (set to 1); (e.g., 0.2 indicates 20% of the average expression in control -treated cells). DEGs that represent DUX4 targets or members of DUX4 target families are shown for each FSHD line (FIG. 41), with genes common to multiple lines highlighted, i: immortalized; pr: primary. Adjusted volcano scatter plots (FIGs. 4J-4O) show the global transcriptional changes in cells treated with TRD(AS DUX4) vs. TRD(AS NT). Each data point represents the change in expression levels of a specific gene in cells treated wtih TRD(AS DUX4) compared to cells treated with TRD(AS NT). Treated cells are from four FSHD lines (MB073 (FIG. 4J), MB2306 (FIG. 4K), RL-01 (FIG. 4L), and MB2453 (FIG. 4M)) and two healthy lines (MB 135 (FIG. 4N) and RL-06 (FIG. 40)). DEGs that do not correspond to known DUX4 targets or target families are summarized (FIG. 4P), with genes common to multiple lines highlighted.

[0029] FIG. 5A-B show charts representing relative enrichment of factors at the DUX4 locus and at the MYODI locus (as a negative control) following transduction of FSHD 1 (FIG. 5A) or FSHD2 (FIG. 5B) myocytes with TRD(AS DUX4) or TRD(AS NT). Chromatin was immunoprecipitated using antibodies to SaCas9, KAP1, H3K9me3, or H3K27me3, and analyzed by qPCR using primers to DUX4 or MYODI (as a negative control). Data are presented as fold enrichment of the target region by each specific antibody in TRD(AS DUX4)-treated cells normalized to enrichment in TRD(AS NT)-treated cells, with all samples normalized to input DNA. Each bar represents the average of four (for FSHD1 cells) or three (for FSHD2 cells) independent ChIP experiments. *p<0.05, **p<0.01 are from comparing to enrichment MYODl.

[0030] FIG. 6 shows a chart representing the expression levels of DUX4-fl and the DUX4- FL target MBD3L2 from tibialis anterior (TA) xenografts of FSHD xenografted mice treated as indicated. Gene expression was assessed by qRT-PCR and normalized to levels of RPL13A. Data are plotted as the mean + / - SEM from at least two independent experiments, with average expression from mice injected with each control vector set to 1.

[0031] FIG. 7 shows a chart representing the expression levels of DUX4-fl and the DUX4-FL mouse target Wfdc3 from tamoxifen (TMX)-inducible ACTA1-MCM; FLExD moderate pathology transgenic mice treated as indicated. Gene expression from TA muscles was assessed by qRT-PCR and normalized to levels of Rpl37. Data are plotted as the mean + / - SEM from at least two independent experiments, with average expression from mice injected with each control vector set to 1.

[0032] FIG. 8 shows a chart representing the physical performance as assessed by a treadmill stress test at 9 days post-TMX injection in mice from FIG. 7. Wild-type performance is the 20-min duration of the test, indicated by the dotted line. Data are plotted as the mean + / - SEM from at least two independent experiments.

[0033] FIGs. 9A-G show charts depicting the efficacy of the CRISPRi treatment in the TMX-inducible ACTA1-MCM; FLExD moderate pathology transgenic mouse model. TRD(AS DUX4) was injected systemically in the LICA1 rAAV vector (LICA1.TRD[AS DUX4] at 1E13 or 5E13 vg / kg. Mice were weighed 7 days prior to TMX induction of DUX4- I expression, and 0, 3, 7, and 10 days post-induction (FIGs. 9A-9C). Treadmill tests were conducted at 9 days post-TMX induction (FIG. 9D). Dotted line at 20 min indicates time of fatigue in uninduced (unaffected) littermates. Vector copy number (VCN) per diploid genome (FIG. 9E) and dSaCas9 expression (FIG. 9F) in EDL muscles are shown. Expression of DUX4-fl and DUX4-FL mouse target genes Wfdc3 and Slcl5a2 in extensor digitorum longus (EDL) muscles were assessed by qRT-PCR and normalized to levels of Rpll3a (FIG. 9G). *p<0.05, **p<0.01 are from comparing to the TRD(AS NT) control.

[0034] FIGs. 10A-D show charts representing the expression levels of DUX4-fI (FIGs. 10A and 10C) and the DUX4-FL target MBD3L2 (FIGs. 10B and 10D) as assessed by qRT-PCR in FSHD1 myocytes transduced as indicated. TRD: MeCP2 TRD; HPla: chromo shadow domain and C-terminal extension; Ml 1 : Ml 1 hybrid promoter driving sgRNA. Data are plotted as the mean + / - SEM from at least four independent experiments, with average expression from cells infected with each control vector set to 1. **p<0.01, ***p<0.001 are from comparing to the equivalent control vector.DETAILED DESCRIPTION OF THE DISCLOSUREDefinitions

[0035] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0036] It is to be noted that, as used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component; for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences, unless stated otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0037] Furthermore, "and / or," where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0038] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and / or "consisting essentially of' are also provided.

[0039] As used herein, the term "about" refers to a value that is within 10% above or below the value being described.

[0040] As used herein, the term "at least" prior to a value or series of values is understood to include the values adjacent to the term "at least," and all subsequent values (numbers, integers, or fractions) that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer, e.g., "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).

[0041] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0042] As used herein, the term "derived from," refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.

[0043] A "chemical bond" is a link between atoms or ions. Chemical bonds can be "covalent bonds" or "noncovalent bonds." A covalent bond refers to the mutual sharing of one or more pairs of electrons between two atoms. A noncovalent bond refers to a relatively weak bond / interaction formed between molecules without sharing electrons. Exemplary noncovalent bonds are ionic interactions, hydrophobic interactions, and hydrogen bonds.

[0044] "Nucleic acid," "polynucleotide," and "oligonucleotide," are used interchangeably in the present application. These terms refer only to the primary structure of the molecule. Thus, unless specified otherwise, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA (e.g., messenger RNAs (mRNAs), guide RNAs (gRNAs), genomic DNAs, plasmid DNAs (pDNAs), or complementary DNAs (cDNAs)). The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as it is generally understood by the person skilled in the art as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Polynucleotides can be made recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis. When referring to the sequence of a polynucleotide, reference is made to the order of the nucleobase (or modifications thereof) comprised in the covalently linked nucleosides (or nucleotides) comprised in the polynucleotide.

[0045] By "isolated" polynucleotide is intended a polynucleotide, DNA or RNA, which has been removed from its native environment. An isolated polynucleotide includes polynucleotides produced and maintained (e.g., upon purification) in vitro, or recombinant polynucleotides maintained in host cells or purified (partially or substantially) from the host cell and maintained in solution. Non-limiting examples of isolated polynucleotides include molecules produced synthetically (z.e., by chemical synthesis), by PCR, by enzymatic digestion (e.g., produced by digestion with restriction enzymes), by ligation, by molecular cloning of a nucleic acid sequences in a vector (e.g. , a viral or a non-viral vector), or, in the case of RNA, transcripts produced by invitro transcription or transcripts produced in host cells (e.g, a heterologous host cells) and maintained in the host cell or purified (partially or substantially) from the host cell and maintained in solution.

[0046] Polynucleotides can be or can include a regulatory element such as a promoter, enhancer, silencer, ribosome binding site, internal ribosomal entry site (IRES), or a transcription terminator (e.g., polyadenylation signal). Further, polynucleotides can comprise sequence elements encoding for regulatory amino acid sequences in the encoded protein product. Non-limiting examples of such regulatory amino acid sequences are intracellular localization signals (e.g., nuclear localization signals, NLS) and self-processing cleavage sites (e.g., 2A peptides such as P2A, E2A, F2A, and T2A, or furin cleavage sites).

[0047] As used herein, the term "specifically hybridizes" refers to the ability of a nucleic acid molecule (e.g., a gRNA) to hybridize to another nucleic acid molecule (e.g., a target genomic region) with greater affinity than it hybridizes to another nucleic acid sequence. An oligonucleotide can specifically hybridizes to more than one target sequence. A portion of a nucleic acid molecule can also specifically hybridize to another portion of the same nucleic acid molecule (intramolecular hybridization).

[0048] As used herein, the term "modulate," or "modulation" refers to a change of amount or quality of a function or activity when compared to the function or activity prior to modulation. Modulation includes either an increase (stimulation or induction) or a decrease (inhibition or reduction).

[0049] As used herein, the term "nucleotide" refers to monomeric units of nucleic acid polymers (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). Naturally occurring nucleotides are composed of three subunit molecules: a naturally occurring nucleobase, a naturally occurring five-carbon sugar (ribose or deoxyribose), and a naturally occurring phosphate group consisting of one to three phosphates. A nucleotide can also be a modified nucleotide, which is a nucleotide comprising a modified nucleobase, a modified sugar moiety, a modified backbone, or any combination thereof.

[0050] As used herein, the term "nucleobases," also known as "nitrogenous bases," or "bases," refers to biological compounds that form nucleosides, which, in turn, are components of nucleotides. A nucleobase may be naturally occurring (such as adenine, guanine, cytosine, thymine, and uracil), synthetic, or modified that is capable of hydrogen bonding with another nucleobase (naturally occurring, synthetic, or modified). Examples of modified nucleobasesinclude, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5- methylcytosine, and 5-hydroxymethoylcytosine.

[0051] A "nucleoside" comprises a nucleobase and a sugar moiety. Both the nucleobase and the sugar moiety can be naturally occurring or modified. Naturally occurring nucleosides includes deoxyribonucleosides and ribonucleosides. Modified nucleosides includes nucleosides with modified or substituted sugar groups, and / or modified nucleobases.

[0052] A "sugar moiety" may comprise ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog. Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-0-Me), 2'-O- methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'^P5' phosphoramidate,2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. A sugar moiety modification may include an extra bridge bond, such as in a locked nucleic acid (LNA). A sugar analog may contain a morpholine ring, such as phosphorodiamidate morpholino (PMO). A sugar moiety may comprise a ribofuransyl or 2'deoxyribofuransyl modification. A sugar moiety may comprise 2 '4 '-constrained 2'0-methyloxyethyl (cMOE) modifications. A sugar moiety may comprise cEt 2', 4' constrained 2'-0 ethyl BNA modifications. A sugar moiety may comprise tricycloDNA (tcDNA) modifications. A sugar moiety may comprise ethylene nucleic acid (ENA) modifications. A sugar moiety may comprise MCE modifications. Modifications are described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1): 37-47, incorporated by reference for this purpose herein.

[0053] As used herein, the terms "backbone" and "backbone structure" refer to the connection between monomers of a nucleic acid. In naturally occurring oligonucleotides, the backbone comprises a 3 '-5' phosphodiester linkage connecting sugar moieties of the oligomer. The backbone structure may include, for example, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See, e.g., LaPlanche et al. Nucleic Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucleic Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). A backbonestructure may not contain phosphorous but rather peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. A backbone modification can be a phosphothioate linkage, or a phosphoramidate linkage.

[0054] The term "vector," as used herein, includes any vector known to the skilled person including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as retroviral, adenoviral or baculoviral vectors), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC)). Said vectors include expression as well as cloning vectors. Expression vectors generally contain a desired coding sequence and appropriate regulatory sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain nucleotide molecule and may comprise specific functional sequences needed for insertion and / or expression of the desired nucleotide molecule. A "vector" can be any vehicle for the cloning, transfer and / or expression of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. Thus, the term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo, or in vivo. In some aspects, insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide into a chosen vector that may or not have complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that can be used for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes described in the literature include: genes providing resistance to neomycin, ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters described in the literature include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0055] The term "expression," as used herein, refers to a process by which a DNA molecule (such as a gene) produces a biochemical, for example, an RNA transcript or a protein. The process includes any manifestation of the functional presence of the DNA molecule (such as a gene) withinthe cell including, without limitation, gene knock-in, as well as both transient expression and stable expression. Expression may include, without limitation, transcription of the DNA molecule (such as a gene) into messenger RNA (mRNA), and the translation of such mRNA into protein(s). Expression of a DNA molecule (such as a gene) produces an "expression product" (such as a "gene expression product"). As used herein, an "expression product" can be either a nucleic acid, e.g., a messenger RNA, or a non-coding RNA (that is an RNA that is not translated into a protein and that can exert a function in the form of RNA (e.g., a tRNA, or a guide RNA in a CRISPR system)), produced by transcription of the DNA molecule (such as a gene), or a protein which is translated from an mRNA transcript. Expression products described herein further include nucleic acids with post transcriptional modifications, e.g., mRNAs which are processed, for example, by capping, splicing, and / or polyadenylation, or peptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0056] The term "mRNA," as used herein includes mRNA transcribed within a cell, as well as in vitro transcribed RNA (IVT RNA) or synthetic RNA (c.g, produced by chemical synthesis). An mRNA molecule may also contain a 5' untranslated region (5'-UTR), and / or a 3' untranslated region (3'-UTR). An mRNA can be produced by in vitro transcription using a DNA template.

[0057] As used herein, the terms "protein," "polypeptide," and "peptide," are used interchangeably, and are intended to encompass a singular "protein," as well as plural " proteins," and refer to a molecule composed of monomers (amino acids) linked by amide bonds (also known as peptide bonds). The term "protein" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, polypeptide, protein, amino acid chain, or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "protein," and the term "protein" can be used instead of, or interchangeably with, any of these terms. The term "protein" is also intended to refer to the products of post-translational modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A protein can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. Proteins include protein fragments, denatured / unstructured proteins, proteins having a primary, a secondary, a tertiary, or aquaternary or aggregated structure, etc. A protein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Proteins can have a defined three-dimensional structure, although they do not necessarily have such structure. Proteins with a defined three- dimensional structure are referred to as folded, and polypeptides that do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded.

[0058] As used herein, the term "coding sequence" or a sequence "encoding" refers to a nucleic acid that is transcribed and can be translated, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter, to produce an expression product. A DNA molecule that is transcribed into a RNA molecule (such as a messenger RNA, or a non-coding RNA) is a DNA molecule encoding such RNA molecule. A DNA molecule that is transcribed in a messenger RNA that is translated into a protein is a DNA molecule encoding such protein.

[0059] As used herein, the term "exon" refers to a coding section of a DNA molecule, or of an RNA molecule which is transcribed from a DNA molecule, that is translated into protein. Exons can be separated by intervening sections of DNA that do not code for proteins, known as "introns." Therefore, the term "intron," as used herein, refers to a segment of nucleic acid that is transcribed and is present in the "pre-mRNA" but excised by the splicing machinery and therefore not present in the mature mRNA transcript. Following transcription, new, immature strands of messenger RNA, called "pre-mRNA," may contain both introns and exons. These pre-mRNA molecules go through a modification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain in the "mature mRNA." Splicing produces a mature messenger RNA molecule that is then translated into a protein. The term "first exon" refers to a coding sequence or sequence of nucleic acids that encodes a polypeptide or polypeptide region and the term "second exon" refers to a different second coding sequence or sequence of nucleic acids that encodes a second polypeptide region. Where the two exons are separated by an intervening intron in the pre-mRNA, the splicing machinery operates to remove the intervening intron and join the two exons in the mature mRNA.

[0060] The term "polyadenylation signal," refers to a nucleic acid sequence present in the RNA transcript that allows for the transcript, when in the presence of the enzyme polyadenyl transferase, to be polyadenylated.

[0061] The term "nuclear localization signal" or NLS refers to an amino acids sequence that mediates the transport of a protein into the nucleus.

[0062] The term "promoter," as used herein in, refers to a nucleotide sequence capable of directing transcription of a DNA sequence operably linked thereto, in a cell or in vitro. A promoter is intended as a DNA region to which RNA polymerases bind and that directs the enzymes to transcribe the operably linked DNA sequence. A DNA sequence and a promoter are "operably linked" if the promoter is capable of directing transcription of that DNA sequence. A promoter can be a "constitutive" promoter that, when operably linked to a polynucleotide encoding an expression product, results in the production of the expression product in a cell under most or all conditions of the cell. A promoter can be a "regulatable promoter" whose activity is affected by a cis or trans acting factor (e.g., an inducible promoter). The term "inducible" promoter means that when the promoter is operably linked to a polynucleotide encoding a specified expression product, it results in the production of the expression product in the cell basically only when the inducer corresponding to the promoter is present in the cell. A promoter can be a "ubiquitous promoter" that is a promoter that is active in a wide range of cells, tissues, and cell cycles, or a "tissue-specific promoter" that is a promoter that has activity only or mostly in certain cell types, z.e., drives the expression of the operably linked nucleotide sequence only or mostly in certain cell types. A promoter can be a "bidirectional promoter" that is a promoter that is an intergenic region between two divergent genes located on complementary strands of the DNA, and drives their coordinated transcription in opposite directions. Promoters include naturally occurring promoters, as well as functional fragments thereof that retain (at least in part) the capacity of directing transcription of a DNA sequence operably linked thereto, such as minimal promoters. Promoters can also include artificially designed and constructed promoters, which can include naturally occurring promoters, modifications thereof, as well as functional fragments thereof that retain (at least in part) the capacity of directing transcription of a DNA sequence operably linked thereto.

[0063] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence capable of regulating the expression of a nucleic acid sequence operably linked to said regulatory sequence. Regulatory sequences are or include, for example, promoters, enhancers (a DNA sequence that increases the level of transcription of an operably linked gene), and silencers (a DNA sequence that decreases the level of transcription of an operably linked gene), or combinations thereof. A regulatory sequence comprises for example, a promoter (or a functional fragment thereof) and one or more enhancers (or a functional fragment thereof). Enhancers and silencersinclude naturally occurring enhancers and silencers, as well as functional fragments thereof that retain (at least in part) the capacity of regulating transcription of a DNA sequence operably linked thereto. Enhancers and silencers can also include artificially designed and constructed enhancers and silencers, which can include naturally occurring enhancers and silencers, modifications thereof, as well as functional fragments thereof that retain (at least in part) the capacity of regulating transcription of a DNA sequence operably linked thereto.

[0064] The term "operably linked" means that a coding nucleic acid sequence and a regulatory sequence(s) are arranged in such a way as to permit the expression of the coding nucleic acid sequence when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).

[0065] As used herein, the term "expression cassette" refers to a polynucleotide flanked by two inverted terminal repeats (ITRs). In one embodiment, the two ITRs are parts of an expression cassette.

[0066] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside of an organism. This technology comprises, but is not limited to, combining nucleic acid sequences (e.g., coding sequences, regulatory elements (e.g., promoters, enhancers, silencers, termination sequences), linkers (e.g, spacers, internal ribosome entry sites, cleavage sites)) derived from a variety of sources, inserting nucleic acid sequences from a variety of sources in appropriate vectors (e.g., delivery vectors, expression vectors, integrating vectors), modifying or altering nucleotide sequences (e.g., by mutagenesis, insertion of modified nucleotides, 5'-capping, polyadenylation), synthesizing artificial nucleotide sequence. A variety of techniques described in the literature (e.g., molecular cloning, polymerase chain reaction (PCR), digestion with restriction enzymes, in vitro ligation, mutagenesis, site-directed mutagenesis, prokaryotic and eukaryotic cell transformation or transduction, in vitro DNA / RNA synthesis, in vitro RNA-5'-capping, in vitro RNA-polyadenylation, complementary DNA (cDNA) synthesis, nucleic acid isolation, and the like) can be used to manipulate nucleic acid sequences outside an organism (see for example Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4thedition).

[0067] As used herein, the term "recombinant," refers to any nucleic acid (e.g., DNA, or RNA), peptide (e.g., oligopeptide, polypeptide, or protein), cell, or organism, which is made by modification of nucleic acid sequences. For example, "recombinant DNA" molecules are DNA molecules derived from one organism and inserted in a host organism to produce new geneticcombinations. For example, "recombinant RNA" molecule (e.g., recombinant mRNA molecules) are RNA molecules derived from one organism and inserted in a host organism to produce the expression of a desired genetic product in the host organism.

[0068] As used herein, the term "genetic locus, " refers to the physical site or location within a genome of a specific DNA sequence, for example a gene.

[0069] As used herein, the term "cell" or "cells" refers not only to the particular subject cell, but also to the progeny or to the potential progeny of such cell(s). The scope of the term as used herein also encompasses the progeny that may or may not in fact be identical to the parent cell because certain modifications may occur in succeeding generations due to either mutation or environmental influences. In one embodiment, the cell comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell. In another embodiment, the mammalian cell comprises HEK293 or HeLa cell.

[0070] As used herein, the term "testing sample" refers to biological material, maintained in culture or derived from an organism, which is tested to assay a quality or a parameter of interest (e.g., the expression level of a gene). Non-limiting examples of such biological materials are cells maintained in culture; whole non-human organisms or any portion or fragment thereof (e.g., an organ, a tissue, a cell, extracellular material, or any fragment or combination thereof); as well as organ or tissue fragments (e.g., biopsies), cells, extracellular material, or any combination thereof, derived from a human subject.

[0071] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modified in Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology,266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity "X" of a first nucleotide (or amino acid) sequence to a second nucleotide (or amino acid) sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide (or amino acid) residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence.

[0072] As used herein, the term "flank," with respect to a sequence that is flanked by other elements, indicates the presence of one or more of the elements that are upstream and / or downstream (z.e., 5' and / or 3', or N-terminal and / or C-terminal) relative to the sequence or that are part of the sequence and at 5' and / or 3' of such an sequence. The term "flank" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding a transgene and a flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e.g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences between.

[0073] As used herein, the terms "AAV virion," "AAV vector," "AAV viral particle," or "AAV particle" refer to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector genome. The particle can comprises a heterologous polynucleotide (z.e., a polynucleotide other than a wild-type AAV genome such as a transgene).An AAV particle comprising a heterologous polynucleotide can be also referred to as a "recombinant AAV particle," "recombinant AAV vector," "rAAV particle," or "rAAV vector."

[0074] As used herein, the term "epigenetic" refers to the changes in gene function that do not entail change in DNA sequence.

[0075] As used herein, the term "administration" refers to the administration of a composition or substance to a subject or system. Administration to an animal subject (e.g., to a human) can be by any appropriate route. "Administering" refers to the physical introduction of a composition or substance, which may comprise a therapeutic agent, to a subject, using any of the various methods and delivery systems known to those skilled in the art. Non-limiting examples of routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Administration can also be via a non-parenteral route, for example, orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, aborally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0076] As used herein, the terms "treat," "treated," and "treating" mean both therapeutic and prophylactic treatment or preventative measures wherein the object is to reverse, alleviate, ameliorate, lessen, inhibit, slow down progression, development, severity or recurrence of an undesired symptom, complication, condition, biochemical indicia of a disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (ie., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. In some aspects,treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g, "prophylactic agent," "prophylactic treatment," "prophylactically effective amount"), refers to any complete or partial prevention of a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect and / or symptom attributable to the disease.

[0077] As used herein, the term "gene therapy" refers to the administration into an individual's cells and / or tissues of an exogenous molecule (e.g., a nucleic acid sequence) to treat, reduce the symptoms of, or reduce the likelihood of a disease, disorder, syndrome, or condition. An exogenous molecule or sequence is understood to be a molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

[0078] As used herein, the term "subject" refers to any organism to which a composition or a substance (e.g., a nucleotide molecule) can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0080] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had byreference to the specification as a whole. Accordingly, the terms defined immediately below and above are more fully defined by reference to the specification in its entirety.

[0081] Various aspects of the invention are described in further detail in the following subsections.Expression Cassettes

[0082] In some aspects, disclosed herein are polynucleotides (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) for the efficient expression of CRISPR systems. In some aspects, the CRISPR system is a CRISPRi system. In some aspects, a CRISPRi system comprises a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD). In some aspects, a CRISPRi system comprises a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional activator domain. Such fusion proteins are herein also referred to as dCas-TRD fusion proteins. In some aspects, the CRISPRi system further comprises a single guide RNA capable of binding to the dCas. In some aspects, the single guide RNA targets (i.e. , specifically hybridizes with, binds to) a specific locus in the genome of a cell. In some aspects, the RNA targets (ie., specifically hybridizes with, binds to) a specific locus in the genome of a cell and thereby directs the dCas-TDR fusion protein to the specific locus in the genome of the cell. In some aspects, the specific locus in the genome of a cell comprises a. DUX-l gene.

[0083] In some aspects, the polynucleotides disclosed herein are expression cassette comprising: (a) a first polynucleotide comprising a regulatory sequence operably linked to a first nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); (b) a second polynucleotide comprising a first promoter operably linked to a second nucleotide sequence encoding a single guide RNA; wherein the first polynucleotide and the second polynucleotide are placed in different orientation from each other, and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b). In some aspects, the first polynucleotide sequence is located 5' to the second polynucleotide. In some aspects, the second polynucleotide sequence is located 5' to the first polynucleotide.

[0084] In some aspects, the polynucleotides disclosed herein are expression cassettes comprising: (a) a first polynucleotide comprising a regulatory sequence operably linked to a nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); and (b) a second polynucleotide comprisingthe antisense of a nucleotide sequence, wherein the nucleotide sequence comprises a first promoter operably linked to a nucleotide sequence encoding a single guide RNA. In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other. In one embodiment, the expression cassette is singlestranded. In another embodiment, the expression cassette is double-stranded or self- complementary.

[0085] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein comprise, from 5' to 3':(a) a first polynucleotide comprising:(i) a regulatory sequence,(ii) a nucleotide sequence encoding a first NLS,(iii) a nucleotide sequence encoding a dCas,(iv) a nucleotide sequence encoding a second NLS,(v) a nucleotide sequence encoding a transcriptional repressor domain, and(vi) a polyadenylation signal; and(b) a second polynucleotide comprising:(i) the antisense of a nucleotide sequence encoding a single guide RNA, and(ii) the antisense nucleotide sequence of a promoter.

[0086] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein comprise, from 5' to 3':(a) a first polynucleotide comprising:(i) a HLH regulatory cassette,(ii) a first portion of a nucleotide sequence encoding a bipartite SV40 NLS,(iii) a nucleotide sequence encoding a dSaCas9,(iv) a second portion of a nucleotide sequence encoding a bipartite SV40 NLS,(v) a nucleotide sequence encoding a MeCP2 transcriptional repressor domain, and(vi) an SV40 polyadenylation signal; and(b) a second polynucleotide comprising:(i) the antisense of a nucleotide sequence encoding a single guide RNA targeting the DUX4 gene, and(ii) the antisense nucleotide sequence of a U6 promoter.

[0087] In some aspects, the polynucleotides disclosed herein are expression cassettes comprising: (a) a first polynucleotide comprising a first promoter operably linked to a nucleotide sequence encoding a single guide RNA; (b) a second polynucleotide comprising the antisense of a nucleotide sequence, wherein the nucleotide sequence comprises a regulatory sequence operably linked to a nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD). In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other. In one embodiment, the expression cassette is single- stranded. In another embodiment, the expression cassette is double-stranded or self-complementary.

[0088] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein comprise, from 5' to 3':(a) a first polynucleotide comprising:(i) a promoter,(ii) a nucleotide sequence encoding a single guide RNA; and(b) a second polynucleotide comprising(i) the antisense nucleotide sequence of a polyadenylation signal,(ii) the antisense of a nucleotide sequence encoding a transcriptional repressor domain,(iii) the antisense of a nucleotide sequence encoding a first NLS,(iv) the antisense of a nucleotide sequence encoding a dCas,(v) the antisense of a nucleotide sequence encoding a second NLS,(vi) the antisense of a regulatory cassette.

[0089] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein comprise, from 5' to 3':(a) a first polynucleotide comprising:(i) a U6 promoter,(ii) a nucleotide sequence encoding a single guide RNA targeting the DUX4 gene; and(b) a second polynucleotide comprising(i) the antisense nucleotide sequence of an SV40 polyadenylation signal,(ii) the antisense of a nucleotide sequence encoding a MeCP2 transcriptional repressor domain,(iii) the antisense of a nucleotide sequence encoding a first portion of a bipartite SV40 NLS,(iv) the antisense of a nucleotide sequence encoding a dSaCas9,(v) the antisense of a nucleotide sequence encoding a second portion of a bipartite SV40 NLS,(vi) the antisense of a HLH regulatory cassette.

[0090] In some aspects, the first polynucleotide is located 5' to the second polynucleotide. In some embodiments, the expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b). In some embodiments, the first polynucleotide and the second polynucleotide are placed in different orientation from each other. In another embodiment, the first polynucleotide and the second polynucleotide are placed in the same orientation from each other.

[0091] In some aspects, transcription of the nucleotide sequence encoding the dCas-TRD fusion protein and of the nucleotide sequence encoding the sgRNA proceeds in opposite directions on the expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein.

[0092] A single-stranded expression cassette disclosed herein can be converted in a double stranded expression cassette. For example, a single-stranded expression cassette disclosed herein can be converted in a double stranded expression cassette by DNA polymerases within a cell or in vitro, by using the single-stranded expression cassette as a template for the synthesis of the complementary strand. Double-stranded expression cassettes comprising the single-stranded expression cassettes disclosed herein can also be generated by any means know to those skilled in the art. For example, a double-stranded expression cassette comprising the single-stranded expression cassettes disclosed herein can be generated by designing and generating any doublestranded vector (e.g., a plasmid vector) comprising two strands: one identical to the single-stranded expression cassette and one complementary to the single-stranded expression cassette.

[0093] The resulting double stranded expression cassette comprises two complementary strands: one identical to the single-stranded expression cassette and one complementary to the single-stranded expression cassette. The strand that is complementary to the single-stranded expression cassette comprise the sense nucleotide sequence of the antisense nucleotide sequence comprised in the single-stranded expression cassette. Therefore, the strand identical to the singlestranded expression cassette comprises the sense sequence of the first polynucleotide (a) and thestrand that is complementary to the single-stranded expression cassette comprises the sense sequence of the second polynucleotide (b). RNA transcription proceeds in opposite directions on the two complementary strands of the double stranded expression cassette.

[0094] A single-stranded expression cassette disclosed herein can be represented by the structure:(sense)Rl-(sense)Cl-(antisense)C2-(antisense)R2 wherein:(sense)Rl is a first regulatory sequence in the sense orientation;(sense)Cl is a first coding nucleotide sequence in the sense orientation;(antisense)C2 is the antisense of a second coding nucleotide sequence; and(antisense)R2 is the antisense of a second regulatory sequence.

[0095] When a single- stranded expression cassette disclosed herein is converted in a double stranded expression cassette , the double stranded expression cassette comprises:A. a first strand identical to the single-stranded expression cassette, comprising: (sense)Rl-(sense)Cl-(antisense)C2-(antisense)R2; andB. a second strand that is complementary to the single-stranded expression cassette, comprising: (anti sense)Rl -(anti sense)Cl-(sense)C2-(sense)R2.Transcription of Cl and C2 proceeds in opposite directions on the two A and B strands of the double stranded expression cassette. That is, transcription of the nucleotide sequence encoding the dCas-TRD fusion protein and of the nucleotide sequence encoding the sgRNA proceeds in opposite directions on the two strands of the double stranded expression cassette disclosed herein.

[0096] The expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) disclosed herein result in increased expression of the dCas- TRD fusion protein and / or of the sgRNA compared to expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) comprising a strand represented by the structure:(sense)Rl-(sense)Cl-(sense)C2-(sense)R2.

[0097] That is, the expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) disclosed herein result in increased expression of either the dCas-TRD fusion protein or of the sgRNA, or both, as compared to expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) wherein transcription of the nucleotide sequence encoding the dCas-TRD fusion protein and of thenucleotide sequence encoding the sgRNA are in the same orientation. In some embodiments, the expression of the dCas-TRD fusion protein is increased. In some embodiments, the expression of the sgRNA is increased. In some embodiments, the expression of the dCas-TRD fusion protein and of the sgRNA is increased. In some embodiments, the expression of the dCas-TRD fusion protein, the expression of the sgRNA, or of both, is increased of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 ? 4>, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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.

[0098] As used herein, the terms "sense" and "antisense," are defined as it is generally understood by the person skilled in the art. It is understood that a nucleic acid molecule (e.g., a DNA molecule encoding a polypeptide or a non-coding RNA) can have a double stranded structure (z.e., consists of two strands of polynucleotides). Based on the strand that serves as a template, for example for mRNA synthesis (z.e., transcription), one strand is called "sense strand" and the other strand is called "antisense strand."

[0099] The sense strand, (or coding strand, plus strand, or non-template strand), comprises a nucleotide sequence (the sense nucleotide sequence) which is the same as the nucleotide sequence comprised in the transcribed sequence (e.g., pre-mRNA or mRNA except the thymine is DNA is replaced by uracil in RNA). The antisense strand (complementary strand, non-coding strand, minus strand, or template strand), comprises a nucleotide sequence (the antisense nucleotide sequence) which is complementary to the nucleotide sequence comprised in the transcribed sequence (e.g.,pre-mRNA or mRNA). The antisense strand acts as a template for the synthesis of the pre-mRNA, mRNA, or non-coding RNAs.

[0100] As used herein, the term "complementary to," when used to describe a nucleic acid molecule, means being able to hybridize (e.g., bind) to another nucleic acid molecule by Watson- Crick base pairing, and / or non-Watson-Crick base pairing, and / or base pairing formed by nonnatural and alternative nucleotides or nucleosides. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. Additionally, it is to be understood that thymidine (T) of any given DNA sequence is replaced by uridine (U) in its corresponding RNA transcript and that this difference does not alter the understanding of the term "complementarity."

[0101] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein comprise a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.I. CRISPRi Systems

[0102] CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR-associated proteins) systems are adaptive immunity systems that are encoded by most archaea and many bacteria and that act against invading genetic element, such as viruses and plasmids.

[0103] CRISPR-Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI), and several subtypes. Different classes, types, and subtypes are distinguished by the repertoires of sequences in the CRISPR system, the organization and sequence of Cas genes, and the structure of repeats within CRISPR matrices. For example, Class I, grouping together types I, III and IV, use a multimeric effector module, and class II grouping together types II, V and VI, use a monomeric effector module. Non-limiting examples of Class II and type II CRISPR systems are Cas9 enzymes (e.g., SpCas9, SaCas9, and NmeCas9), and non- limiting examples of Class II and type V CRISPR systems are Cas 12 enzymes (e.g., Casl2fl, AsCasl2fl, CasMINI, and SpCasl2fl).

[0104] CRISPR-Cas systems use RNA-guided nucleases to cleave genetic elements. Each system comprises a cluster of CRISPR-associated (Cas) genes, and a distinctive array of repetitive elements (direct repeats) interspaced by short variable sequences (spacers) derived from exogenous DNA targets (protospacers), and together they constitute the CRISPR RNA array. Within the target DNA, each protospacer can be associated with a protospacer adjacent motif (PAM), which canvary depending on the specific CRISPR system. Not all CRISPR-Cas systems require a PAM to be present in the target DNA, for example in type I and type II CRISPR-Cas systems, but not in type III systems, the selection of protospacers in invading nucleic acid depends on the PAM.

[0105] The CRISPR locus is transcribed in a long primary CRISPR transcript, which is then cleaved resulting in the crRNAs, each crRNA unit contains a guide nucleotide sequence and a partial direct repeat, where the former directs the Cas enzyme (or its fragment) to a target DNA sequence via Watson-Crick base pairing. Most of the type II systems and some CRISPR systems of other types (e.g., Cas 12b, Cas 12c, and Casl2fl) additionally use a trans-encoded small RNA (transcrRNA or tracrRNA), a small RNA molecule that is required for the maturation of the pre- crRNA and for interference.

[0106] Different Cas enzymes can cleave double-stranded DNA and single-stranded DNA or RNA. Cas enzymes (or their fragments) can, for example, cleave dsDNA and generate sequencespecific nuclease-induced DNA nicking or double-strand breaks (DSBs). Upon cleavage by Cas enzymes, the target locus typically undergoes one of two major pathways for DNA damage repair: the error-prone nonhomologous end-joining (NHEJ) or the high-fidelity homology-directed repair (HDR) pathway, both of which can be used to achieve a desired editing outcome. In the absence of a repair template, DSBs are re-ligated through the NHEJ process, which leaves scars in the form of insertion / deletion (indel) mutations. NHEJ can be harnessed to mediate gene knockouts, as indels occurring within a coding exon can lead to frameshift mutations and premature stop codons. Multiple DSBs can additionally be exploited to mediate larger deletions in the genome. HDR is an alternative major DNA repair pathway. Although HDR typically occurs at lower and substantially more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. The repair template can either be in the form of conventional double-stranded DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides. The latter provides an effective and simple method for making small edits in the genome, such as the introduction of single-nucleotide mutations for probing causal genetic variation. Unlike NHEJ, HDR is generally active only in dividing cells, and its efficiency can vary widely depending on the cell type and state, as well as the genomic locus and repair template.

[0107] The RNA-guided nuclease function of CRISPR-Cas can be reconstituted in mammalian cells through the heterologous expression of (human codon-optimized) Cas enzymes and the requisite RNA components. Furthermore, the crRNA and tracrRNA can be fused togetherto create a chimeric, single-guide RNA (sgRNA). Cas enzymes can thus be re-directed toward almost any target of interest (in immediate vicinity of a PAM sequence for certain CRISPR-Cas systems) by altering the nucleotide guide sequence within the sgRNA. Thus, CRISPR-Cas can be used as a programmable nuclease-based genome editing technology.

[0108] Cas enzymes comprise at least one nuclease domain having endonuclease activity. For example, a Cas9 nuclease comprises a HNH domain, which cleaves the guide RNA complementary strand, and a RuvC domain, which cleaves the non-complementary strand; a Casl2a nuclease comprises a RuvC domain and aNUC domain; and a Casl3a nuclease comprises two HNEPN domains. When both nuclease domains are active the CRISPR nuclease has doublestranded cleavage activity (ie., cleaves both strands of a double-stranded nucleic acid sequence).

[0109] One or both of the nuclease domains in a Cas enzyme can be inactivated by one or more mutations and / or deletions, and the resulting Cas enzyme variant is a nickase that cleaves one strand of a double-stranded nucleic acid sequence or a catalytically inactive Cas enzyme. For example, one or more mutations in the RuvC domain of Cas9 protein (e.g., D10A, D8A, E762A, and / or D986A) results in an HNH nickase that nicks the guide RNA complementary strand; and one or more mutations in the HNH domain of Cas9 protein (e.g., H840A, H559A, N854A, N856A, and / or N863A) results in a RuvC nickase that nicks the guide RNA non-complementary strand. Comparable mutations can convert Casl2a and Casl3a nucleases to nickases. Wherein both the nuclease domains in a Cas enzyme are inactivated, the Cas enzyme is a catalytically inactive Cas protein (or "dead Cas," "dCas"), which does not possess nuclease activity. dCas proteins can retain their ability to bind to a specific target DNA molecule when associated to a guide RNA.

[0110] Chimeric dCas-X molecules, in which X is, in principle, any functional domain, can be used to deliver virtually any cargo (e.g., a functional domain) to a specific locus in the genome. For example, transcriptional effector domains can be fused to dCas proteins to repress / reduce or activate / increase expression of a target gene in a genome, wherein the dCas-effector domain fusion protein is targeted to the specific gene in the genome via a single guide RNA associated with the dCas protein. The term CRISPR interference or CRISPRi refers to a CRISPR system wherein a dCas protein is fused to a transcriptional repressor domain, and the term CRISPR activation or CRISPRa refers to a CRISPR system wherein a dCas protein is fused to a transcriptional activator. The dCas-sgRNA complex binds to a genomic locus via basepairing between the sgRNA (i.e., the targeting sequence of the sgRNA, the spacer portion of the sgRNA) and the DNA in the genomic locus, thereby delivering the transcriptional repressor or activator fused to the dCas to the genomiclocus. The transcriptional repressor or activator leads to downregulation or upregulation of the gene in the genomic locus, respectively.[OHl] Modified CRISPRi and CRISPRa systems have been developed. For example, in the SunTag system dCas is fused to a GCN4 peptide array that attracts scFv-linked transcriptional effector domains. In the Scaffold system, modified gRNAs carry aptamer sequences (e.g., MS2, PP7, or Com) and attract aptamer-specific proteins (MCP, PCP, Com) fused to transcriptional effector domains. The Casilio system is an upgrade of Scaffold and introduces the shorter Casilio aptamers into gRNA, improving gRNA stability and potency. SAM combines dCas proteins linked to transcriptional effector domains and Scaffold technology with modified gRNAs, thus enabling transcriptional regulation both by transcriptional effector domains linked to dCas and domains recruited by gRNA aptamers. The TREE system combines SunTag and Scaffold, enabling recruitment of multiple transcriptional effector domains.II. Catalytically Inactive Cas Enzyme

[0112] As described above, both of the nuclease domains in a Cas enzyme can be inactivated by one or more mutations and / or deletions, to obtain a catalytically inactive Cas enzyme (or "dead Cas," "dCas"), which does not possess nuclease activity, but retains its ability to bind to a specific target DNA molecule when associated to a guide RNA. For example, mutations that can be introduced in the RuvC domain of Cas9 protein are D10A, D8A, E762A, and D986A, mutations; and mutations that can be introduced in the HNH domain of Cas9 protein are H840A, H559A, N854A, N856A, and / or N863 A mutations. Comparable mutations can convert Casl2a and Casl3a nucleases to Cas 12a and Cas 13a nickases or dCasl2a and dCasl3a. In principle, any Cas enzyme can be modified to obtain a corresponding dCas protein by introduction of mutations in the nuclease domains. Non limiting examples of Cas enzymes that can be modified to obtain dCas proteins to be used in this invention are Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2, Casl2a (also known as Cpfl), Casl2e, Casl2f, Casl2fl (also known as Casl4al), Casl3a, Casl3b, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, CasX, CasY, Cas 14, natural and synthetic variants thereof, homologues thereof, and modified versions thereof (e.g., CasMINI, a protein engineered from L / « / Casl2f). In some embodiments, the dCas is derived from a Cas9, a Cas6, a Cpfl, a Casl2a, a Casl3a, a CasX, or aCasY. In some embodiments, the dCas is a dCas9, a dCas6, a dCpfl, a dCasl2a, a dCasl3a, a dCasX, or a dCasY. In some embodiments, the dCas is a dCas9.

[0113] Cas enzymes that can be modified to obtain dCas proteins can be derived from any source. For example, Cas enzymes can be derived from Streptococcus pyogenes, Staphylococcus aureus, Campylobacter jejuni, Corynebacterium diphtheria, Eubacterium ventriosum, Lactobacillus farciminis, Sphaerochaeta globus, Azospirillum (e.g., strain B510), Gluconacetobacter diazotrophicus, Neisseria cinerea, Roseburia intestinalis, Parvibaculum lavamentivorans, Nitratifractor salsuginis (e.g., strain DSM 16511), Campylobacter lari (e.g., strain CF89-12), or Streptococcus thermophilus (e.g., strain EMD-9), Streptococcus salivarius, Streptococcus pasteurianus, Streptococcus mutans, Streptococcus mitis, Streptococcus infantarius, Streptococcus intermedins, Streptococcus equ, Streptococcus agalactiae, Streptococcus anginosus, Bacillus thuringiensis, Finitimus, Streptococcus dysgalactiae, Streptococcus gallolyticus, Streptococcus macedonicus, Streptococcus gordonii, Streptococcus suis, Streptococcus iniae, Neisseria meningitides, Lactobacillus casei, Lactobacillus salivarius, Listeria innocua, Listeria monocytogenes, Lactobacillus buchneri, Lactobacillus paracasei, Lactobacillus sanfranciscensis, Lactobacillus fermentum, Listeria innocua serovar, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus sanfranciscensis, Haemophilus sputorum, Geobacillus, Enterococcus hirae, Enterococcus faecalis, Bacillus cereus, Treponema socranskii, Finegoldia magna, Acidaminococcus, Lachnospiraceae bacterium, Planctomycetes , and others. In some embodiments, the Cas enzymes is derived from Staphylococcus aureus.

[0114] For example, Streptococcus pyogenes Cas9 may be rendered catalytically dead by mutations of DIO and at least one of E762, H840, N854, N863, or D986, typically H840 and / or N863. Mutations in corresponding orthologues are known, such as N580 in Staphylococcus aureus Cas9. Thus, for example, Cas9 derived from Staphylococcus aureus (SaCas9) can be mutated to obtain a catalytically inactive SaCas9 (dSaCas9) by introducing a D10A and a N580A mutation in the SaCas9 nuclease domains; Cas9 derived from Streptococcus pyogenes (SpCas9) can be mutated to obtain a catalytically inactive SpCas9 (dSpCas9) by introducing a D10A and a H840A mutation in the SpCas9 nuclease domains; Casl2f can be mutated to obtain a catalytically inactive Casl2f (dCasl2f) by introducing a D326A and a D510A mutation in the Casl2f nuclease domains. Oftentimes, such mutations cause catalytically dead Cas proteins to possess no more than 3% of the normal nuclease activity.

[0115] In some embodiments, the dCas is a dCas9. In some embodiments, the dCas9 is derived from Staphylococcus aureus (dSaCas9).

[0116] In some embodiments, the dCas is a full length dCas. In some embodiments, the dCas is a functional fragment of a dCas. It is to be understood that the expression "functional fragment" refers to a portion of a molecule that is capable of exerting (at least in part) a certain function of the molecule. For example, in a dCas-TRD fusion protein the dCas directs the TRD to the target site via binding to a sgRNA, thus a "functional fragment" of a dCas that can be used in a dCas-TRD fusion protein is a portion of a dCas capable of directing the TRD to the target site via binding to a sgRNA.

[0117] In some embodiments, the dCas9 comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 45. In some embodiments, the dCas9 is encoded by a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 44.III. Transcriptional Repressor

[0118] As used herein, the term "transcriptional repressor" refers to any molecule that can repress, decrease, or inhibit, expression or function of a coding nucleic acid sequence (e.g., a gene). Repression, decrease, or inhibition of expression of a coding nucleic acid sequence can be achieved by any mechanism by the transcriptional repressor. For example, repression can be achieved by competition between an activator and a repressor for a common binding site in the genome; by the sequestration of an activator by a repressor into an inactive complex; by inhibitory contacts between the repressor and components of the basal transcription machinery; by inhibition (e.g., chemical modification) of the basal transcription machinery; by ablation of activator function; by chromatin remodeling (e.g., chemical modification of genomic DNA and / or of proteins associated thereto, such histones); etc. It is to be understood that the mechanisms of repression of gene expression are not yet fully elucidated and any classification of repression mechanisms is further complicated by the complexity and diversity of intermolecular interactions involved in the regulation of transcription. In addition, a same repressor can use multiple and different mechanisms to repress the expression of different genes and / or a combination of mechanisms to repress a single gene.

[0119] Transcriptional repressors that act by chromatin remodeling may chemically alter the structure of the DNA backbone or post-translationally modify histone proteins. DNA methylation regulates many biological processes in mammalian cells and can be targeted usingepigenome engineering. In mammalian cells, the methyltransferases DNMT3A and DNMT3B catalyze methylation of unmethylated CpGs, with the co-factor DNMT3L as an important stimulatory factor for DNMT3 A. dCas molecules can be fused to the catalytic domain of DNA methyltransferase (DNMT3 A), which allows site-specific methylation of the CpG islands around the dCas target site and repression of nearby genes. Posttranslational modifications of histones are important epigenomic features that are associated with the gene expression. There are at least eight types of characterized histone modifications that are involved in transcription regulation, including acetylation, methylation (lysine and arginine), phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deamination, and proline isomerization. dCas9 has can be fused to different histone- modifying enzymes. For example, histone demethylase LSD1 can be coupled to dCas to repress gene expression around the dCas target site by decreasing the epigenetic modification level of H3K4Me2 and H3K27Ac.

[0120] It is to be understood that, the transcriptional repressor domain to be used in the present invention can be a full length transcriptional repressor as well as a functional fragment thereof. As explained above, the expression "functional fragment" refers to a portion of a molecule that is capable of exerting (at least in part) a certain function of the molecule. In a dCas-TRD fusion protein, the TRD represses, decreases, or inhibits expression of a target coding nucleotide sequences (e.g., a gene), thus a "functional fragment" of a transcriptional repressor that can be used in a dCas-TRD fusion protein is a portion of a transcriptional repressor capable of repressing, decreasing, or inhibiting expression of the target coding nucleotide sequences (e.g., a gene). Any portion of a transcriptional repressor that retains the capability of repressing, decreasing, or inhibiting expression of a target coding nucleotide sequences (e.g., a gene) can be used as TRD in the present invention. For example, SUV39H1 is a histone methyltransferase that establishes constitutive heterochromatin at pericentric and telomeric regions. The SET domain of SUV39H1 participates in stable binding to heterochromatin and mediates H3K9 trimethylation, a repressive mark that recruits HP1. The SET domain contains the active site of enzymatic activity, and both pre-SET and post-SET domains contribute to methyltransferase activity. The methyl-CpG-binding protein MeCP2 plays diverse roles in chromatin regulation, but its transcriptional repressors domain is all that is required for binding repressive histone marks and co-repressor complexes to enable efficient gene silencing.

[0121] Examples of the transcriptional repressors that can be used in the present invention of from which the TRDs to be used in the present invention can be derived include, but are notlimited to, Kriippel associated box (KRAB); MAX interactor- 1 (MXI1); Sin3a Interacting Domain 4X (SID4X); Lysine-specific demethylase 1 (LSD1); DNA methyltransferase (DNMT) 3A, 3B, or 3L; Histone deacetylases 3 (HDAC3); Heterochromatin Protein 1 (HP1) family proteins, including HP la and HPIy; methyl-CpG-binding protein MeCP2 (MeCP2); histone-lysine N- methyltransferase protein SUV39H1; or any combinations thereof.

[0122] Examples of TRDs to be used in the present invention include, but are not limited to Kriippel associated box (KRAB) transcriptional repressor domain (KRAB TRD); the chromo shadow domain and C-terminal extension regions of HP la and HPIy (HP la and HPIy TRDs); the transcription repression domain of the methyl-CpG-binding protein MeCP2 (MeCP2 TRD); the SET domain of the histone-lysine N-methyltransferase protein SUV39H1 and the pre- and postSET domains of SUV39H1 in addition to the enzymatically active SET domain (SUV39H1 TRD), or any combinations thereof.

[0123] In some embodiments, the TRD is SUV39H1 pre-SET, SET, and post-SET domain, KRAB TRD, MeCP2 TRD, HPla TRD, or HPIy TRD. In some embodiments, the TRD is HPla TRD or HPIy TRD. In some embodiments, the TRD is MeCP2 TRD. In some embodiments, the TRD comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 47 or 49. In some embodiments, the TRD is encoded by a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 46 or 48.IV. Linkers

[0124] In some embodiments, in the dCas-TRD fusion protein, the dCas and the TRD are linked directly by a peptide bond. A peptide bond is a covalent bond that forms when the carbon atom from the carboxyl group of one amino acid shares electrons with the nitrogen atom from the amino group of a second amino acid. In some embodiments, the dCas and the TRD are linked by a peptide linker. Peptide linkers can be flexible linkers, rigid linker, or cleavable linkers. The linker can improve the folding or the stability of a fusion protein, increase expression, improve biological activity, enable targeting, or alter the chemical properties of a fusion protein. Non-limiting examples of peptide linkers are (G)n, (GSG)n, (GGGGS)n (SEQ ID NO: 22), (EAAK)n(SEQ ID NO: 23), or (XP)n, wherein n is any integer, e.g., any integer between 1 and 35, and X is any amino acid. For example, a linker can be (G)e-s, (G4S)I-9, (GSG)i-s, (GGGGS)I-4 (SEQ ID NO: 22), (EAAK)I-3 (SEQ ID NO: 23), or (XP)nwherein X is A, L, or E, for example (AP)?. In some aspects,n is an integer between 1 and 31. In some aspects, n is an integer that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In addition, the dCas and the TRD can be further linked by one or more noncovalent bonds, for example as a result of the tridimensional structure (e.g., folding) of the dCas-TRD fusion protein. Non-limiting examples of noncovalent bonds are ionic interactions, hydrophobic interactions, and hydrogen bonds.V. Nuclear Localization Signals (NLS)

[0125] In some embodiments, the dCas-TRD fusion proteins further comprise at least one nuclear localization signal. Nuclear localization signals (NLS) are generally short peptides that act as a signal that mediates the transport of proteins from the cytoplasm into the nucleus. This NLS- dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex, is facilitated by members of the importin superfamily.

[0126] In some embodiments, the dCas-TRD fusion protein further comprises one NLS. In some embodiments, the dCas-TRD fusion protein further comprises more than one NLSs. The more than one NLSs can be the same or different. In some embodiments, the dCas-TRD fusion protein further comprises a bipartite NLS. NLS can be localized N-terminal to the dCas, C-terminal of the dCas, N-terminal to the TRD, C-terminal to the TRD, or any combination thereof. In some embodiments, the dCas-TRD fusion protein further comprises more than one NLSs and the more than one NLSs are localized N-terminal and C-terminal to the dCas. In some embodiments, the NLS is a bipartite NLS and a first amino acid cluster of the NLS is localized N-terminal to the dCas and a second acid cluster of the NLS is localized C-terminal to the dCas.

[0127] Non-limiting examples of NLS are: a 53BP1 NLS, a CXCR4 NLS, an ERK5 NLS, an EWS NLS, an IER5 NLS, an ING4 NLS, an Hrpl NLS, an MSX1 NLS, an NLS-RARa NLS, a nucleoplasmin NLS, a Pho4 NLS, a PTHrP NLS, an rpL23a NLS, a simian virus 40 (SV40) large T antigen NLS, a UL79 NLS, a VACM-1 / CUL5 NLS, a VP1 NLS, a PABPN1 NLS, a STAT1 NLS, a FGF2 NLS, a RARa NLS, a tandem phenylalanine-glycine repeat (FG repeat) from a nuclear pore complex, a histone H2a NLS, or a histone H2b NLS.

[0128] In some embodiments, the NLS is a SV40 NLS. In some embodiments, the NLS is a bipartite SV40 NLS.

[0129] In some embodiments, the NLS comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 24-42. In someembodiments, the NLS comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 41.VI. Polyadenylation Signals

[0130] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein further comprise a polyadenylation signal. In some aspects, the polyadenylation signal can range from absent to about 500 nucleotides in length. In some aspects, the polyadenylation signal is about 10-100, about 10- 90, about 10-80, about 10-70, about 10-60, about 10-55, about 10-50, about 20-100, about 20-90, about 20-80, about 20-70, about 20-60, about 20-55, about 20-50, about 30-100, about 30-90, about 30-80, about 30-70, about 30-60, about 30-55, about 30-50, about 40-100, about 40-90, about 40- 80, about 40-70, about 40-60, about 40-55, about 40-50, about 45-100, about 45-90, about 45-80, or about 45-70 about 45-60, about 45-55, about 45-50 nucleotides in length.

[0131] Non-limiting examples of polyadenylation signals are a human growth hormone polyadenylation (poly A) (hGHpA) signal, bovine growth hormone polyadenylation (poly A) (bGHpA) signal, synthetic (SYN) polyadenylation (poly A) (SYNpA) signal, SV40 early polyadenylation signal, and SV40 polyadenylation signal sequence (SV40pA). In some embodiments, the polyadenylation signal is a SV40pA.

[0132] In some embodiments, the polyadenylation signal comprises a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 43.VII. Single Guide RNAs

[0133] A guide RNA disclosed herein (gRNA) may be a crRNA, a tracrRNA, or a crRNA / tracrRNA (or single guide RNA, sgRNA). A gRNA may be a naturally occurring or a non- naturally occurring gRNA. In some embodiments, a sgRNA disclosed herein comprises a spacer portion that hybridizes to, and / or is complementary to (partially or completely) at least a portion of a target endogenous genomic locus (e.g., a gene); and a scaffold portion that mediates binding to the Cas protein.

[0134] A sgRNA or portion thereof that hybridizes to a target endogenous genomic locus e.g., a gene) (a target site) may be any length necessary. For example, a spacer portion of a sgRNA may be any length necessary for specific hybridization. In some embodiments, the spacer portion of a sgRNA is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides long. In some embodiments, the spacer portion of a sgRNA is 19 or 21 nucleotides long. sgRNA(s) can be between about 5 andabout 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 92, 93, 94, 95, 96, 97,98, 99, or 100 nucleotides in length, or longer).

[0135] To facilitate sgRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan 21 (2014)); Heigwer et al. (Nat Methods, 11(2): 122-123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases.

[0136] In some embodiments, two or more sgRNA (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) are directed to each target endogenous genomic locus e.g., a gene).

[0137] In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) a portion of a target endogenous genomic locus comprising a DUX4 gene. In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) a portion of a DUX4 gene. In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) a promoter of &DUX4 gene. In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) an intron of a DUX4 gene. In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) an exon of & DUX4 gene. In some embodiments, the sgRNA disclosed herein hybridizes to, and / or is complementary to (partially or completely) an exon 1 of a. DUX4 gene. In some embodiments, the DUX4 gene is a human DUX4 gene. In some embodiments, the spacer portion of the sgRNA comprises a nucleotide sequence of 12-21 nucleotides in length. In some embodiments, the spacer portion of the sgRNA comprises a nucleotide sequence of 12-21 nucleotides in length, wherein the spacer portion of the sgRNA comprises a region of at least 12 contiguous nucleotides at least 70%,at least 80%, at least 90%, at least 95%, or 100% identical to any one of amino acid sequences SEQ ID NOs: 1-8. In some embodiments, the spacer portion of the sgRNA comprises a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of nucleotide sequences SEQ ID NOs: 1-8. In some embodiments, the scaffold portion of the sgRNA comprises a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of amino acid sequences SEQ ID NOs: 50.VIII. Promoters

[0138] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein further comprise one or more promoters.

[0139] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein further comprise a Polymerase II promoter. The promoter can be any Polymerase II promoter capable of driving the transcription of a DNA coding sequence into mRNA. In some aspects, the Polymerase II promoter is a constitutive promoter, a regulatable promoter, an ubiquitous promoter, or a tissue-specific promoter. In some aspects, the Polymerase II promoter is a constitutive promoter. Non-limiting examples of constitutive Polymerase II promoters are a CBA promoter, a CMV promoter, an EFla promoter, a CAG promoter, an UbC promoter, and CBh promoter. In some aspects, the Polymerase II promoter is a muscle tissue-specific Polymerase II promoter. Non-limiting examples of muscle tissue-specific Polymerase II promoters are a MHCK7 promoter, a CK8 promoter, a CK8e promoter, a CKM promoter, a dMCK promoter, a tMCK promoter, a DES promoter, a HSA promoter, a SPc5-12 promoter, a SP-301 promoter, a MHC promoter, a Sk-CRM promoter, a Sk- CRM4 promoter, or a functional fragment thereof. In some aspects, the muscle tissue-specific promoter is a CKM promoter or a functional fragment thereof.

[0140] In some aspects, the expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein further comprises one or more enhancers, or a functional fragments thereof, such as a MCK enhancer or one or more or a functional fragments thereof.

[0141] In some aspects, the Polymerase II promoter, or a functional fragment thereof, and the one or more enhancers, or functional fragments thereof, are comprised in a regulatory sequence. For example, in some embodiments, the regulatory sequence is a No Heart (NH) regulatory sequence comprising: three copies of a modified Ckm (mouse or human) enhancer in tandem,upstream of an 80-bp basal Ckm promoter (mouse or human). The NH regulatory sequence comprises Trex sequence, an AT-rich sequence, two right E-box sequences, and aMEF2 sequence. With respect to certain Ckm sequences (tMCK), the main features of the NH regulatory sequence are: 1) Left E-box mutated to Right E-box; 2) enhancer CArG and AP2 sites removed; 3) 63 bp between Right E-box and MEF2 site removed; 4) sequence between transcription factor binding motifs minimized; 5) -80 to +50 promoter sequence used; and 6) consensus Initiator element (Inr) added. In some embodiments, the regulatory sequence is a Have a Little Heart (HLH) regulatory sequence. The HLH regulatory sequence is based on the NH regulatory sequence, with the following modification: the additional Right E-box is replaced with the original Left E-box from the Ckm enhancer.

[0142] In some aspects, the regulatory sequence comprises a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 9 or 10.

[0143] In some embodiments, the expression cassettes (e.g., single-stranded, doublestranded, or self-complementary expression cassettes) disclosed herein further comprise a Polymerase III promoter or a functional fragment thereof. The promoter can be any Polymerase III promoter or a functional fragment thereof capable of driving the transcription of a DNA coding sequence into a non-coding RNA. Non-limiting examples of Polymerase III promoters are 7SK, U6, Mi l (e.g., Mi l hybrid promoter), Hl, and functional fragments thereof. In some embodiments, the Polymerase III promoter is a U6 promoter. In some embodiments, the Polymerase III promoter is a Ml 1 hybrid promoter. In some embodiments, the Polymerase III promoter comprises a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 51.

[0144] In some aspects, the Polymerase II promoter or functional fragment thereof drives the expression of the dCas-TRD fusion protein. In some aspects, the Polymerase III promoter or functional fragment thereof drives the expression of the sgRNA. In some aspects, the Polymerase II promoter drives the expression of the dCas-TRD fusion protein and the Polymerase III promoter drives the expression of the sgRNA. In some aspects, the NH regulatory sequence drives the expression of the dCas-TRD fusion protein. In some aspects, the HLH regulatory sequence drives the expression of the dCas-TRD fusion protein. In some aspects, the U6 promoter drives the expression of the sgRNA. In some aspects, the Ml 1 hybrid promoter drives the expression of the sgRNA. In some aspects, the NH regulatory sequence drives the expression of the dCas-TRD fusion protein and the U6 promoter drives the expression of the sgRNA. In some aspects, the NHregulatory sequence drives the expression of the dCas-TRD fusion protein and the Mi l hybrid promoter drives the expression of the sgRNA. In some aspects, the HLH regulatory sequence drives the expression of the dCas-TRD fusion protein and the U6 promoter drives the expression of the sgRNA. In some aspects, the HLH regulatory sequence drives the expression of the dCas-TRD fusion protein and the Ml 1 hybrid promoter drives the expression of the sgRNA.IX. Inverted Terminal Repeats (ITRs)

[0145] In some embodiments, expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein further comprise a pair of inverted terminal repeats (ITRs). ITRs comprise sequence regions, which can be complementary and symmetrically arranged. ITRs comprises, in some embodiment, of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences. In some aspects, the ITRs are of the same serotype as one another. In some aspects, the ITRs are of different serotypes. Independently, each ITR can be about 75 to about 175 nucleotides in length. An ITR can be about 100-105 nucleotides in length, about 106-110 nucleotides in length, about 111-115 nucleotides in length, about 116-120 nucleotides in length, about 121-125 nucleotides in length, about 126-130 nucleotides in length, about 131-135 nucleotides in length, about 136-140 nucleotides in length, about 141-145 nucleotides in length or about 146-150 nucleotides in length. In some aspects, the ITR serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVrh47, AAV11, AAV12 serotype, or a variant thereof. In some aspects, the ITR serotype is AAV2.

[0146] In some embodiments, the serotype of the ITRs is AAV2.

[0147] The expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) disclosed herein can be comprised in an AAV vector. The expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein can be comprised in an AAV genome, and the AAV genome can be encapsidated in an AAV capsid. The ITRs comprised in the expression cassettes (e.g., singlestranded, double-stranded, or self-complementary expression cassettes) disclosed herein can be derived from the same serotype as the capsid, selected from any of the serotypes listed herein, or a derivative thereof. The ITR can be of a different serotype from the capsid. Zero, one or both of the ITRs can have the same serotype as the capsid.X. AAV vectors

[0148] The expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) disclosed herein can be comprised in an AAV genome, and the AAV genome can be encapsidated in an AAV capsid. As such, also provided herein are AAV vectors comprising the expression cassettes (e.g., single-stranded, double-stranded, or self- complementary expression cassettes) disclosed herein as well the AAV capsid.

[0149] AAVs vectors comprise an AAV genome containing approximately 4700 or less nucleotides. In general, an AAV genome comprises two open reading frames (ORFs) flanked by a pair of inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length and have multiple functions, such as serving as origins of replication, and as packaging signals for the viral genome. The ORFs: replication (rep) and capsid (cap), encode proteins that allow for the replication, assembly, and packaging of a complete AAV virion. At least four viral proteins are expressed from the rep ORF: Rep 78, Rep 68, Rep 52, and Rep 40; and the cap ORF encodes at least three proteins: VP1, VP2, and VP3 that assemble to form the AAV capsid. AAV is a helperdependent virus, requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion.

[0150] Different AAV serotypes have been described, non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, AAV-PHP.B, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMY03 (AAVS10P1), AAV9-RGD (e g., MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E), AAV-SLB101, and LICA1. The AAV vectors of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype.

[0151] It is to be understood that when an expression cassette (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein is to be comprised in an AAV vector, the expression cassette (e.g, single-stranded, double-stranded, or self-complementary expression cassettes) is of a length suitable to be comprised in an AAV vector. In general, an AAV vector contains a genome of approximately 4700 or less nucleotides, therefore, an expression cassette (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) is of a length of no more than about 4700 nucleotides where to be comprised in an AAV vector. Different combinations of the elements comprised in the expression cassettes (e.g., singlestranded, double-stranded, or self-complementary expression cassettes) disclosed herein (e.g., regulatory sequences, dCas, TRD) can be selected to achieve a length of the expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) suitable to be comprised in an AAV vector. Non-limiting examples of such combinations of elements are disclosed elsewhere herein.Pharmaceutical Compositions

[0152] In some aspects, the expression cassettes (e.g., single-stranded, double-stranded, or self-complementary expression cassettes) disclosed herein, or any combination thereof, are complexed, or packaged in a liposome, a nanoliposome, a lipid nanoparticle (LNP), a lipoplex, a micell, a nanomicell, a nanoemulsion, an oil-in-water emulsions, a PEG-conjugated lipid nanoparticle, a polymeric nanoparticle, a lipid-polymer hybrid nanoparticle, a polysaccharidic nanocarrier, an RNA / DNA-peptide nanoparticle, an RNA / DNA-peptide nanocomplex, a biomimetic nanovesicle, a lipidoid-RNA / DNA complex, a virus-like particle, dendrimer nanoparticle, a nanogel, a metallic nanoparticle, a gold nanoparticle (AuPNs), a magnetic nanoparticle, a theranostic nanoparticle, or any combination thereof. As such, also provided herein are LPN comprising the single- or double-stranded expression cassettes disclosed herein, or any combination thereof.

[0153] In some aspects, disclosed herein are pharmaceutical compositions comprising the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, and a pharmaceutically acceptable carrier. In some aspects, the combination of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes ), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, with one or more pharmaceutically acceptable carrier facilitates, enhances or enables administration of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes ), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, to a subject. In some aspects, the combination of the polynucleotides (e.g., single- stranded, double-stranded, or self-complementary expression cassettes ), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, with one or more pharmaceutically acceptable carrier facilitates, enhances or enables the delivery of the polynucleotides (e.g., single-stranded, doublestranded, or self-complementary expression cassettes ), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, to a target cell. In some aspects, after administration to the subject of the pharmaceutical compositions disclosed herein, at least a portion of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, is delivered to a target cell.

[0154] Provided herein are also pharmaceutical compositions comprising the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the cells, disclosed herein, or any combination thereof, and a pharmaceutically acceptable excipients or adjuvants, or any combination thereof.

[0155] Examples of pharmaceutically acceptable excipients are, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, colorants, amino acids, stabilizers, bulking agents, surfactants, antimicrobials, preservatives, metal ions, chelators, cyclodextrin-based excipients, polyanions, polycations, salts, solubilizers, detergents, compatible solid or liquid fillers, encapsulating substances, or any combination thereof, which are suitable for administration to a subject. Specific examples of excipient include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers, or any combination thereof.

[0156] Examples of pharmaceutically acceptable carriers are, but are not limited to, lipids, polymers, polysaccharides, peptides, proteins, lipidoids, and any combination thereof. In some aspects, the lipid is selected from the group consisting of: cationic lipids, non-cationic lipids, steroid lipids, ionizable lipids, PEG-conjugated lipids, and any combination thereof.

[0157] Pharmaceutically acceptable carriesrs, adjuvants, and excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical carriesrs, adjuvants, and excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice.Cells

[0158] Provided herein are also cells comprising the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. The cells can be maintained in culture (in vitro), or comprised in an individual (in vivo), such as a human. The cells can be a muscle cells. In some aspects, the muscle cells are derived from skeletal muscle or smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue. In some aspects, the muscle cells are a myoblast, a myocyte, a myotube, a cardiomyocyte, or a cardiomyoblast. In some aspects, the cells comprise a genome comprising a DUX4 open reading frame embedded within a 3.3 kb large and CpG-rich D4Z4 unit on chromosome 4. In some aspects, the cells comprises a 4qA or a 4qB chromosome 4. In some aspects, DUX4 is expressed in the cells. In some aspects, DUX4 is overexpressed in the cells. In one embodiment, the cell comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell. In another embodiment, the mammalian cell comprises HEK293 or HeLa cell.

[0159] Non-limiting examples of cells for AAV production include Sf9 insect cells and HEK 293T cells. In one embodiment, the cell described herein comprises an insect cell, e.g., a Drosophila cell e.g., an S2 cell or Kc cell), a silkworm cell e.g., a Bme21 cell), or a mosquito cell e.g., a C6 / 36 cell); or a mammalian cell (preferably a human cell, e.g., a human primary cell or an established cell line). In one embodiment, the mammalian cell comprises a 293 cell, a COS cell, a HeLa cells, or a KB cell.Methods of Treatment and Use

[0160] Provided herein are also methods of inducing transcriptional repression of a. DUX4 gene in a cell or in a population of cells comprising contacting the cell or the population of cells with the polynucleotides e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof.

[0161] In some aspect, the transcription level of the DUX4 gene in the cell or in the population of cells is reduced compared to the transcription level of the DUX4 gene in the same cell or population of cells before contacting the cell or population of cells with the polynucleotides e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combinationthereof. In some aspect, the transcription level of the DUX4 gene in the cell or in the population of cells is reduced of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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 %in the cell upon contacting the cell or the population of cells with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof.

[0162] In some aspect, the transcription level of the DUX4 gene in the cell or in the population of cells is reduced compared to the transcription level of the DUX4 gene in a same cell or population of cells not contacted with the polynucleotides (e.g., single-stranded, doublestranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. In some aspect, the transcription level of the DUX4 gene in the cell or in the population of cells is reduced of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least 61 %,at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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 % in a cell or population of cells contacted with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof, compared to a same cell or population of cells not contacted with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof.

[0163] In some aspects, provided herein are also methods of inducing transcriptional repression of at least one DUX4 target gene in a cell or in a population of cells comprising contacting the cell or the population of cells with the polynucleotides (e.g., single-stranded, doublestranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. DUX4 target genes include, but are not limited to ZSCAN4, TR1M43 RFPL , RFPL2, RFPL4B, PRAMEF1, PRAMEF2, PRAMEF12, TRIM43, MDB3L2; and Wfdc3, Agtr2, Myolg, Azi2, Cdkiila, and Zscan4c.

[0164] In some aspect, the transcription level of the at least one DUX4 target gene in the cell or in the population of cells is reduced compared to the transcription level of the at least one DUX4 target gene in the same cell or population of cells before contacting the cell or population of cells with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. In some aspect, the transcription level of at least one DUX4 target gene in the cell or in the population of cells is reduced of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %,at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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 % in the cell or in the population of cells upon contacting the cell or the population of cells with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. In some aspect, the transcription level of the at least one DUX4 target gene in the cell or in the population of cells is reduced compared to the transcription level of the at least one DUX4 target gene in a same cell or population of cells not contacted with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. In some aspect, the transcription level of the at least one DUX4 target gene in the cell or in the population of cells is reduced of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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 % in a cell or population of cells contacted with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof, compared to a same cell or population of cells not contacted with the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, or the pharmaceutical compositions disclosed herein, or any combination thereof. In some aspects, the cell or the population of cells is a cell or a population of cells maintained in vitro, such as a population of cells maintained in culture. In some aspects the cell or the population of cells is a cell or a population of cells comprised in a testing sample derived from an organism. In some aspects, the cell is a muscle cell. In some aspects, the population of cells is a heterogeneous population of cells comprising one or more muscle cells. In some aspects, the population of cells is a homogeneous population of muscle cells.

[0165] In some aspects, the transcription level of the DUX4 gene and / or of the at least one DUX4 target gene is measured by qRT-PCR.

[0166] Provided herein are also method of treating FSHD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof.

[0167] In some aspect, the FSHD is FSHD1. In some aspect, the FSHD is FSHD2. In some aspect, the subject is a human. In some aspect, the cells of the subject comprise a genome comprising & DUX4 open reading frame embedded within a 3.3 kb large and CpG-rich D4Z4 unit on chromosome 4. In some aspects, the cells of the subject comprises a 4qA or a 4qB chromosome 4. In some aspects, DUX4 is expressed in the cells of the subject before administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof. In some aspects, DUX4 is expressed in the muscle cells of the subject before administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self- complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof. In some aspects, DUX4 is overexpressed in the cells of the subject before administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof. In some aspects, DUX4 is overexpressed in the muscle cells of the subject before administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof.

[0168] In some aspects, administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof, results in repression of DUX4 transcription (expression) in the cells of the subject, thereby ameliorating, reducing or reversing the severity of the FSHD condition in the subject. In some aspects, administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof, results in repression of DUX4 transcription (expression) in the muscle cells of the subject, thereby ameliorating, reducing or reversing the severity of the FSHD condition in the subject.

[0169] In some aspects, the subject is a male subject and administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof, does not results in repression of DUX4 transcription (expression) in the testis cells of the subject

[0170] In some aspects, administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof, results in a reduction of DUX4 transcription (expression) in the cells of the subject of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17%, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24%, at least 25 %, at least 26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31%, at least 32 %, at least 33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38%, at least 39 %, at least 40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45%, at least 46 %, at least 47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52%, at least 53 %, at least 54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59%, at least 60 %, at least 61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66%, at least 67 %, at least 68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73%, at least 74 %, at least 75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, 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 %. In some aspects, administering to the subject a therapeutically effective amount of the polynucleotides (e.g., singlestranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof, results in a reduction of DUX4 transcription (expression) in the muscle cells of the subject of at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 %, at least 31 %, at least 32 %, at least33 %, at least 34 %, at least 35 %, at least 36 %, at least 37 %, at least 38 %, at least 39 %, at least40 %, at least 41 %, at least 42 %, at least 43 %, at least 44 %, at least 45 %, at least 46 %, at least47 %, at least 48 %, at least 49 %, at least 50 %, at least 51 %, at least 52 %, at least 53 %, at least54 %, at least 55 %, at least 56 %, at least 57 %, at least 58 %, at least 59 %, at least 60 %, at least61 %, at least 62 %, at least 63 %, at least 64 %, at least 65 %, at least 66 %, at least 67 %, at least68 %, at least 69 %, at least 70 %, at least 71 %, at least 72 %, at least 73 %, at least 74 %, at least75 %, at least 76 %, at least 77 %, at least 78 %, at least 79 %, at least 80 %, at least 81 %, at least82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least96 %, at least 97 %, at least 98 %, or at least 99 %.

[0171] In some aspects, cells of the subject are comprised in a testing sample derived from the subject after administering to the subject a therapeutically effective amount of the polynucleotides (e.g., single-stranded, double-stranded, or self-complementary expression cassettes), the AAV vectors, the LNPs, the pharmaceutical compositions, or the cells disclosed herein, or any combination thereof.Table 1 SequencesWORKING EXAMPLESExample 1 : Generation and in vitro testing of modified CRISPRi single-vector constructsResults

[0172] A dSaCas9-TRD single-vector construct (therapeutic cassette TRD+HA[DUX4]) comprising: an HLH regulatory cassette, a first portion of a bipartite SV40 NLS, a nucleotide sequence encoding a dSaCas9, a second portion of a bipartite SV40 NLS, a nucleotide sequenceencoding a TRD transcriptional repressor domain, a nucleotide sequence encoding an hemagglutinin (HA) tag, an SV40 polyadenylation signal, a U6 promoter, and a nucleotide sequence encoding a single guide RNA, was modified as follows:1) removal of the hemagglutinin (HA) epitope tag to avoid issues of potential immunogenicity to generate therapeutic cassette TRD[DUX4];2) changing the orientation of the sgRNA cassette (U6 promoter + nucleotide sequence encoding a single guide RNA) to an antisense configuration to generate therapeutic cassette TRD+HA[AS DUX4]) (FIG. 1);3) both 1) and 2) to generate therapeutic cassette TRD(AS DUX4).

[0173] Therapeutic cassettes shown in FIG. 1 were synthesized and fully sequenced by GenScript before cloning into a lentiviral (LV) vector for testing in vitro, or into an adeno- associated viral (AAV) vector for testing in vivo.

[0174] Immortalized FSHD1 muscle cells (MB073) were transduced via 3 LV infections with centrifugation, switched to differentiation conditions after the last infection, and harvested after 2 days of differentiation. Efficacy of CRISPRi repression was assessed by comparing levels of DUX4-fl and the direct DUX4-FL target MBD3L2 in treated cells (FIG. 2A-2B). Removal of the HA tag did not affect the efficacy of the construct; TRD+HA(DUX4) and TRD(DUX4) were equally effective at repressing DUX4- 1 and MBD3L2 (FIG. 2A-2B). Likewise, TRD(AS DUX4) showed similarly effective repression compared to TRD(DUX4) (FIG. 2A-2B).

[0175] The ability of the therapeutic cassette to repress DUX4-fl and its downstream target genes was validated by qRT-PCR in four patient myoblast lines (two FSHD1 immortalized, one FSHD1 primary, and one FSHD2 immortalized). All cells were transduced as above with LV vectors expressing TRD(AS DUX4) or the non-targeting control TRD(AS NT). As levels of DUX4-FL protein are low and difficult to assess in FSHD myocytes, DUX4-FL target gene expression is routinely assessed as a relevant functional readout of DUX4 activity. Thus, relative mRNA levels of DUX4-fl, the DUX4-FL targets MBD3L2 and TRIM43, and the dSaCas9 cargo were assessed by qRT-PCR (FIG. 3A-3Q). In all FSHD lines, DUX4- / 1 and DUX4-FL target genes were consistently repressed to similar levels (-50%) by the CRISPRi treatment (FIG. 3A-3C, 3E- 3G, 3I-3K, 3M-3P). Interestingly, the FSHD2 immortalized line contains two FSHD-permissive alleles (4A and 4AL); thus, DUX4-fl expression was assessed using assays specific for each allele. Strikingly, DUX4-fl was repressed at both alleles and levels of both target genes were reduced (FIG. 3M-N). In all lines, dSaCas9 levels were equivalent in cells treated with TRD(AS DUX4)vs. TRD(AS NT) (FIG. 3D, 3H, 3L, 3Q), and the morphology, fusion, and apparent health of the myotubes was unaffected.

[0176] Global transcriptomic effects of the CRISPRi treatment were assessed in four FSHD lines and two healthy lines. Cells were transduced as above with LV vectors expressing TRD(AS DUX4) or the non-targeting control TRD(AS NT). RNA-seq analysis was performed with 5 biological replicates of TRD(AS DUX4) vs. TRD(AS NT) in each line by GENEWIZ (Azenta Life Sciences) using their Illumina HiSeq 2 x 100 bp platform. Raw data was analyzed in-house for differentially expressed genes (DEGs) that represent amelioration of DUX4-mediated dysregulation or off-target effects of the CRISPRi treatment. Gene sets were mined for direct / indirect DUX4 targets in the literature (Jagannathan 2016, Hum Mol Genet 25, 4419-4431; Geng 2012, Dev Cell 22, 38-51). In all FSHD lines, the CRISPRi treatment resulted in altered expression of many genes that are deregulated by DUX4 misexpression in FSHD (FIG. 4A-4O). These include factors involved in early development, germ cell function, immune modulation, and stress response (reviewed in Himeda 2019, Annu Rev Genomics Hum Genet 20, 265; Campbell 2018, Hum Mol Genet 27(R2), R153). While DUX4 is thought to be a transcriptional activator, its signature includes both upregulated and downregulated genes; the latter are likely to be indirect targets. Many of the DEGs were common to multiple lines (FIG. 41), and their differential expression represents a return to a more healthy pattern of gene expression. For example, DUX4 increases expression levels of MBD3L2, DUXA, LEUTX, KHDC1L, and LCNL1 in multiple independent studies (compared in Jagannathan 2016, Hum Mol Genet 25, 4419-4431); consistent with this, our CRISPRi treatment led to a decrease in expression of these genes. Conversely, levels of MYODI, LY6E, RARRES3, and SERPINGJ, which are downregulated by DUX4 expression, were increased by the CRISPRi treatment. In the primary FSHD1 line (RL-01), some targets (e.g., transcription factors such as MBD3L2 and TRIM43') were expressed at levels beneath detection; however, qRT-PCR analysis (FIG. 3J-3K) revealed that these genes were also consistently repressed by the CRISPRi treatment.

[0177] Of the very few non-DUX4 targets that showed altered expression (FIG. 4P), none are known to play critical roles in skeletal myocytes. Importantly, levels of myogenic genes other than the DUX4 target MYODI were not significantly altered in any of the cell lines tested. Both healthy lines showed very few DEGs in response to the CRISPRi treatment (FIG. 4N-4O); these include unannotated genes, pseudogenes, ncRNAs of unknown function, and, interestingly, severalDUX4 targets. The latter is perhaps not surprising, since DUX4 can be expressed at very low levels in healthy myocytes (Jones 2012, Hum Mol Genet 21, 4419-4430).

[0178] Overall, the transcriptomic analysis indicates that this single-vector system for CRISPRi is both effective at reversing the DUX4 molecular signature and highly specific in human myocytes.

[0179] Repressive chromatin changes at the target locus were assessed in the MB073 FSHD1 line and the MB2453 FSHD2 line. Cells were transduced as above with LV vectors expressing TRD(AS DUX4) or the non-targeting control TRD(AS NT). Chromatin immunoprecipitation (ChIP) assays were used to assess dSaCas9 binding and several marks of repressive chromatin (KAP1, H3K27me3, and H3K9me3) at DUX4 (FIG. 5A-5B). In FSHD1, such changes are extremely difficult to detect, considering that the disease locus is a single epigenetically de-repressed repeat among hundreds of identical repeats that are actively repressed. Likewise, Cas9 has more difficulty accessing targets in heterochromatin (Kallimasioti-Pazi 2018, PLoS Biol 16, e2005595); thus, enrichment of dSaCas9-TRD at the pathogenic repeat is dampened by the presence of hundreds of relatively inaccessible repeats of identical sequence. Despite these caveats, dSaCas9 and KAP1 displayed ~2-fold enrichment at DUX4 in TRD(AS DUX4) treated FSHD1 myocytes, overcoming the high background (FIG. 5A). Even H3K9me3 and H3K27me3 were detectably elevated \.DUX4, although enrichment of the latter was not significant (FIG. 5A). In FSHD2, both 4q and lOq D4Z4 alleles are epigenetically de-repressed; thus, an increase in repression at the pathogenic allele should, in theory, be easier to detect; however, although dSaCas9 and KAP1 were enriched at the locus, there were no detectable changes in H3K27me3 or H3K9me3 (FIG. 5B).Example 2: In vivo testing of modified CRISPRi single-vector constructsResults

[0180] To test the CRISPRi modifications in vivo, a human xenograft mouse model containing mature FSHD myofibers was utilized (Mueller 2019, Exp Neurol 320, 113011; Sakellariou 2016, Skelet Muscle 6, 4). As these mice contain a pathogenic D4Z4 array from an FSHD patient which maintains the endogenous nuclear context of the human FSHD locus, they are the best available in vivo model for assessing the effects of epigenetic repressors targeted to the disease locus. Immortalized FSHD1 myoblasts were injected into the TA muscle compartment of immunodeficient NRG mice. At 4 weeks post-engraftment, CRISPRi constructs were injectedsystemically in MyoAAV2A vectors at 5E13 vg / kg. Four weeks post-injection, xenografts were assessed for expression of DUX4- / 1 an . MBD3L2 by qRT-PCR.

[0181] A confounding factor in determining the efficacy of any DUX4-targeted treatment is the high variability in DUX4-fl levels across samples and over time. This has been well- documented in primary FSHD myocytes (Jones 2012, Hum Mol Genet 21, 4419-4430; Jones 2015, Clinical Epigenetics 7, 37) and in xenograft studies using cells and tissue from multiple sources (immortalized FSHD1 and FSHD2 myoblasts, and muscle from biopsy and autopsy) (Mueller 2019, Exp Neurol 320, 113011; Chen 2016, Mol Then 10.1038; Oliva 2019, J Pharmacol Exp Ther 370, 219-230). Thus, it was expected that there would be extreme variability in the expression of DUX4-fl in control-treated xenografts. In TRD(DUX4)-treated xenografts, the measure of successful inhibition would be consistent clustering of DUX4-fl levels at the low end of this range. Indeed, despite the expected high variability in levels of DUX4-fl and MBD3L2 in xenografts treated with TRD+HA(NT), treatment with TRD+HA(AS DUX4) yielded fairly consistent repression of both genes, and better repression than TRD+HA(DUX4) (FIG. 6).

[0182] This result was further validated in vivo using ACTAl-MCM;FLExD FSHD-like bi-transgenic mice, which can be induced to express DUX4-fl and develop a moderate pathology in response to a low dose of tamoxifen (TMX) (Jones 2018, PLoS ONE 13, e0192657; Jones 2020, Skelet. Muscle 10,8). These mice carry one human D4Z4 repeat from which DUX4- 1 is expressed and can be targeted by the sgRNA to exon 1. Mice were injected systemically with CRISPRi constructs in MyoAAV2A at 1.5E14 vg / kg. At 3-4 weeks post-AAV injection, DUX4-fl expression was induced by 5mg / kg TMX IP injection. At 10 days post TMX injection, expression levels of DUX4-fl and the DUX4-FL mouse target Wfdc3 from TA muscles were assessed by qRT-PCR. As in the xenografted mice, treatment with TRD+HA(AS DUX4) yielded fairly consistent repression of both genes, and better repression than TRD+HA(DUX4) (FIG. 7).

[0183] Physical performance of these mice was assessed by a treadmill stress test at 9 days post- TMX injection. While wild-type mice run for the full 20-min duration of the test, control- treated ACTAl-MCM;FLExD mice reached fatigue after only 2 min. Strikingly, ACTA1- MCM;FLExD mice treated with TRD+HA(DUX4) ran for 10 min, and those treated with TRD+HA(AS DUX4) had greatly improved performance, with time to fatigue reaching nearly wild-type levels (FIG. 8).

[0184] To validate this result using another myotropic vector, ACTAl-MCM;FLExD mice were injected systemically with TRD(AS DUX4) in the LICA1 rAAV vector (LICA1.TRD[ASDUX4]) at 1E13 or 5E13 vg / kg (FIGs. 9A-9G). At 4 weeks post- AAV injection, DUX4- 1 expression was induced by 5mg / kg TMX IP injection. In a dose-dependent fashion, the CRISPRi treatment prevented the consistent decline in body weight normally seen in this moderate pathology model (FIGs. 9A-9C) and greatly improved treadmill performance (FIG. 9D). At 10 days post TMX injection, EDL muscles were harvested for assessment of viral genomes and gene expression by qRT-PCR. Both vector copy number (FIG. 9E) and levels of dSaCas9 (FIG. 9F) showed a dose-dependent increase. Strikingly, DUX4-fl and two mouse-specific DUX4 target genes (Wfdc3 and Slcl5a2) were consistently reduced by both doses (FIG. 9G).

[0185] Taken together, the molecular and functional data indicated that placing the sgRNA cassette in the antisense orientation is beneficial for this single-vector CRISPRi system in the therapeutically relevant context of an AAV vector delivered to skeletal muscle.Example 3 : In vitro testing of other CRISPRi single-vector constructsResults

[0186] Other variations in CRISPRi platform repress DVX4- / 1 in vitro. (FIGs. 10A- 10D) The single-vector CRISPRi system was tested in the context of a different repressor fused to dSaCas9 and a different promoter driving the sgRNA. Replacement of the MeCP2 TRD with the chromo shadow domain and C-terminal extension of HP la resulted in equivalent repression of DUX4-fl and MBD3L2 in FSHD1 myocytes (FIG. 10A-10B). Likewise, replacement of the sgRNA-driving U6 promoter with the Ml 1 promoter, a 100-bp H1 / 7SK hybrid mini promoter that confers greater Pol III activity and enhanced specificity for Pol III vs. Pol II (Gao 2021, J Biol Chem 296, 100026), yielded equivalent repression (FIG. 10C-10D).Materials and Methods

[0187] Antibodies and plasmids. The ChlP-grade antibodies used in this study, a-KAP I (ab3831), oc-H3K27me3 (ab6002), and oc-H3K9me3 (ab8898), were purchased from Abeam, and a-SaCas9 (11E7-9) was purchased from MBL Life Science. Single-vector CRISPRi constructs were designed with the following modifications: 1) removal of the HA tag and 2) orientation of the sgRNA expression cassette in the antisense ( s. sense) configuration. Constructs contain the Ckm-based HLH regulatory cassette driving dSaCas9 flanked by the SV40 bipartite nuclear localization signal and fused in-frame to the MeCP2 transcriptional repressor domain (TRD) + / - the HA tag, followed by the SV40 late pA signal. This was followed immediately by a sgRNAexpression cassette (the U6 promoter driving a sgRNA targeted to the DUX4 promoter / exon 1 [or a non-targeting control sgRNA], the SaCas9-optimized scaffold, and cPPT / CTS) in either the sense or antisense configuration. Therapeutic cassettes were synthesized in pUC57 and sequenced by GenScript Biotech, then cloned into a pRRLSIN LV vector for transduction of FSHD myocytes. For testing in vivo, therapeutic cassettes were cloned between the AAV2 ITRs (using Mlul and RsrII) of the pAAV-CA plasmid, a gift from Naoshige Uchida (Addgene plasmid # 69616; http: / / n2t.nct / addgene:69616; RRID:Addgene_69616). pRRLSIN.cPPT.PGK-GFP.WPRE was a gift from Didier Trono (Addgene plasmid # 12252; http: / / n2t.nct / addgene: 12252; RRID:Addgene_12252). MyoAAV2A vectors were produced by Vector Biolabs and LICA1 vectors were produced by Genethon.

[0188] Cell culture, transient transfection, and LV transduction. Immortalized FSHD1 (MB073), FSHD2 (MB2453), and healthy (MB 135) myoblast lines were obtained from the Fred Hutchinson Cancer Center. The immortalized MB2306 FSHD1 myoblast line was obtained from the Leiden University Medical Center. Primary FSHD1 (RL-01) and healthy (RL-06) myoblasts were obtained from the Jones lab at the University of Nevada, Reno School of Medicine. All cells were grown as described (Campbell 2018, eLife 7, e31023). For xenograft transplantation, myoblasts were expanded in culture prior to injection (Mueller 2019, Exp Neurol 320, 113011; Sakellariou 2016, Skelet Muscle 6, 4). LV particles were generated using 293 T packaging cells, as described (Himeda 2016, Mol Ther 24, 527-535). At -70% confluency, myoblasts were subjected to three serial infections, as described (Himeda 2016, Mol Ther 24, 527-535), and switched to low- serum differentiation medium, then harvested 2 days following the last round of infection.

[0189] Quantitative reverse transcriptase PCR (qRT-PCR). Total RNAs were extracted using TRIzol (Invitrogen) and purified using the RNeasy Mini kit (Qiagen) after on-column DNase I digestion. From cultured myocytes, total RNA (2 pg) was used for cDNA synthesis using oligo dT and Superscript III Reverse Transcriptase (Invitrogen); DUX4-fl was amplified by qPCR using 200 ng of cDNA, and other genes were amplified using 10-20 ng of cDNA, as described (Himeda 2020, Mol Ther Methods Clin Dev. 20:298-311). Oligonucleotide primer sequences are as reported (Himeda 2020, Mol Ther Methods Clin Dev. 20:298-311).

[0190] RNA-seq analysis. Myoblasts from four FSHD lines and two healthy lines were transduced with TRD(AS DUX4) or the non-targeting control TRD(AS NT). RNA-seq was performed with 5 biological replicates of TRD(AS DUX4) vs. TRD(AS NT) in each line by GENEWIZ (Azenta Life Sciences) using their Illumina HiSeq 2 x 100 bp platform, as described(Himeda 2020, Mol Ther Methods Clin Dev. 20:298-311). Heat maps and volcano plots of differentially expressed genes (DEGs) were generated using Prism 7 (Graphpad).

[0191] Chromatin immunoprecipitation (ChIP). ChIP assays were performed with LV- transduced differentiated myotubes using the Fast ChIP method (Nelson 2006, Nat Protoc 1, 179- 185) as described (Himeda 2016, Mol Ther 24, 527-535). Chromatin was immunoprecipitated using 2 pg of specific antibodies. SYBR green quantitative PCR assays were performed as described (Himeda 2016, Mol Ther 24, 527-535).

[0192] AAV transduction in FSHD1 mouse models. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Nevada, Reno. All mice were anesthetized prior to inj ection. Human xenografted mice were created essentially as described (Mueller 2019, Exp Neurol 320, 113011; Sakellariou 2016, Skelet Muscle 6, 4). Briefly, the hindlimbs of 8-week-old immunodeficient NRG mice were X-irradiated to prevent regeneration of murine muscle. Five days later, the irradiated Tibialis anterior (TA) muscles were injected with BaC12 to eliminate the majority of mouse muscle tissue. The following day, 2E6 immortalized FSHD1 myoblasts were injected into the TA compartments. Over the next 4 weeks, intermittent neuromuscular electrical stimulation (iNMES) of the peroneal nerve was performed to induce repeated maximal contractions of the engrafted TA muscles. At 4 weeks post-engraftment, CRISPRi constructs were injected systemically, via retro-orbital injection, in MyoAAV2A vectors at 5E13 vg / kg. Four weeks post-injection, engrafted TA muscles were dissected for analysis of gene expression. CRISPRi constructs were delivered systemically, via retro-orbital injection (ROI), in MyoAAV2A at 1.5E14 vg / kg or in LICA1 rAAV vectors at 1E13 or 5E13 vg / kg to 4- week-old male ACTAl-MCM;FLExD moderate pathology FSHD-like transgenic mice (Jones 2020, Skelet. Muscle 10, 8). At 3-4 weeks post-AAV injection, mice were subjected to intraperitoneal injections of 5 mg / kg TMX to induce DUX4-fl expression in skeletal muscles. For MyoAAV2A-injected mice, TA muscles were harvested at 10 days post-TMX injection for analysis of gene expression. For LICA1 -injected mice, EDL muscles were harvested at 10 days post TMX injection for analysis of vector genomes and gene expression. Physical performance of all mice was assessed by a treadmill stress test at 9 days post-TMX injection.

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Claims

CLAIMSWHAT IS CLAIMED IS:

1. An expression cassette comprising:(a) a first polynucleotide comprising a regulatory sequence operably linked to a first nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD);(b) a second polynucleotide comprising a first promoter operably linked to a second nucleotide sequence encoding a single guide RNA; wherein the first polynucleotide and the second polynucleotide are placed in different orientation from each other, and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b).

2. The expression cassette of claim 1, wherein the first polynucleotide sequence is located 5' to the second polynucleotide.

3. The expression cassette of claim 1, wherein the second polynucleotide sequence is located 5' to the first polynucleotide.

4. The expression cassette of any one of claims 1-3, wherein the nucleotide sequence encoding the fusion protein further comprises a polyadenylation signal.

5. The expression cassette of claim 4, wherein the polyadenylation signal is a SV40, or a bGH polyadenylation signal.

6. The expression cassette of any one of claims 1-5, wherein the fusion protein further comprises at least one nuclear localization signal (NLS).

7. The expression cassette of claim 6, wherein the same or different NLS are located N- terminal and C-terminal to the dCas.

8. The expression cassette of claim 6 or 7, wherein the at least one NLS is a SV40, or a nucleoplasmin NLS.

9. The expression cassette of any one of claims 1-8, wherein the regulatory sequence comprises a second promoter.

10. The expression cassette of claim 9, wherein the second promoter is a Muscle Creatine Kinase (Ckm) promoter,.

11. The expression cassette of any one of claims 1-10, wherein the regulatory sequence further comprises one or more enhancers.

12. The expression cassette of claim 11, wherein the one or more enhancers is a Ckm, enhancer.

13. The expression cassette of any one of claims 1-12, wherein the regulatory sequence is aNH or a HLH regulatory sequence.

14. The expression cassette of any one of claims 1-13, wherein the TRD is a SUV39H1 preSET, SET, and post-SET domain; a KRAB TRD; a MeCP2 TRD; a HPla TRD; or a HPly TRD;.

15. The expression cassette of any one of claims 1-14, wherein the first promoter is a U6 promoter, a Ml 1 hybrid promoter, a 7SK promoter, or a Hl promoter.

16. The expression cassette of any one of claims 1-15, wherein the nucleotide sequence encoding the single guide RNA comprises a spacer portion and a scaffold portion.

17. The expression cassette of claim 16, wherein the scaffold portion of the single guide RNA is a scaffold portion capable of binding to a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY.

18. The expression cassette of claim 16 or 17, wherein the spacer portion of the single guide RNA comprises a sequence targeting a DUX4 gene promoter and / or a DUX4 gene exon 1.

19. The expression cassette of any one of claims 1-18, wherein the dCas enzyme is a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY.

20. The expression cassette of any one of claims 1-19, wherein the ITRs in the pair of ITRs are the same or different.

21. The expression cassette of any one of claims 1-20, wherein the ITRs are derived from an AAV genome of serotype AAV2.

22. The expression cassette of any one of claims 1-21, wherein in the fusion protein the dCas is located N-terminal to the TRD.

23. The expression cassette of claim 22, wherein:(c) the first polynucleotide comprises:(i) a HLH regulatory cassette,(ii) a first portion of a nucleotide sequence encoding a bipartite SV40 NLS,(iii) a nucleotide sequence encoding a dSaCas9,(iv) a second portion of a nucleotide sequence encoding a bipartite SV40 NLS,(v) a nucleotide sequence encoding a MeCP2 TRD, and(vi) an SV40 polyadenylation signal; and(d) the second polynucleotide comprises:(i) a nucleotide sequence encoding a single guide RNA targeting the DUX4 gene, and(ii) a U6 promoter.

24. The expression cassette of claim 23, wherein the expression cassette comprises a nucleotide sequence at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.

25. A single-stranded expression cassette comprising:(a) a first polynucleotide comprising a regulatory sequence operably linked to a first nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD);(b) a second polynucleotide comprising the antisense of a second nucleotide sequence, wherein the second nucleotide sequence comprises a first promoter operably linked to a third nucleotide sequence encoding a single guide RNA; wherein the first polynucleotide is located 5' to the second polynucleotide; and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b); and wherein the first polynucleotide and the second polynucleotide are placed in different orientation from each other.

26. A single-stranded expression cassette comprising:(a) a first polynucleotide comprising a first promoter operably linked to a first nucleotide sequence encoding a single guide RNA;(b) a second polynucleotide comprising the antisense of a second nucleotide sequence, wherein the second nucleotide sequence comprises a regulatory sequence operably linked to a third nucleotide sequence encoding a fusion protein comprising a catalytically inactive Cas enzyme (dCas) and a transcriptional repressor domain (TRD); wherein the first polynucleotide is located 5' to the second polynucleotide; and wherein the cassette further comprises a pair of inverted terminal repeats (ITRs) flanking (a) and (b); and wherein the first polynucleotide and the second polynucleotide are placed in different orientation from each other.

27. The single strand expression cassette of claim 25 or 26, wherein:(a) the nucleotide sequence encoding the fusion protein further comprises a SV40 or a bGH polyadenylation signal;(b) the fusion protein further comprises at least one SV40 or nucleoplasmin nuclear localization signal (NLS);(c) the regulatory sequence comprises a Muscle Creatine Kinase (Ckm) promoter;(d) the regulatory sequence comprises a Ckm enhancer;(e) the TRD is a SUV39H1 pre-SET, SET, and post-SET domain; a KRAB TRD; a MeCP2 TRD; a HPla TRD; a HPly TRD;(f) the nucleotide sequence encoding the single guide RNA comprises a scaffold portion capable of binding to a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY, , and a spacer portion comprising a sequence targeting a DUX4 gene promoter and / or &DUX4 gene exon 1;(g) the dCas enzyme is a dCas9, dCas6, dCpfl, dCasl2a, dCasl3a, dCasX, or dCasY;(h) the ITRs are derived from an AAV genome of serotype AAV2; and(i) in the fusion protein the dCas is located N-terminal to the TRD.

28. A self-complementary expression cassette comprising the expression cassette of anyone of claims 1-24 and its complementary sequence.

29. An AAV vector comprising the expression cassette of any one of claims 1-24, the singlestranded expression cassette of any one of claims 25-27, or the self-complementary expression cassette of claim 28.

30. A lipid nanoparticle (LNP) comprising the expression cassette of any one of claims 1-24, the single-stranded expression cassette of any one of claims 25-27, or the self- complementary expression cassette of claim 28.

31. A cell comprising the expression cassette of any one of claims 1-24, the single-stranded expression cassette of any one of claims 25-27, the self-complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, or any combination thereof.

32. A pharmaceutical composition comprising the expression cassette of any one of claims 1- 24, the single-stranded expression cassette of any one of claims 25-27, the self- complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, the cell of claim 31, or any combination thereof, and a pharmaceutically acceptable carrier.

33. A method of inducing transcriptional repression of a DUX4 gene in a population of cells comprising contacting the population of cells with the expression cassette of any one of claims 1-24, the single-stranded expression cassette of any one of claims 25-27, the self-complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, the pharmaceutical composition of claim 32, or any combination thereof.

34. The method of claim 33, wherein transcription level of the DUX4 gene in the population of cells is reduced compared to the transcription level of the DUX4 gene in the same population of cells before contacting the population of cells with the expression cassette of any one of claims 1-24, the single-stranded expression cassette of any one of claims 25-27, the self-complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, the pharmaceutical composition of claim 32, or any combination thereof.

35. The method of claim 33 or 34, wherein transcription level of the DUX4 gene in the population of cells is reduced compared to the transcription level of the DUX4 gene in a same population of cells not contacted with the expression cassette of any one of claims 1- 24, the single-stranded expression cassette of any one of claims 25-27, the self- complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, the pharmaceutical composition of claim 32, or any combination thereof.

36. A method of treating FSHD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the expression cassette of any one of claims 1-24, the single-stranded expression cassette of any one of claims 25-27, the self- complementary expression cassette of claim 28, the AAV vector of claim 29, the LNP of claim 30, the cell of claim 31, the pharmaceutical composition of claim 32, or any combination thereof.

37. The method of claim 36, wherein the FSHD is FSHD1 or FSHD2.

38. The method of claim 36 or 37, wherein the subject is a human.

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