Oligonucleotides and compositions thereof for neuromuscular disorders

Abstract

Disclosed herein is an engineered DUX4-targeting oligonucleotide for selectively inhibiting the RNA transcript associated with neuromuscular disease, such as facioscapulohumeral muscular dystrophy.Disclosed herein is also a vector containing any of these, a pharmaceutical preparation containing any of these, and a kit containing any of these.Disclosed herein is also a method for selectively inhibiting the expression and activity of polypeptide by contacting the RNA transcript associated with neuromuscular disease, such as facioscapulohumeral muscular dystrophy, with DUX4-targeting oligonucleotide.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 221,568, filed July 14, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. Summary of the Invention [Means for solving the problem]

[0003] Certain aspects of the present disclosure relate to engineered DUX4-targeting oligonucleotides that are about 15 to about 25 nucleotides in length, and the engineered DUX4-targeting oligonucleotides comprise at least about: 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 20,962-42,138. Additionally, the engineered DUX4-targeting oligonucleotides can be about 15 to about 25 nucleotides in length and comprise at least about 80%, 85%, 90%, or 95% sequence identity to any one of SEQ ID NOs: 42,006-42,138.

[0004] In certain cases, the engineered DUX4-targeting oligonucleotide comprises DNA nucleotides and RNA nucleotides. In some cases, the oligonucleotide comprises DNA nucleotides. In some cases, the oligonucleotide comprises RNA nucleotides. In certain cases, the oligonucleotide is a small interfering RNA (siRNA), a microRNA (miRNA), a small nuclear RNA (snRNA), a U-spliceosomal RNA (U-RNA), a small nucleolar RNA (snoRNA), a Piwi-interacting RNA (piRNA), a repeat-associated small interfering RNA (rasiRNA), a small rDNA-derived RNA (srRNA), a transfer RNA-derived small RNA (tsRNA), a ribosomal RNA-derived small RNA (rsRNA), a long non-coding RNA-derived small RNA (lncsRNA), or a messenger RNA-derived small RNA (msRNA). In certain cases, the oligonucleotide can comprise at least one nucleobase of a locked nucleic acid.

[0005] The above DUX4-targeting oligonucleotides are capable of binding to a DUX4 coding sequence in an aqueous solution with a predicted melting temperature (Tm) of from about 45 to about 65° C., where the aqueous solution has a pH range of from about 7.2 to about 7.6.

[0006] Another aspect of the present disclosure is a method for producing a method for manufacturing a semiconductor device comprising the steps of: i) a conjugate of the DUX4-targeting oligonucleotide as described above, wherein the conjugate comprises an oligonucleotide and with an antibody, antibody fragment, single monomeric variable antibody domain, naturally occurring ligand, small molecule, or peptide; Optionally, iii) a linker connecting i) to ii). Includes.

[0007] Another aspect of the present disclosure relates to a vector that contains or codes for the conjugate described herein or the oligonucleotide described herein.In certain cases, the vector can include a viral vector, a nanoparticle vector, a liposomal vector, an exosomal vector, an extracellular vesicle vector, or a combination thereof.The vector can be a liposomal vector.The vector can be a nanoparticle vector.The vector can be an exosomal vector.The vector can be an extracellular vector.

[0008] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an engineered DUX4-targeting oligonucleotide as described herein, a conjugate as described herein, a vector as described herein, which is a vector as described in any one of claims 10-15, and a pharma- ceutically acceptable: excipient, diluent, carrier, or combination thereof. In certain cases, the pharma- ceutically acceptable excipient comprises a buffering agent, a stabilizer, an antioxidant, a diluent, or any combination thereof. In certain examples, the pharma- ceutical acceptable diluent comprises distilled water, deionized water, saline, Ringer's solution, dextrose solution, cell growth medium, phosphate buffered saline (PBS), or any combination thereof. The pharmaceutical composition described herein can be in unit dose form.

[0009] Another aspect of the present disclosure relates to a kit comprising an engineered DUX4-targeting oligonucleotide as described herein, a conjugate as described herein, a vector as described herein, or a pharmaceutical composition as described herein, and a container. In certain cases, the container can include a jar, an ampoule, a syringe, a bag, a box, or a combination thereof.

[0010] Another aspect of the present disclosure is a method for treating disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.The disease or condition is a DUX4-mediated disease or condition.The DUX4-mediated disease or condition is facioscapulohumeral muscular dystrophy.The subject can be a subject in need thereof.The subject can be a human subject in need thereof.

[0011] In this method, administration is in an amount of about 0.001 mg to about 10,000 mg of the pharmaceutical formulation per kg of the subject's body weight. Administration can be oral, intranasal, rectal, topical, intraocular, intramuscular, intravenous, intraperitoneal, intracardiac, subcutaneous, intracranial, intrathecal, or any combination thereof.

[0012] The method can employ a pharmaceutical composition, wherein the pharmaceutical composition is a liquid dosage form administered in a volume of about 1 ml to about 5 ml, about 5 ml to about 10 ml, about 15 ml to about 20 ml, about 25 ml to about 30 ml, about 30 ml to about 50 ml, about 50 ml to about 100 ml, about 100 ml to about 150 ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400 ml, about 400 ml to about 450 ml, about 450 ml to about 500 ml, about 500 ml to about 750 ml, or about 750 ml to about 1000 ml. In certain cases, the pharmaceutical composition is a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, a pill form, a capsule form, a gel, or any combination thereof.

[0013] In certain cases, administration includes systemic administration or local administration. Administration can be systemic administration, where systemic administration includes at least one of: parenteral administration, intravenous administration, subcutaneous administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intravascular administration, injection, oral administration, inhalation administration, intraduodenal administration, rectal administration, or any combination thereof.

[0014] In certain cases, the methods further comprise administering a combination therapy, either simultaneously or sequentially.

[0015] Another aspect of the present disclosure relates to a method of administering an engineered DUX-4 targeting oligonucleotide as described herein, wherein after administration, the engineered DUX-4 targeting oligonucleotide selectively hybridizes to two different endogenous disease-associated RNAs, where one of the two different endogenous disease-associated RNAs is a DUX4 RNA transcribed from a first locus, and one of the two different endogenous disease-associated RNAs is transcribed from a locus different from the first locus.Furthermore, in certain cases, the engineered DUX4-targeting oligonucleotide hybridizes to an endogenous disease-associated RNA transcribed from a locus different from the first locus, such that at least 10 consecutive oligonucleotides of the engineered DUX4 targeting oligonucleotide hybridize to at least two different consecutive sections of consecutive bases, interrupted by at least one nucleic acid base.The method can be a method of treating a disease or condition that is a DUX4-mediated disease or condition.The disease or condition can be facioscapulohumeral muscular dystrophy. Upon hybridization between the engineered DUX4-targeting oligonucleotide and the second RNA, the predicted thermal melting point can be from about 40°C to about 65°C.

[0016] Another aspect of the present disclosure is a composition for use in treating a neuromuscular disease, comprising an engineered DUX4-targeting oligonucleotide as described herein, a conjugate as described herein, a vector as described herein, a pharmaceutical composition as described herein, and a pharma- ceutically acceptable excipient, diluent, or carrier. The composition can be for use where the neuromuscular disease is facioscapulohumeral muscular dystrophy. [Brief description of the drawings]

[0017] [Figure 1]FIG. 1 shows genetic modifications that lead to FSHD. [Diagram 2] FIG. 1 shows alternatively spliced ​​DUX4 transcripts originating from the D4Z4 region. [Diagram 3] FIG. 1 shows an overview of read coverage from RNA-Seq data of alternatively spliced ​​DUX4 transcripts from FSHD and healthy muscle biopsy tissue. [Figure 4] FIG. 1 shows an overview of read coverage from RNA-Seq data of alternatively spliced ​​DUX4 transcripts from testis. [Diagram 5] FIG. 1 shows the serum stability of chemically modified anti-DUX4 ASOs compared to unmodified oligos. [Figure 6A]

[0023] Figures 6A and 6B illustrate the reduction in innate immune activation. Figure 6A depicts the reduction in innate immune production of IFNα and TNFα following exposure of PBMCs to engineered anti-DUX4 ASOs. [Figure 6B] Figure 6A illustrates the reduction in innate immune activation by engineered DUX4 ASOs using a Raw-blue cell assay. [Figure 7] FIG. 1 shows the DUX4 ASO HTS assay design using stable human or mouse myoblasts expressing eGFP with the coding sequence of DUX4 in the 3'UTR. [Figure 8A] Figure 8A shows knockdown of DUX4 mRNA.Figure 8B shows that therapeutic ASOs have potent knockdown of DUX4 in FSHD myotubes. [Figure 8B] Figure 8B shows knockdown of DUX4 mRNA.Figure 8B shows knockdown of DUX4 and DUX4-inducible genes ZSCAN4 and SLC34A2 in FSHD myotubes. [Figure 9] FIG. 1 shows simultaneous knockdown of DUX4 and DBET RNA transcripts in FSHD patient myoblasts by multi-targeted ASOs. [Figure 10]FIG. 1 shows a schematic overview of the data analysis to identify FSHD-associated genes and pathways. [Figure 11] FIG. 1 shows the expression of genes representing six FSHD-related biological functions separated by horizontal gaps (from top to bottom): DUX4 regulation, extracellular matrix, cell cycle, immune / inflammatory response, immunoglobulin and muscle development related. [Figure 12A] Figure 12 shows exemplary pathways of potential effects of identified co-targets. Figure 12A shows pathway regulation of Ki-67 on cell proliferation according to Xie et al. (18). [Figure 12B] FIG. 12B is a diagram showing exemplary pathways of potential effects of identified co-targets. FIG. 12B is a diagram showing induction of IRF5 in inflammatory signaling according to Elkon et al. (19). [Figure 13] FIG. 1 shows RNA expression of IRF5 and MKI67 in patient biopsy samples. [Figure 14A] Figure 14 shows validation of co-targeted transcripts with multiple targeting ASOs. Figure 14A shows myoblasts after treatment with ASOs. [Figure 14B] Figure 14B shows validation of co-targeted transcripts by multiplex targeting ASOs. Figure 14B shows qRT-PCT results from RNA (DUX4, DBET, IRF5, and MKI67) obtained from myoblasts treated with ASOs. [Figure 15] 1 is a diagram illustrating the methods and systems disclosed herein. [Figure 16] FIG. 1 shows a computer controlled system programmed to analyze genetic material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] overview Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of muscular dystrophy (MD), affecting approximately 40,000 symptomatic patients in the United States (1, 2). FSHD type 1 (FSHD1), which accounts for 95% of all FSHD patients, is the result of a reduction in the number of D4Z4 repeats on chromosome 4q35 from approximately 100 to fewer than 11 (3). FSHD type 2 (SHD2) is the result of a loss-of-function mutation in an epigenetic factor, structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) (3) (Figure 1). Both genetic mutations result in hypomethylation of the D4Z4 region, which allows for inappropriate expression of the dual homeobox 4 protein (DUX4) gene encoded within D4Z4. Aberrant expression of DUX4 is severely toxic to muscle tissue, thus resulting in oxidative stress and apoptosis of muscle cells, leading to impaired muscle function (4, 5). FSHD results in progressive muscle weakness of the face, shoulders, arms, abdomen, and legs. Approximately 20% of patients are eventually wheelchair-bound (6). If only one to three D4Z4 repeats remain, a more severe and rapidly progressive disability results (7), often with childhood onset (8) and loss of hearing and vision (9). There is a broad scientific consensus in the field that if DUX4 expression could be eliminated in muscle tissue, it would be possible to halt the progression of FSHD1 and 2 (10-12). Several studies have shown that RNA oligonucleotide therapeutics have the potential to directly suppress DUX4 and reverse muscle pathology in vitro and in mouse models (13-16). However, preservation of the complementary binding site for oligonucleotide therapeutics targeting DUX4 remains an issue.

[0019] Oligonucleotide therapeutics (ONTs) designed to treat any disorder will be most effective in regulating the targeted transcript if they are perfectly complementary to the target RNA binding site of the disease transcript. In addition, the targeted binding sequence needs to have low variability among patients with the disorder. Otherwise, patients with SNPs or mutations in the sequence of the disease gene at the target binding site may not be perfectly complementary to the therapeutic oligonucleotide, thus resulting in less than complete silencing of the disease gene by ONT. This application is the first to solve the problem of determining conserved variant sequences within the DUX4 gene / exon, identify RNA therapeutics that target clinically relevant DUX4 variants, and create RNA therapeutics with superior structural modifications for efficacy and stability.

[0020] Typically, sequence databases containing hundreds to thousands of individuals are used to select highly conserved binding sites for oligonucleotide therapeutics (ONTs) (20). However, these databases cannot be used to accurately predict the variability of the DUX4 gene. The challenge is to find conserved therapeutic targets for DUX4. A solution and the generation and validation of DUX4-targeting oligonucleotides are disclosed herein. Most public sequence databases utilize DNA fragment sequencing technology to efficiently and inexpensively collect sequence data from populations. This involves fragmenting long genomic DNA into pieces several hundred bases long that are cloned, amplified, and sequenced. Individual fragments are then mapped to a larger known reference genomic sequence. Such techniques are known to be ineffective at precisely distinguishing or mapping repetitive sequences (21).

[0021] The coding region of the DUX4 gene is present in each D4Z4 repeat on chromosome 4. DNA from normal individuals contains 11–200 copies of D4Z4 on each chromosome 4 (12). In addition, DUX4-containing D4Z4 repeats are also found on chromosome 10. However, deletion of the D4Z4 repeat on chromosome 10 is not associated with the development of facioscapulohumeral muscular dystrophy (FSHD) due to the absence of downstream exons 3–5 of the DUX4 coding sequence. Thus, the sequence variation found in the DUX4 coding sequence on chromosome 10 does not appear to be important for the design of ONTs. Furthermore, D4Z4 pseudogenes are found throughout the human genome as well (22), and significant sequence overlap exists between the DUX4 sequence of D4Z4 and other repetitive DNA sequences encoding the DUX family members DUX1–DUX5 (23). This genomic complexity results in poor mapping of sequenced DNA fragments that overlap with D4Z4 repeats, and little confidence in which genomic loci they originate from. In predicting DUX4 coding sequence mutations in FSHD patients, genomic complexity creates a problem in that there is little confidence that sequence data and listed mutations can accurately predict the conserved sequence of DUX4, because most of the data is mixed with sequence mutations from other genomic locations that are not related to the disease-causing truncated D4Z4 repeat array present on one copy of chromosome 4 in FSHD patients. One logical solution would be to use RNA-seq data from muscle biopsies of FSHD patients. As shown in Example 1, such an approach does not provide sufficient data to allow prediction of DUX4 coding sequence variation.

[0022] This disclosure, for the first time, solves the problem of determining conserved variant sequences within the DUX4 gene / exon, identifies ONT therapeutics that target clinically relevant DUX4 variants, and generates ONT therapeutics with superior structural modifications for efficacy and stability.

[0023] Disclosed herein are sequences representing all regions of the DUX4 coding sequence that are >85% conserved among 206 subjects (Table 4). To identify these regions, the inventors surprisingly discovered that sufficient reads could be identified in the RNA-seq data by combining RNA-seq data from muscle biopsies of patients into a database combined with RNA-seq from testis samples, as shown in Example 3. While it is known in the art that low levels of DUX4 expression are observed in gametocytes of the testis (24), one skilled in the art would not have expected to be able to predict variability in DUX4 disease transcripts from testis RNA sequences, because testis-expressed DUX4 transcripts have been reported to be differentially spliced ​​and lack exon 1, exon 2, and exon 3 regions contained in the predicted disease-causing DUX4 muscle-specific transcript (25) (Figure 2). Combining RNA-seq data from these two tissues into a single dataset, we were able to generate sufficient read coverage across the DUX4 disease gene, thereby predicting regions that are >85% conserved and likely to effectively treat most patients.

[0024] Antisense oligonucleotides (ASOs), which rely on cleavage of complementary RNA and subsequent RNase H for degradation, can and do silence many RNAs besides the intended RNA target (26, 27). These non-targeted RNAs are often referred to as off-target effects. In the case of gapmer ASOs, this occurs when the DNA portion of the oligonucleotide binds to a partially complementary target site and induces RNAse H cleavage, thereby causing degradation of the unintended RNA off-target. Careful sequence analysis can identify many of these potential interactions. However, simple sequence alignment often does not accurately predict true off-target interactions. We have developed a data analysis pipeline for predicting and tracking off-target effects for RNA therapeutics that also considers structural motifs and binding energies, thereby improving predictions (WO2021203043).

[0025] A common practice in the field is to avoid off-target effects as much as possible in the design of oligonucleotides. The novel approach described herein is to instead look at off-targets holistically. We first look for those that are likely to be potentially harmful and cause toxicity by filtering predicted targets through toxicity databases such as Toxnet and Ingenuity Pathway Analysis (IPA). We also consider off-targets that may be related to disease pathways through analysis of the transcriptome profile of muscle biopsies from FSHD patients, or may be related to known disease pathways such as inflammation, muscle cell division or cell death pathways, by looking for genes that are grossly overexpressed in a subset.

[0026] This information allows for prioritization of which ASO sequences to synthesize, test, and validate. ASOs that demonstrate high knockdown capacity and high disease relevance off-targets are then used to validate knockdown of off-target transcripts in differentiated myotubes in vitro by qRT-PCR.

[0027] definition Unless otherwise expressly stated, open-ended terms such as "contain," "containing," "include," "including," etc. mean "comprising."

[0028] The singular forms "a," "an," and "the" are used herein to include plural references unless the context clearly dictates otherwise. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this application are approximations that may vary depending upon the desired properties sought to be obtained.

[0029] As used herein, the term "about" can mean a referenced numerical indication, plus or minus: 5%, 10%, 15%, or 20% of the referenced numerical indication. In some cases, "about" can mean a referenced numerical indication, plus or minus 15% of the referenced numerical indication. In some cases, "about" can mean a referenced numerical indication, plus or minus 20% of the referenced numerical indication. In the context of biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold of a value. When specific values ​​are described in the present application and claims, the term "about" should be assumed to mean within an acceptable range of error for the particular value, unless otherwise indicated. Also, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0030] As used herein, the term "substantially" can refer to a value approaching 100% of a given value. In some cases, the term can refer to an amount that can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or about 100% of the total amount.

[0031] The term "homology" can refer to the % identity of a sequence to a reference sequence. In practice, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (can correspond to a particular nucleic acid sequence described herein), such a particular polypeptide sequence can be conventionally determined using known computer programs such as Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence, parameters can be set so that the percentage of identity is calculated over the full length of the reference sequence and that the gap of homology of up to 5% of the entire reference sequence is allowed. Any of the sequences disclosed herein also include sequences with about: 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the disclosed sequences.

[0032] The term "oligonucleotide" can refer to DNA, RNA or hybrid nucleic acid sequence, whether chemically modified or not, where, for example, single strand, as in the case of DNA, is usually bound to target RNA in reverse complementarity.In the case of RNA, oligonucleotide can be single stranded, as in the case of miRNA, where single strand is bound to target RNA sequence in reverse complementarity.In another example, RNA oligonucleotide can be double stranded, as in the case of siRNA, where one strand is bound to target RNA sequence in reverse complementarity.

[0033] As used herein, in some instances, the terms "targeting" and "targeted" may be used interchangeably, for example, an oligonucleotide targeting DUX4 may be a DUX4-targeting oligonucleotide or an oligonucleotide targeting DUX4. An oligonucleotide targeting DUX4 may be a DUX4-targeting oligonucleotide. The targeting sequence may have reverse complementarity to a DUX4 transcript. In some cases, the targeting sequence may have at least partial reverse complementarity to a DUX4 transcript and one or more additional loci or transcripts thereof. In some cases, a locus may have at least partial reverse complementarity to a DUX4 transcript and one or more additional loci or transcripts thereof.

[0034] The term "fragment" as used herein can be a portion of a sequence, a subset that may be shorter than the full length sequence. A fragment can be a portion of a gene. A fragment can be a portion of a peptide or a portion of a protein. A fragment can be a portion of an amino acid sequence. A fragment can be a portion of an oligonucleotide sequence. A fragment can be less than about: 20, 30, 40, 50 amino acids in length. A fragment can be less than about: 2, 5, 10, 20, 30, 40, 50 oligonucleotides in length.

[0035] As used herein, the term "epigenetic marker" can be any covalent modification of a nucleic acid base.

[0036] The terms "administer," "administering," "administration," and the like, as used herein, can refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological action. The term "delivery" includes direct application to the affected tissue or area of ​​the body.

[0037] The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to an animal, typically a mammal.

[0038] The terms "treat," "treating," and "treatment," as used herein, may imply, at least in part,: alleviating, relieving, or ameliorating the symptoms of a disease or condition; preventing further symptoms; ameliorating or preventing the underlying cause of the symptoms; preventing the recurrence of the symptoms; inhibiting the disease or condition, e.g., at least partially arresting the onset of the disease or condition; relieving the disease or condition; causing regression of the disease or condition; alleviating the conditions caused by the disease or condition; or halting the symptoms of the disease or condition, either prophylactically, therapeutically, or both.

[0039] As used herein, an "agent" or "biologically active agent" can refer to a biological, pharmaceutical, or chemical compound, or a salt of any of these structures.

[0040] As used herein, the term "tissue" can refer to any tissue sample. The tissue can be a tissue suspected or confirmed to have a disease or condition.

[0041] The term "mammalian cell" can refer to any mammalian cell, typically a human cell.

[0042] Engineered DUX4-targeting oligonucleotides The disclosure herein provides therapeutic targeting of RNA transcripts containing selected DUX4 target loci. Two main methods are used in RNA medicine: double-stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASOs). Overall, RNAi can function by activating ribonucleases, which, together with other enzymes and complexes, coordinately degrade RNA after the original RNA target has been cleaved into smaller pieces. Antisense oligonucleotides can bind to their target nucleic acids via Watson-Crick base pairing and can inhibit or alter gene expression through steric hindrance, alteration of splicing, initiation of target degradation, or other events.

[0043] In certain embodiments of the present disclosure, oligonucleotide therapeutics (ONT) can be designed to treat any disorder that can be applied to regulate targeted transcripts.In certain embodiments, the treatment is with one or more substantially or completely complementary ASOs with respect to the target RNA binding site of the disease that has the transcript that needs to be downregulated.In certain cases, the oligonucleotide therapeutics is mainly DNA, and in other cases, the oligonucleotide is mainly RNA.In general, the ASO that effectively targets DUX4 can bind to fusion transcripts and induce degradation through RNAse H.

[0044] In other embodiments of the present disclosure, an interfering RNA such as an siRNA or miRNA that contains a sequence that is complementary to the DUX4 RNA transcript can be designed to treat any disorder for which regulating such a targeted transcript is applicable. In certain embodiments, the siRNA is double-stranded, where one strand is complementary. The RISC uses the guide strand of the miRNA or siRNA to target the complementary 3'-untranslated region (3'UTR) of the mRNA transcript via Watson-Crick base pairing, thereby allowing the RISC to regulate gene expression of the mRNA transcript in several ways, such as mRNA degradation, thereby preventing or reducing protein expression of the selected mRNA.

[0045] The mentioned oligonucleotides can include miRNA. Such miRNA can contain one or more sequence modifications, one or more chemical modifications, or a combination thereof, which can: enhance the stability of miRNA; substantially reduce or eliminate immune activation (such as via the innate immune response); improve the pharmacological activity of miRNA; retain the poly-targeting effect of miRNA; or any combination thereof.

[0046] The nucleic acid sequences provided herein, including but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to those nucleic acids with modified nucleic acid bases. As a further example and without limitation, an oligonucleotide having the nucleic acid base sequence "ATCGATCG" encompasses any oligomeric compound having such a nucleic acid base sequence, whether modified or unmodified, including but not limited to those compounds containing RNA bases, such as those having the sequence "AUCGAUCG", and those having some DNA bases and some RNA bases, such as "AUCGATCG", as well as oligomeric compounds having other modified or naturally occurring bases. Similarly, an RNA transcript containing the sequence "AUCGAUCG" encompasses any corresponding DNA sequence, such as "ATCGATCG". Nucleic acid sequences herein also include sequences that contain at least about: 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the disclosed sequences.

[0047] In certain cases, the oligonucleotide construct can include a first strand that includes a DUX4-targeting oligonucleotide and a second strand that includes a sequence complementary to at least a portion of the DUX4-targeting oligonucleotide. The second strand can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more complementary to the first strand. The second strand can be at least about: 5, 10, 15, or 20 consecutive bases complementary to the first strand. The oligonucleotide can include a terminal overhang, such as a 5' or 3' end. The first strand, the second strand, or a combination thereof can include one or more chemical modifications. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the bases of the first strand, the second strand, or a combination thereof can include a chemical modification. The first strand, the second strand, or a combination thereof can include one or more sugar modifications. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the bases of the first strand, the second strand, or a combination thereof can include a sugar modification. The sugar modification can include a glycosylated base. In some cases, the base of the nucleotide can be glycosylated with a glycan. The first strand, the second strand, or a combination thereof can include a combination of bases with chemical modifications and sugar modifications.

[0048] In some cases, the oligonucleotides described herein, such as DUX4-targeting oligonucleotides or salts thereof, can be about 5 to about 50 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 40 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 30 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 25 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 60 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 80 nucleotides in length. In some cases, the DUX4-targeting oligonucleotides or salts thereof can be about 5 to about 100 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof can be from about 5 to about 200 nucleotides in length.

[0049] In certain other cases, the interfering RNA can be a regulatory non-coding RNA (ncRNA), which includes short non-coding RNA sequences expressed in the genome that regulate the expression or function of other biomolecules in mammalian cells. ncRNAs are generally <200 nucleotides in length, can be single-stranded or double-stranded, and can form non-linear secondary or tertiary structures. ncRNAs can include exogenously derived small interfering RNAs (siRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), U-spliceosomal RNAs (U-RNAs), small nucleolar RNAs (snoRNAs), Piwi-interacting RNAs (piRNAs), repeat-associated small interfering RNAs (rasiRNAs), small rDNA-derived RNAs (srRNAs), transfer RNA-derived small RNAs (tsRNAs), ribosomal RNA-derived small RNAs (rsRNAs), long non-coding RNA-derived small RNAs (lncsRNAs), or messenger RNA-derived small RNAs (msRNAs).

[0050] DUX4-targeting oligonucleotides can include DNA, RNA, or mixtures thereof. In some cases, DUX4-targeting oligonucleotides can include multiple nucleotides. In some cases, DUX4-targeting oligonucleotides can include artificial nucleic acid analogs. In some cases, DUX4-targeting oligonucleotides can include DNA, cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA. In some cases, DUX4-targeting oligonucleotides can include shRNA, or siRNA, ncRNA mimics, small hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotides (ASO), gapmers, mixmers, double-stranded RNA (dsRNA), single-stranded RNAi, (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotides (RNAa), or exon skipping oligonucleotides. In some cases, DUX4-targeting oligonucleotides can include fully synthetic miRNA. Fully synthetic miRNAs are not derived from or based on ncRNA. Alternatively, a fully synthetic miRNA may be based on an analysis of multiple potential target sequences, or may be based on an isolated naturally occurring non-coding sequence that is not an ncRNA.

[0051] Modified Oligonucleotides In some cases, the second strand can comprise chemically modified bases of nucleotides.In some cases, a subset of the bases of the second strand can be chemically modified, such as about 1% to about 5% bases, about 1% to about 10% bases, about 1% to about 20% bases, about 1% to about 30% bases, about 1% to about 40% bases, about 1% to about 50% bases, about 1% to about 60% bases, about 1% to about 70% bases, about 1% to about 80% bases, or about 1% to about 90% bases or more.The second strand described herein can be chemically modified in the same manner as described herein for DUX4-targeting oligonucleotides.

[0052] The oligonucleotide can include a sugar modification. The oligonucleotide can include multiple sugar modifications. The sugar modification can include glucose or a derivative thereof. The sugar modification can include ribose or deoxyribose. The sugar modification can include a monosaccharide, a disaccharide, a trisaccharide, or any combination thereof.

[0053] In some cases, ribonucleotides or deoxynucleotides can be modified by chemical modifications described herein, for example, at the base moiety, sugar (ribose) moiety, phosphate moiety, or any combination thereof that forms the backbone of a DUX4-targeting oligonucleotide.

[0054] Oligonucleotides, such as DUX4-targeting oligonucleotides, can include chemical modifications. Oligonucleotides can include multiple chemical modifications. Oligonucleotides can include multiple chemical modifications within a portion of the oligonucleotide, such as the termini. Chemical modifications can include methyl groups, fluoro groups, methoxyethyl groups, ethyl groups, amide groups, ester groups, any two or more of these, or any combination thereof. Chemical modifications can include chemically modified nucleotides, such as guanosine, uridine, adenosine, thymidine, or cytidine, including any naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytidine, chemically altered, for example, by acetylation, methylation, hydroxylation, or the like, including 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine riboside, 2-aminopurine riboside, 2'-araadenosine, 2'-aracytidine, 2'-arauridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro Fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'-O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2-thiocytidine, 2-thiocytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-Dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-uracil, 5-chloro-ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thiouridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine riboside, 7-deaza-2'-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine and 8-oxoguanosine, benzimidazole riboside, beta-D-mannosyl-queosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, xyloadenosine, or any combination thereof. The production of such variants is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 or 5,700,642.

[0055] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can contain chemically modified nucleotides, such as 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate, 2-aminoadenosine-5-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O- Methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5- Iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7- Deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O-6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, xanthosine-5'-triphosphate,or any combination thereof.

[0056] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can include chemically modified nucleotides, such as pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudour ... thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, or any combination thereof.

[0057] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can contain chemically modified nucleotides, such as 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, or any combination thereof.

[0058] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can contain chemically modified nucleotides, such as 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6 ... adenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, or any combination thereof.

[0059] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can contain chemically modified nucleotides, such as inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, or any combination thereof.

[0060] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can contain chemically modified nucleotides, such as 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thio-uridine, 5-amino-iso-cytidine, 5-amino-iso ... amidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, or any combination thereof.

[0061] In some cases, oligonucleotides such as DUX4-targeting oligonucleotides can include chemically modified nucleotides, which can be chemically modified at the 2' position. Chemically modified oligonucleotides can include a substituent at the 2' carbon atom, where the substituent can include 2'-hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-methoxyethyl, 2'-fluoro, halogen, alkoxy group, hydrogen, aryloxy group, amino group or aminoalkoxy group, locked nucleic acid (LNA), or any combination thereof.

[0062] Another chemical modification to oligonucleotides such as DUX4-targeting oligonucleotides (such as those involving the 2' position of the nucleotide) can be a locked nucleic acid (LNA) nucleotide, an ethylene-bridged nucleic acid (ENA) nucleotide, an (S)-constrained ethyl (cEt) nucleotide, a bridged nucleic acid (BNA) nucleotide, or any combination thereof. The backbone modification can lock the sugar of the modified nucleotide into a preferred Northern structure. In some cases, the presence of this type of modification in the target sequence of the DUX4-targeting oligonucleotide can allow stronger and faster binding of the DUX4-targeting oligonucleotide sequence to the target site.

[0063] In some cases, oligonucleotides such as DUX4-targeting oligonucleotides can include at least one chemically modified nucleotide, in which case the phosphate backbone that can be incorporated into DUX4-targeting oligonucleotide can be modified. One or more phosphate groups of the backbone can be modified, for example, by replacing one or more of the oxygen atoms with different substituents. In addition, modified nucleotides can include a complete replacement of unmodified phosphate moieties with modified phosphates, as described herein. Examples of modified phosphate groups can include phosphorothioates, methyl phosphonates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl phosphonates, aryl phosphonates, or hosto triesters. Phosphate linkers can also be modified by replacing the oxygens linked by nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene phosphonates).

[0064] In some cases, an oligonucleotide, such as a DUX4-targeting oligonucleotide, can include a sugar modification. The sugar modification can include a conjugate, such as a linker. In some cases, a DUX4-targeting oligonucleotide can include one or more linker groups. A DUX4-targeting oligonucleotide can be linked to an antibody, a protein, a lipid, an aptamer, a small molecule, a drug, or any combination thereof. The linker can form a covalent bond. A DUX4-targeting oligonucleotide can be linked to one or more oligonucleotides, such as a second DUX4-targeting oligonucleotide, via a linker. In some cases, the linker can be a cleavable linker. In some cases, the linker can include an azido linker. A DUX4-targeting oligonucleotide can include a base of a nucleotide that is glycosylated with a glycan. In some cases, a DUX4-targeting oligonucleotide can include an abasic site, such as a nucleotide that lacks an organic base. In some cases, an abasic nucleotide can include a chemical modification as described herein, such as at the 2' position of the ribose. In some cases, the 2'C atom of ribose can be substituted with a substituent such as a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group, or an aminoalkoxy group, in some cases from 2'-hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl, or 2'-fluoro. In some cases, the abasic site nucleotide can include structure 1A and can include structure 1B: [ka]

[0065] In some cases, oligonucleotides such as DUX4-targeting oligonucleotides can be modified by the addition of a "5'-CAP" structure. The 5'-cap can be an entity such as a modified nucleotide entity that can "cap" the 5'-end of a mature miRNA. The 5'-cap can typically be formed by a modified nucleotide, in particular a derivative of a guanine nucleotide. In some cases, the 5'-cap can be linked to the 5'-end of a DUX4-targeting oligonucleotide via a 5'-5'-triphosphate linkage. The 5'-cap can be methylated, for example m7GpppN, where N can be the terminal 5'-nucleotide of the nucleic acid that carries the 5'-cap, such as the 5'-end of an RNA. 5'-Cap structures include: glyceryl, inverted deoxy abasic residue (moiety), 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydro hydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or a bridged or non-bridged methylphosphonate moiety.In some cases, the modified 5'-CAP structure can include: CAP1 (methylation of the ribose of the nucleotide adjacent to m7G), CAP2 (methylation of the ribose of the second nucleotide downstream of m7G), CAP3 (methylation of the ribose of the third nucleotide downstream of m7G), CAP4 (methylation of the ribose of the fourth nucleotide downstream of m7G), ARCA (anti-reverse CAP analog), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.

[0066] In some cases, oligonucleotides, such as DUX4-targeting oligonucleotides, can include covalent modifications that can include adding a methyl group, a hydroxymethyl group, a carbon atom, an oxygen atom, or any combination thereof to one or more bases of a nucleic acid sequence. In some cases, the covalent modification can include changing the oxidation state of a molecule associated with the nucleic acid sequence, such as an oxygen atom, or a combination thereof. The covalent modification can be performed on any base, such as cytosine, thymine, uracil, adenine, guanine, or a combination thereof. In some cases, the epigenetic modification can include oxidation and can also include reduction. The nucleic acid sequence can include one or more epigenetically modified bases. The epigenetically modified bases can include any base, such as cytosine, uracil, thymine, adenine, or guanine. The epigenetically modified bases can include methylated bases, hydroxymethylated bases, formylated bases, or carboxylic acid-containing bases or salts thereof. The epigenetically modified bases can include 5-methylated bases, such as 5-methylated cytosine (5-mC). The epigenetically modified base can include a 5-hydroxymethylated base, such as 5-hydroxymethylated cytosine (5-hmC). The epigenetically modified base can include a 5-formylated base, such as 5-formylated cytosine (5-fC). The epigenetically modified base can include a 5-carboxylated base, such as 5-carboxylated cytosine (5-caC), or a salt thereof. In some cases, the epigenetically modified base can include a methyltransferase-directed transfer of an activating group (mTAG).

[0067] The epigenetically modified bases can include one or more bases or purines (as in Structure 1) or one or more bases of pyrimidines (as in Structure 2). The epigenetic modification can be made at one or more of any position. For example, the epigenetic modification can be made at one or more of the purines, including positions 1, 2, 3, 4, 5, 6, 7, 8, 9, as shown in Structure 1. In some cases, the epigenetic modification can be made at one or more of the pyrimidines, including positions 1, 2, 3, 4, 5, 6, as shown in Structure 2.

[0068] [ka]

[0069] The nucleic acid sequence can include epigenetically modified bases. The nucleic acid sequence can include multiple epigenetically modified bases. The nucleic acid sequence can include epigenetically modified bases located within CG sites, CpG islands, or combinations thereof. The nucleic acid sequence can include a variety of epigenetically modified bases, such as methylated bases, hydroxymethylated bases, formylated bases, carboxylic acid-containing bases or salts thereof, any of a plurality of these, or any combination thereof.

[0070] In some cases, when chemically modified, the DUX4-targeting oligonucleotide or salt thereof can be of the formula: guide pattern 1, guide pattern 2, or guide pattern 3, as shown in Table 1.

[0071] [Table 1]

[0072] As shown in Table 1, N and n can be any nucleotide, natural or unnatural; {N} can be an LNA; [N] can be a BNA; <n>can be a 2'-methyloxyethyl modified uracil, guanine, adenine, or cytosine; * can be a phosphothionate modified backbone; mp can be a methylphosphonate modified backbone; CAP can be an alkylamino group such as a 5'-terminal methyl group (5'-Omethyl) or an amino-carbon 6 chain (5'-aminoC6); a can be 10-26; b can be 8-24; c can be 4-20; d can be 5-22; e can be 9-25.

[0073] In some cases, oligonucleotides such as DUX4-targeting oligonucleotides can include chemical modifications to the base or sugar of the DUX4-targeting oligonucleotide compared to the natural base or sugar. In some cases, DUX4-targeting oligonucleotides can include two or more chemical modifications, such as multiple chemical modifications. A portion of the base or a portion of the sugar of the DUX4-targeting oligonucleotide can include one or more chemical modifications. In some cases, about: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the base or sugar of the DUX4-targeting oligonucleotide can be chemically modified.

[0074] In some cases, the DUX4-targeting oligonucleotide may be engineered or modified to improve specificity for the RNA sequence of the plurality of RNA sequences. The DUX4-targeting oligonucleotide may be modified to significantly improve specificity for the RNA sequence of the plurality of RNA sequences. The improved specificity may be compared to a corresponding oligonucleotide that may not be engineered, or may be compared to a corresponding oligonucleotide that may be engineered or modified in a different way. The specificity may be improved by at least about: 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the corresponding oligonucleotide. The DUX4-targeting oligonucleotide may be engineered or modified to improve specificity for a first RNA sequence compared to a second RNA sequence.

[0075] DUX4-Targeting Oligonucleotide Research and Discovery To identify the target DUX4 variant, the identity between the reference sequence (query sequence, i.e., the sequence described herein) and the subject sequence, also called global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some cases, the parameters for certain embodiments in which identity is interpreted narrowly, used in the FASTDB amino acid alignment, include: scoring scheme=PAM (percentage of mutations allowed) 0, k-tuple=2, mismatch penalty=1, linkage penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, but not due to internal deletions, the results can be manually corrected to take into account that the FASTDB program does not account for the N- and C-terminal truncations of the subject sequence when calculating the global percent identity. For subject sequences that are N- and C-terminally truncated compared to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that lie at the N- and C-termini of the subject sequence that are not matched / aligned with the corresponding subject residues as a percentage of the total bases of the query sequence. The determination of whether a residue is matched / aligned can be determined by the results of FASTDB sequence alignment. This percentage can then be subtracted from the percent identity calculated by the FASTDB program using the specified parameters to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only the residues to the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score.That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence are considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, so the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since the 10 unpaired residues are 10% of the sequence (number of residues at the N- and C-terminus that are not matched / total number of residues in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90-residue subject sequence is compared with a 100-residue query sequence. In this case, the deletion is an internal deletion, so there are no residues at the N- or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected, again only those residue positions outside the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence, as displayed in the FASTDB alignment, are manually corrected.

[0076] To evaluate all the different OTN positions, windows of size 15bp, 16bp, 17bp, 18bp, 19, and 20bp were created with 1bp slides within the reference sequence spanning the DUX4 gene chr4:190,173,774-190,185,942. For each window, the reverse complement (antisense) sequence of the reference was also reported, so this could be directly used for OTN design. The RNA-Seq BAM files of all samples were merged into a single BAM file using the Pysamstats vl.1.2 tool https: / / github.com / alimanfoo / pysamstats, and a custom Python script was used to obtain the reference base frequency and read depth at each genomic position in the merged BAM file. The average coverage was defined as the average number of reads covering each base in the window. A minimum conservation score was calculated for each OTN window representing the least conserved base. Mean melting temperatures (Tm) were calculated for the resulting OTN / target RNA duplexes using the Primer3 v2.4.0 R tool (39) with default parameters and using the nearest neighbor model. Two melting temperature (TM) values ​​were reported based on different salt correction formulas defined by SantaLucia 1998 (40) and Owczarzy et al. 2004 (41). We then filtered the data for OTN and OTN binding sites of DUX4 of 15-20 bp in length with the following criteria: average coverage >50, minimum conservation >85% among individuals in this study, and average TM 45-65°C. All resulting OTN sequences and paired DUX4 target site sequences, all represented in DNA form, are submitted as a sequence listing file encompassing SEQ ID NOs: 1-2X,XXX. We have included them in this disclosure as they will be a useful resource for any efforts to develop OTN to treat DUX4-mediated disorders. These DUX4-targeting oligonucleotides or salts thereof, when chemically modified or unmodified, can have at least 90% sequence identity with any one of SEQ ID NOs: 41,923 to 42,115.In certain cases, the DUX4-targeting oligonucleotide or its salt can have at least about 80% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115. For example, the DUX4-targeting oligonucleotide or its salt can have at least about 90% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide or its salt can have at least about 80%-100% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide or its salt can have at least about 85%-100% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide or its salt can have at least 80% sequence identity with at least about 10 consecutive bases of any one of SEQ ID NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide or salt thereof can comprise at least 85% sequence identity with at least about 10 consecutive bases of any one of SEQ ID NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide can comprise at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or any combination thereof, with any one of SEQ ID NOs: 41,923-42,115.

[0077] Additionally, the analysis provided the ability to generate a large number of DUX4-targeting oligonucleotides without (SEQ ID NOs: 41,923-41,982) and with (SEQ ID NOs: 41,983-42,115) chemical modifications, all represented in DNA form, as shown in Table 2. For chemically modified DUX4-targeting oligonucleotides, as shown in Table 2, {N} can be an LNA; [N] can be a BNA; (N) can be a 2'-methyloxyethyl modified uracil, guanine, adenine, or cytosine; * can be a phosphothionate modified backbone; mp can be a methylphosphonate modified backbone; aminoC6- can be a 5'-amino-carbon6 strand. In addition, as shown in Table 2, certain DUX4-targeting oligonucleotides could interact with multiple subsequences of the target DUX4 mRNA as shown in Table 3, also submitted in an xml file. In addition, any chemically modified oligonucleotide, even if not shown in the table, could be synthesized with a 5'amino-carbon6 strand while retaining activity. Although the additional target RNAs are only listed next to the unmodified sequence of the oligonucleotide, and the additional target RNAs are not repeated with chemically modified versions of the same sequence, they would still be targeted by that sequence.

[0078] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

[0079] In the far right column of Table 2 are RNAs that are partially complementary to the listed DUX4-targeted oligonucleotides, but originate from different loci. Using a modified script from GGGenome (https: / / gggenome.dbcls.jp / ), we allow for rapid alignment of our oligonucleotide sequences to the human transcriptome (Human RNA Refseq release 205, March 2021). This script identified all transcripts that are partially complementary to each potential DUX4-targeted oligonucleotide, but contain no more than four mismatches, bulges, insertions or deletions, contain two regions of complementarity at least seven consecutive bases in length, or contain one region at least ten consecutive bases in length. These interactions can also have a predicted TM of about 40°C to about 65°C.

[0080] In addition, to understand which other transcripts may be relevant to FSHD and may be advantageous to target, we have assembled a database of 10 studies with strict criteria for sample handling, transcriptome profiling by microarray and RNAseq, and important patient information. We identified genes that are commonly upregulated in FSHD versus control muscles within public datasets or using our own RNA-seq analysis. Interestingly, the clusters match well with clinical severity scores (i.e., mild, moderate, or severe disease). In support of our analysis, a similar analysis from a subset of samples included in our larger meta-analysis yielded similar results, as shown in Figure 11. From this analysis, we have created a database of upregulated genes in FSHD, while maintaining the supporting evidence of this dysregulation and any relevant clinical correlations with each gene. From this database, we then performed pathway enrichment analysis using GO pathway analysis (12). The most upregulated pathways include inflammatory response and other immune regulatory pathways, cell proliferation, and cell cycle regulation. Additional targeted RNAs represent upregulated or otherwise disease-associated transcripts that may be advantageous for knockdown in addition to DUX4.

[0081] In addition, numerous mRNA subsequences of additional genes were associated with FSHD. For example, AS-DX-007 (SEQ ID NO: 23,789) is predicted to target three co-targets associated with FSHD, such as DBET, MKI67, and IRF5. DBET is a non-coding RNA associated with the opening of D4Z4 repeats and the expression of DUX4 (38). MKI67 encodes the Ki-67 protein, which is upregulated in FSHD muscle tissue, and may be involved in DUX4 induction of myofibroblast proliferation and injury. (Figure 12A). IRF5 (interferon regulatory factor 5) encodes a transcription factor that is upregulated by several inflammatory signals, and leads to the expression of several cytokines, such as TNF, and the induction of intracellular interferon responses (Figure 12B). Target subsequences within the mRNA transcripts of these genes (including that of DUX4 itself) are provided herein in Table 3, as shown in RNA form.

[0082] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16]

[0083] Consequences of DUX4-targeting oligonucleotide interaction with RNA targets In some cases, a DUX4-targeting oligonucleotide or salt thereof containing a modification when contacted with a DUX4 mRNA sequence can produce a polypeptide encoded by the DUX4 mRNA sequence that is less active than an equivalent amount of an otherwise equivalent oligonucleotide lacking the modification when contacted with the DUX4 mRNA sequence. In some cases, the less activity can be about 1.2-fold or less. In some cases, the less activity can be about 1.5-fold or less. In some cases, the less activity can be about 1.7-fold or less. In some cases, the less activity can be about 2.0-fold or less. In some cases, the less activity can be about: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5-fold less. In some cases, the less activity can be about 1.2-fold to about 2.0-fold less. In some cases, the lower activity can be about 1.1 to about 1.5 fold lower. In some cases, the lower activity can be about 1.1 to about 2.5 fold lower. In some cases, the lower activity can be about 1.2 to about 3.0 fold lower. In some cases, the lower activity can be about 1.2 to about 10 fold lower expression. In some cases, the lower activity can be about 14 to about 14 fold lower. In some cases, the lower expression can be about 18 to about 18 fold lower expression. In some cases, the lower activity can be about 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold lower. In some cases, the lower activity can be about 1.2 to about 14 fold lower. In some cases, the lower activity can be from about 1.1-fold to about 20-fold lower. In some cases, the lower activity can be from about 1.2-fold to about 30-fold lower.

[0084] In some cases, a DUX4-targeting oligonucleotide or salt thereof when contacted with an mRNA sequence can produce about: 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold lower expression of a polypeptide encoded by the mRNA sequence compared to contacting an equal amount of an otherwise equivalent oligonucleotide lacking the modification with the mRNA sequence. Lower expression can be from about 1.2 fold to about 10 fold lower expression.

[0085] In some cases, a DUX4-targeting oligonucleotide or salt thereof when contacted with an mRNA sequence can produce about: 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold less activity of a polypeptide encoded by the mRNA sequence compared to contacting an equal amount of an otherwise equivalent oligonucleotide lacking the modification with the mRNA sequence. The lesser activity can be from about 1.2-fold to about 10-fold less expression.

[0086] In some cases, the DUX4-targeting oligonucleotide or salt thereof can include a predicted thermal melting temperature at physiological salt and pH of at least about 45-65° C. In some cases, the DUX4-targeting oligonucleotide or salt thereof can bind to an RNA sequence at about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C. In some cases, the DUX4-targeting oligonucleotide or salt thereof can bind to an RNA sequence at a pH of about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, or 7.8.

[0087] subject In some embodiments, the subject may include a mammal that is adapted to receive the compositions described herein that include engineered DUX4-targeting nucleic acids (such as in the form of oligonucleotides) or that is treated by the methods described herein. Examples of such mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). The mammal may be of any age or at any stage of development, for example, the mammal may be neonatal, infant, adolescent, adult, or in utero. The mammal may be male or female. In some cases, the human can be about: 1 day to about 7 days old, 1 week to about 5 weeks old, 1 month to about 12 months old, 1 year to about 6 years old, 5 years to about 15 years old, 14 years to about 30 years old, 25 years to about 50 years old, 40 years to about 75 years old, 70 years to about 100 years old, 85 years to about 110 years old, or about 100 years to about 130 years old.

[0088] In some cases, the subject may not have been previously diagnosed with a disease or condition. In some cases, the subject may have been diagnosed with a disease or condition. In some cases, the subject may not have received a definitive diagnosis of a disease or condition. The subject may be at risk of developing a disease or condition (such as based at least in part on genetic variants). The subject may have undergone a diagnostic test. Diagnostic tests include imaging, blood count analysis, histopathological analysis, biomarker analysis, or any combination thereof.

[0089] The subject can be a patient, such as a patient undergoing treatment for a condition or disease, such as a neuromuscular disease.In certain cases, the subject may be at risk of developing a condition or disease, such as a neuromuscular disorder.The subject may be in remission from a condition or disease, such as a neuromuscular disorder.The subject may be healthy.

[0090] In some embodiments, the subject can be a subject in need thereof. In some embodiments, the subject can have a disease in which treating facioscapulohumeral muscular dystrophy (FSHD) includes, for example, alleviating the muscle weakness experienced by a mammal suffering from facioscapulohumeral muscular dystrophy (FSHD) and / or causing regression or disappearance of muscle weakness.

[0091] Administration and Treatment In some embodiments, the DUX4-targeting oligonucleotides disclosed herein can be used to treat a subject to result in: reduced fatigue, increased energy, weight gain, weight loss, increased muscle mass, improved physical flexibility, improved posture, improved range of motion, relief of muscle tone, relief of muscle pain, or any combination thereof.

[0092] The subject in need thereof can be treated for disease or condition.The treatment can be pretreatment, prophylactic treatment, or preventive treatment.The treatment includes administering to the subject in need thereof the DUX4-targeting oligonucleotide, nucleic acid construct, vector, or pharmaceutical composition described herein.

[0093] Treating includes administering to the patient an oligonucleotide targeting a highly conserved engineered DUX-4, as well as an oligonucleotide targeting an engineered DUX-4 selected from SEQ ID NOs: 20,962-41,922 and / or SEQ ID NOs: 41,923-42,115 shown in Table 2 of the XML sequence listing file submitted at the time of filing, or any combination thereof.

[0094] Delivery includes direct application to the affected tissue or area of ​​the body. Delivery includes intraparenchymal, intrathecal, intraventricular, or intracapsular injection. The compositions provided herein can be administered by any method. The administration method can be by inhalation, intraarterial, intraventricular, intracapsular, intramuscular, intraorbital, intraparenchymal, intraperitoneal, intrathecal, intravenous, intraventricular, stereotactic, subcutaneous, or any combination thereof. Delivery includes parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular, or infusion), oral administration, inhalation administration, intraduodenal administration, and rectal administration. Delivery includes topical administration (such as lotions, creams, ointments) to external surfaces such as the skin. In some cases, the subject can administer the composition even in the absence of a caregiver. In some cases, the subject may administer the pharmaceutical preparation under the supervision of a medical professional (e.g., a doctor, a nurse, a physician's assistant, a hospital janitor, a hospice worker, etc.). In some cases, the pharmaceutical preparation may be administered by a medical professional. In some cases, the treatment of a neuromuscular disease, such as facioscapulohumeral muscular dystrophy, is by using a composition comprising a DUX4-targeting oligonucleotide, a vector comprising the oligonucleotide, or a pharmaceutical preparation as described below. Furthermore, a medicament can be manufactured using a DUX4-targeting oligonucleotide, a vector comprising the oligonucleotide, or a pharmaceutical preparation as described below. The medicament may be used to treat or prevent facioscapulohumeral muscular dystrophy.

[0095] The method of administration includes in vivo or in vitro delivery methods. The method includes contacting a cell, such as a cell in vivo, with a DUX4-targeting oligonucleotide, a nucleic acid construct, a vector, or a pharmaceutical composition described herein. The method includes contacting a cell, such as an isolated and purified cell (such as a cell in vitro), with a DUX4-targeting oligonucleotide, a nucleic acid construct, a vector, or a pharmaceutical composition described herein. The method includes contacting a tissue, such as a tissue in vivo or an isolated tissue in vitro, with a DUX4-targeting oligonucleotide, a nucleic acid construct, a vector, or a pharmaceutical composition described herein.

[0096] Treatment includes two or more DUX4-targeting oligonucleotides delivered in a single dose.Delivery can be simultaneous, such as delivery of two or more DUX4-targeting oligonucleotides in one injection or in two separate injections at the same time.Delivery can be sequential, such as delivery of a first dose and a second dose that can be separated by a period of time, such as minutes, hours, days, weeks, or months.

[0097] Certain aspects of the present disclosure relate to the administration of DUX4-targeting oligonucleotides.The human cell can be a head and neck tissue cell, a skin cell, a cervical cell, a prostate cell, a stem cell, a bone cell, a blood cell, a muscle cell, a fat cell, a nerve cell, an endothelial cell, a sperm cell, an egg cell, a cancer cell, a barrier cell, a hormone secreting cell, an exocrine cell, an epithelial cell, an oral cell, a sensory transduction cell, an autonomic nerve cell, a peripheral nerve cell, a central nerve cell, a secretory cell, a cardiac muscle cell, a white blood cell, a germ cell, a nurse cell, a kidney cell, or any combination thereof.

[0098] The tissue can be a sample that may be substantially healthy, substantially benign, or otherwise substantially free of disease or condition. The tissue can be tissue removed from a subject, such as a tissue biopsy, tissue resection, aspiration (such as fine needle aspiration), tissue washing, cytology specimen, bodily fluid, or any combination thereof. The tissue can include cancerous cells, tumor cells, non-cancerous cells, or a combination thereof. The tissue can include a blood sample (such as a cell-free DNA sample). The tissue can be a sample that may be genetically modified.

[0099] Treatment includes treatment of conditions associated with neuromuscular diseases such as facioscapulohumeral muscular dystrophy. Treatment can result in reduced fatigue, increased energy, weight gain, weight loss, increased muscle mass, improved physical flexibility, improved posture, improved range of motion, release of muscle tension, relief of muscle pain, or any combination thereof.

[0100] Certain aspects of the present disclosure relate to the delivery of oligonucleotides, such as DUX4-targeting oligonucleotides, using vectors. Vectors can be used to deliver DUX4-targeting oligonucleotides, nucleic acid constructs, or any combination thereof. The vectors can include DNA, such as double-stranded DNA or single-stranded DNA. The vectors can include RNA. In some cases, the RNA can include base modifications. The vectors can include recombinant vectors. The vectors can be vectors that are modified from naturally occurring vectors. The vectors can include at least a portion of a non-naturally occurring vector. In some cases, the vectors can include viral vectors, liposomes, nanoparticles, exosomes, extracellular vesicles, or any combination thereof. In some cases, the viral vectors can include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, retroviral vectors, any portion of any of these, or any combination thereof. In some cases, the nanoparticle vectors can include polymer-based nanoparticles, amino lipid-based nanoparticles, metal nanoparticles (such as gold-based nanoparticles), any portion of any of these, or any combination thereof. In some cases, the vectors can include AAV vectors. The vector can be modified to include a modified VP1 protein (such as an AAV vector modified to include a VP1 protein). The AAV can include - serotypes, such as AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, derivatives of any of these, or any combination thereof.

[0101] In certain embodiments, the delivery of the oligonucleotide intended to be used as engineered DUX4-targeting oligonucleotide is through liposome delivery. In certain examples, the liposome can be a positively charged liposome. In certain examples, the liposome can be a negatively charged liposome. In other examples, the delivery of the engineered DUX4-targeting oligonucleotide is polymeric delivery. In other examples, the delivery of the engineered DUX4-targeting oligonucleotide is dendrimer-mediated delivery. In other examples, the delivery of the engineered DUX4-targeting oligonucleotide is through microinjection, electroporation, ultrasound, gene gun or hydrodynamic application. In other examples, the delivery of the engineered DUX4-targeting oligonucleotide is through conjugation or association with nanoparticles.

[0102] Pharmaceutical preparations In some embodiments, a wide variety of pharmaceutical formulations for delivery of engineered DUX4-targeting oligonucleotide targets can be used.

[0103] The pharmaceutical formulation can include a pharma- ceutically acceptable excipient, diluent, carrier, or combinations thereof.

[0104] The carrier for the pharmaceutical formulation can in certain cases be a solid carrier and can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, etc. In other cases, the carrier can be a liquid carrier and can include phosphate buffered saline, syrup, oil, peanut oil, olive oil, water, emulsions, wetting agents, sterile solutions, or any combination thereof.

[0105] In some aspects relating to pharmaceutical formulations, the pharmaceutical formulation can include a pharma- ceutically acceptable diluent, which can include, for example, sterile distilled water, deionized water, saline, Ringer's solution, dextrose solution, cell growth medium, phosphate buffered saline (PBS), or any combination thereof.

[0106] In some aspects related to pharmaceutical formulations, the pharmaceutical formulations can include excipients. In examples related to excipients, the excipients can include pH agents, stabilizers, buffering agents, solubilizers, or any combination thereof. The excipients can include surfactants, sugars, amino acids, antioxidants, salts, non-ionic surfactants, solubilizers, triglycerides, alcohols, or any combination thereof. The excipients may include sodium carbonate, acetate, citrate, phosphate, polyethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HCl, disodium edetate, lecithin, glycerin, xanthan gum, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. The excipients may be those described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

[0107] The present disclosure can include salts, including pharma- ceutically acceptable salts, of the compositions described herein.Compounds or compositions of the present disclosure that can possess sufficiently acidic, sufficiently basic, or both functional groups can react with any of several inorganic bases, inorganic acids, or organic acids to form salts.Alternatively, compositions that contain compounds that are essentially charged, such as those that have quaternary nitrogen, can form salts with suitable counterions, for example halides such as bromide, chloride, or fluoride, especially bromide.

[0108] The pharmaceutical composition can include a first active ingredient. The first active ingredient can include a DUX4-targeting oligonucleotide as described herein. The pharmaceutical composition can be formulated in a unit dose form. The pharmaceutical composition can include a pharma- ceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition can include a second, third, or fourth active ingredient, such as a second DUX4-targeting oligonucleotide.

[0109] In some cases, when stored in a sealed container placed in a room for a period of time, the engineered DUX4-targeting oligonucleotide or salt thereof containing the modification will remain at least about 80% of the initial amount of the engineered DUX4-targeting oligonucleotide or salt thereof. In some cases, the engineered DUX4-targeting oligonucleotide will remain at least about 70% of the initial amount. In some cases, the engineered DUX4-targeting oligonucleotide will remain at least about 90% of the initial amount. In some cases, the engineered DUX4-targeting oligonucleotide will remain at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%. In some cases, the engineered nucleotide can be at least about 60% to at least about 80%. In some cases, the engineered nucleotide can be at least about 80% to at least about 99%. In some cases, the storage period can be at least 1 month. In some cases, the storage period can be at least about 3 months. In some cases, the storage period can be at least about 1 year. In some cases, the storage period can be at least about 1, 2, 4, 6, 8, 12, 18, 24, 36, 48, or 60 months. In some cases, the storage period can be at least about 1 month to at least about 1 year. In some cases, the storage period can be at least about 6 months to at least about 2 years. In some cases, the storage period can be at least about 1 month to at least about 5 years.

[0110] In some embodiments, the pharmaceutical composition can be administered to the subject in an appropriate unit dose. The pharmaceutical composition can be in unit dose form. In some cases, a unit dose can refer to a form of pharmaceutical product that contains a specific mixture of active and inactive ingredients, diluents, or excipients, in a specific configuration, and is apportioned to a specific dose to be delivered and sold for use. In some cases, a unit dose can also include non-reusable packaging, although the FDA distinguishes between a "package" and a "preparation" of unit doses. Two or more unit doses can refer to separate pharmaceutical products packaged together, or a single pharmaceutical product that contains multiple drugs and / or doses. In some cases, the term unit dose can also refer to particles that contain the pharmaceutical composition, and any mixtures that are included. In some cases, the type of unit dose varies depending on the route of administration of the drug delivery and the substance delivered. In some embodiments, administration can include intravenous, intraperitoneal, intra-arterial, intratumoral, subcutaneous, intramuscular, intranasal, topical, oral, or intradermal administrations. In some cases, administration can be by inhalation. In some embodiments, the dosing regimen can be determined by the attending physician and clinical factors. In some embodiments, the dosage to a subject can depend on many factors, including the subject's size, body surface area, age, sex, general health, the compound administered, the time and route of administration, other drugs administered simultaneously, or any combination thereof. In some embodiments, the dosage range can include 0.001 to 1000 μg. In some embodiments, the dosage can be below or above such ranges. In some embodiments, the periodic administration regimen of the pharmaceutical composition can range from 1 μg to 10 mg. In some embodiments, the periodic administration regimen of the pharmaceutical composition can range from 10 μg to 10 mg per day, per week, or per month. 2 Units ~ 10 12 The dosage may range from 1 μg to 10,000 mg per kilogram of body weight per minute for the pharmaceutical composition or the engineered polynucleotide or the DNA encoding the engineered polynucleotide or the vector containing or encoding the engineered polynucleotide, respectively, if the regimen includes continuous infusion. In certain instances, the range is from 1 mg per kilogram of body weight to 1000 mg per kilogram of body weight. In some embodiments, progress can be monitored by periodic evaluation.

[0111] In some embodiments of the present disclosure, when the pharmaceutical composition is a liquid, the pharmaceutical composition can be administered in a liquid dosage form, such as: about 1 ml to about 5 ml, about 5 ml to about 10 ml, about 15 ml to about 20 ml, about 25 ml to about 30 ml, about 30 ml to about 50 ml, about 50 ml to about 100 ml, about 100 ml to about 150 ml, about 150 ml to about 200 ml, about 200 ml to about 250 ml, about 250 ml to about 300 ml, about 300 ml to about 350 ml, about 350 ml to about 400 ml, about 400 ml to about 450 ml, about 450 ml to about 500 ml, about 500 ml to about 750 ml, or about 750 ml to about 1000 ml.

[0112] In some embodiments, the compositions described herein can be administered to a subject in need thereof for one or more days. In some embodiments, administration can be for about: 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, or about 31 days. In some embodiments, administration can be for about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 months. In some embodiments, administration can be for about: 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or for about 50 or more years. In some cases, administration can be for the rest of one's life. In some embodiments, the pharmaceutical compositions described herein can be administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days. In some cases, the compositions described herein may be administered on consecutive days or on non-consecutive days.In some cases, the compositions described herein may be administered to a subject more than once per day.In some examples, the compositions described herein may be administered to a subject: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0113] In some embodiments, disclosed herein are methods for using the compositions disclosed herein. In some embodiments, the daily oral dosage regimen can be from about 0.1 milligrams per kilogram (mg / kg) to about 80 mg / kg, from about 0.2 mg / kg to about 30 mg / kg, or from about 0.5 mg / kg to about 15 mg / kg of total body weight. In some embodiments, the daily parenteral dosage regimen can include from about 0.1 mg / kg to about 10,000 mg / kg, from about 0.2 mg / kg to about 5,000 mg / kg, or from about 0.5 mg / kg to about 1,000 mg / kg of total body weight. In some embodiments, the daily topical dosage regimen can be from about 0.1 mg to about 500 mg. In some embodiments, the daily dosage regimen can be from about 0.01 mg / kg to about 1,000 mg / kg per day. In some embodiments, the optimal amount and interval of each dosage of the composition can be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular subject being treated, and preferably, such optimal value can be determined by the method described herein. In some embodiments, the number of doses of the composition given per day for a specified number of days can be ascertained by those skilled in the art using a conventional process of treatment determination testing. In some embodiments, the dosage regimen can be determined by the attending physician and other clinical factors. In some embodiments, the dosage for any one subject can depend on many factors. In some embodiments, factors that affect the dosage can include the subject's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, other drugs being administered at the same time, or any combination thereof. In some embodiments, progress can be monitored by regular evaluation.

[0114] The pharmaceutical composition can be administered. The daily oral dosage regimen can be from about 0.1 milligrams per kilogram (mg / kg) to about 80 mg / kg, from about 0.2 mg / kg to about 30 mg / kg, or from about 0.5 mg / kg to about 15 mg / kg of total body weight. In some embodiments, the daily parenteral dosage regimen can include from about 0.1 mg / kg to about 10,000 mg / kg, from about 0.2 mg / kg to about 5,000 mg / kg, or from about 0.5 mg / kg to about 1,000 mg / kg of total body weight. In some embodiments, the daily topical dosage regimen can be from about 0.1 mg to about 500 mg. In some embodiments, the daily dosage regimen can be from about 0.01 mg / kg to about 1,000 mg / kg per day. In some embodiments, the optimal amount and interval of each dosage of the composition can be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular subject being treated, and preferably, such optimal value can be determined by the method described herein. In some embodiments, the number of doses of the composition given per day for a specified number of days can be ascertained by those skilled in the art using a conventional process of treatment determination testing. In some embodiments, the dosage regimen can be determined by the attending physician and other clinical factors. In some embodiments, the dosage for any one subject can depend on many factors. In some embodiments, factors that affect the dosage can include the subject's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, other drugs being administered at the same time, or any combination thereof. In some embodiments, progress can be monitored by regular evaluation.

[0115] The composition or formulation can be used herein to treat or prevent a neuromuscular disease and includes an engineered DUX4-targeting oligonucleotide configured to hybridize to an RNA comprising a portion of an RNA transcript (the engineered DUX4-targeting oligonucleotide comprises at least about 70% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115), a vector encoding or comprising said oligonucleotide, and a pharmaceutically acceptable: excipient, diluent or carrier. In certain cases, the neuromuscular disease is facioscapulohumeral muscular dystrophy. In other embodiments, the use of an engineered DUX4-targeting oligonucleotide configured to hybridize to an RNA comprising a portion of an RNA transcript (the engineered DUX4-targeting oligonucleotide comprises at least about 70% sequence identity with any one of the oligonucleotides of SEQ ID NOs: 41,923-42,115), and a pharmaceutically acceptable: excipient, diluent or carrier may be required in the manufacture of a medicament for the treatment and prevention of facioscapulohumeral muscular dystrophy.

[0116] Combination therapy In some embodiments, a method is disclosed herein for administering a DUX4-targeting oligonucleotide or salt thereof in combination with a combination therapy to a subject. In some embodiments, one or more additional combination therapies can be administered simultaneously. In some embodiments, one or more additional therapeutic agents can be administered sequentially. In some cases, the combination therapy can include immunotherapy, hormonal therapy, cryotherapy, surgical procedures, or any combination thereof. The combination therapy can include administration of a pharmaceutical composition, such as a small molecule. The combination therapy can include administration of a pharmaceutical composition, such as one or more antibiotics. The combination therapy can include administration of a muscle relaxant, an antidepressant, a steroid, an opioid, a cannabis-based therapeutic agent, acetaminophen, a nonsteroidal anti-inflammatory agent, a neuropathic agent, cannabis, a progestin, a progesterone, or any combination thereof. The neuropathic agent can include gabapentin. The nonsteroidal anti-inflammatory agent can include naproxen, ibuprofen, a COX-2 inhibitor, or any combination thereof. The second therapy may include administration of a biopharmaceutical agent, cell therapy, regenerative medicine therapy, tissue engineering approach, stem cell transplantation, or any combination thereof. The combination therapy may include medical treatment. The medical treatment may include epidural injections (such as steroid injections), acupuncture, exercise, physical therapy, ultrasound, surgical therapy, chiropractic manipulation, osteopathic manipulation, chemonucleolysis, or any combination thereof. The combination therapy may include use of a respiratory support device or a ventilator. The combination therapy may include administration of a regenerative or immunotherapy, such as a protein, stem cells, cord blood cells, cord tissue, tissue, or any combination thereof. The second therapy may include an anti-inflammatory or anti-fibrotic compound, such as pirfenidone, nintedanib, tocilizumab, mycophenolate mofetil / prednisone mycophenolate, azathioprine, or a combination thereof. The second therapy may include a biosimilar.

[0117] In some aspects, when the combination therapy is a pharmaceutical agent, the pharmaceutical agent is included in the pharmaceutical composition in the form of a fixed dose combination.

[0118] In some cases, the combined therapeutic dosage regimen can be administered for a period of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or about 12 weeks.In some cases, the dosage regimen can be administered for a period of about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 12 months.In some cases, the dosage regimen can be administered for a period of about 1 year, about 2 years, or more than about 3 years.

[0119] In some embodiments, methods of use for the combination therapy compositions disclosed herein are disclosed herein. In some embodiments, the daily oral dosage regimen can be from about 0.1 milligrams per kilogram (mg / kg) to about 80 mg / kg, from about 0.2 mg / kg to about 30 mg / kg, or from about 0.5 mg / kg to about 15 mg / kg of total body weight. In some embodiments, the daily parenteral dosage regimen can include from about 0.1 mg / kg to about 10,000 mg / kg, from about 0.2 mg / kg to about 5,000 mg / kg, or from about 0.5 mg / kg to about 1,000 mg / kg of total body weight. In some embodiments, the daily topical dosage regimen can be from about 0.1 mg to about 500 mg. In some embodiments, the daily dosage regimen can be from about 0.01 mg / kg to about 1,000 mg / kg per day. In some embodiments, the optimal amount and interval of each dosage of the composition can be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular subject being treated, and preferably, such optimal value can be determined by the method described herein. In some embodiments, the number of doses of the composition given per day for a specified number of days can be ascertained by those skilled in the art using a conventional process of treatment determination testing. In some embodiments, the dosage regimen can be determined by the attending physician and other clinical factors. In some embodiments, the dosage for any one subject can depend on many factors. In some embodiments, factors that affect the dosage can include the subject's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, other drugs being administered at the same time, or any combination thereof. In some embodiments, progress can be monitored by regular evaluation.

[0120] In some embodiments, the combination therapy described herein can be administered to a subject in need thereof for one or more days. In some embodiments, administration can be for about: 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, or about 31 days. In some embodiments, administration can be for about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 months. In some embodiments, administration can be for about: 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, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or for about 50 or more years. In some cases, administration can be for the rest of one's life. In some embodiments, the pharmaceutical compositions described herein can be administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more days. In some cases, the compositions described herein may be administered on consecutive days or on non-consecutive days.In some cases, the compositions described herein may be administered to a subject more than once per day.In some cases, the compositions described herein may be administered to a subject: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0121] In some embodiments, the periodic administration regimen of the pharmaceutical composition can range from 1 μg to 10 mg. In some embodiments, the periodic administration regimen of the pharmaceutical composition can range from 10 mg per day, per week, or per month. 2 Units ~ 10 10 The dosage can range from 1 μg to 10,000 mg of the drug, in some embodiments, where the regimen includes continuous infusion. In certain instances, the range is from 1 mg per kg of body weight to 1000 mg per kg of body weight. In some embodiments, progress can be monitored by periodic evaluation.

[0122] kit The kit can include a DUX4-targeting oligonucleotide in a container, a nucleic acid construct in a container, a vector in a container, or a pharmaceutical composition in a container. The kit can include two or more DUX4-targeting oligonucleotides in a container, two or more vectors in a container, two or more nucleic acid constructs in a container, or two or more pharmaceutical compositions in a container. In some cases, the container can be a plastic container, a glass container, or a metal container. The container can include a syringe, a vial, an ampoule, a bag, a jar, or the like.

[0123] The kit may include multiple containers, each container containing one or more DUX4-targeting oligonucleotides, or nucleic acid constructs, or vectors, or pharmaceutical compositions. The kit may include an excipient or diluent or buffer or liquid or gel-like medium for storage of the DUX4-targeting oligonucleotides, nucleic acid constructs, vectors, or pharmaceutical compositions. The kit may include an excipient or diluent or buffer or liquid or gel-like medium for in vivo delivery of the DUX4-targeting oligonucleotides, nucleic acid constructs, vectors, or pharmaceutical compositions to a subject. The excipient or diluent or buffer or liquid or gel-like medium may be included in the container that contains the DUX4-targeting oligonucleotides (or nucleic acid constructs or vectors or pharmaceutical compositions) or may be contained in a separate container. The kit may include a delivery vehicle, such as a syringe or needle. The kit may include one or more reagents for downstream analysis.

[0124] In some cases, when the DUX4-targeting oligonucleotide or salt thereof may be stored in a sealed container placed in a room at about 21 to about 25° C. (e.g., about 21, 22, 23, 24, 25° C.) for a period of at least about: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, with a relative atmospheric humidity of about 45% to about 55% (such as about: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%), at least about: 70%, 75%, 80%, 85%, 90%, 95% of the initial amount of the DUX4-targeting oligonucleotide or salt thereof remains. In some cases, the period can be from about 1 month to about 1 year. In some cases, the period can be from about 1 month to about 2 years. In some cases, the period can be from about 1 month to about 6 months. In some cases, the period can be from about 1 month to about 3 years. In some cases, the period can be from about 1 month to about 9 months.

[0125] diagnosis In some cases, the method can further include diagnosing the subject as suffering from the disease. In some cases, diagnosing can include using an in vitro diagnostic method. In some cases, the in vitro diagnostic method can be a companion diagnostic method. In other examples, diagnosing can include an in vivo diagnostic method.

[0126] Diagnostic tests can include imaging, blood count analysis, histopathology analysis, biomarker analysis, biopsy, magnetic resonance imaging, physical examination, urinalysis, ultrasound, genetic testing, liver function tests, positron emission tomography, x-ray, serology, angiography, electrocardiography, endoscopy, diagnostic polymerase chain reaction test (PCR), Pap smear, hematocrit test, skin allergy test, urinalysis, colonoscopy, enzyme-linked immunosorbent assay (ELISA), microscopic analysis, bone marrow test, rapid diagnostic test, pregnancy test, visceral function test, toxicology test, infectious disease test, body fluid test, or any combination thereof.

[0127] Computer Control System The present disclosure provides a computer control system programmed to carry out the method of the present disclosure. Figure 15 shows a computer system 101 programmed or otherwise configured to predict or confirm the efficacy of various constructs for therapeutic effects, such as in the treatment of FSDH. The computer system 101 can regulate various aspects of the present disclosure, such as modeling or identifying constructs for various therapeutic targets, modeling efficacy or stability of constructs, or any combination thereof. The computer system 101 can be a user's electronic device or a computer system located remotely with respect to the electronic device. The electronic device can be a mobile electronic device.

[0128] The computer system 101 comprises a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 105, which may be a single-core processor, a multi-core processor, or multiple processors for parallel processing. The computer system 101 also comprises memory or memory locations 110 (e.g., random access memory, read-only memory, flash memory), electronic storage 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripherals 125, such as cache, other memory, data storage, and / or electronic display adapters. The memory 110, storage 115, interface 120, and peripherals 125 are in communication with the CPU 105 through a communication bus (solid lines), e.g., a motherboard. The storage 115 may be a data storage device (or data repository) for storing data. The computer system 101 may be operatively coupled to a computer network ("network") 130 with the aid of the communication interface 120. Network 130 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. Network 130 may in some cases be a telecommunications and / or data network. Network 130 may include one or more computer servers, thereby enabling distributed computing, such as cloud computing. Network 130 may in some cases implement a peer-to-peer network, assisted by computer system 101, thereby enabling devices connected to computer system 101 to operate as clients or servers.

[0129] CPU 105 can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 110. The instructions may be issued to CPU 105, which may then be programmed or otherwise configured to perform the methods of the present disclosure. Examples of operations performed by CPU 105 may include fetch, decode, execute, and writeback.

[0130] The CPU 105 may be part of a circuit, such as an integrated circuit. One or more other components of the system 101 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0131] The storage device 115 can store files, such as drivers, libraries, and saved programs. The storage device 115 can store user data, such as user settings and user programs. The computer system 101 can include one or more additional data storage devices, in some cases external to the computer system 101, such as on a remote server in communication with the computer system 101 through an intranet or the Internet.

[0132] Computer system 101 can communicate with one or more remote computer systems through network 130. For example, computer system 101 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a phone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 101 through network 130.

[0133] The methods described herein may be performed in the form of machine (e.g., computer processor) executable code stored in electronic storage locations of computer system 101, such as memory 110 or electronic storage 115. Machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by processor 105. In some cases, the code may be stored in memory 110 so that it can be retrieved from storage 115 and immediately accessed by processor 105. In some situations, electronic storage 115 may be omitted, with machine executable instructions stored in memory 110.

[0134] The code may be pre-compiled and configured for use by a machine having a processor arranged to execute the code, or may be compiled during run-time. The code may be provided in a programming language that may be selected to allow the code to be executed in a pre-compiled or as-compiled manner.

[0135] Aspects of the systems and methods provided herein, such as the computer system 101, can be embodied in programming. Various aspects of the technology can be considered a "product" or "article of manufacture," which typically takes the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage at any time for software programming. All or a portion of the software can sometimes be communicated over the Internet or various other telecommunications networks. For example, such communication can enable loading of the software from one computer or processor to another, e.g., from an administrative server or host computer to the computer platform of an application server. Thus, other types of media that may carry software elements include light waves, radio waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical terrestrial networks, and across various air links. The physical elements that carry such waves, e.g., wired or wireless links, optical links, etc., may also be considered media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0136] Thus, a machine-readable medium such as a computer executable code can take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of a computer, such as those that can be used to implement the databases, etc., shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that make up a bus in a computer system. Carrier wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium having a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer can read programming code and / or data, etc. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0137] The computer system 101 may include or be in communication with an electronic display 135 that includes a user interface (UI) 140 for providing, for example, one or more results (either immediate results or archived results from a previous method), one or more user inputs, reference values ​​or derivatives thereof from a library or database, or any combination thereof. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0138] In some cases, as shown in FIG. 15, a sample 202 containing genetic material can be obtained from a subject 201, such as a human subject. The sample 202 can be subjected to one or more methods described herein, such as performing an assay. In some cases, the assay can include sequencing (such as nanopore sequencing), genotyping, hybridization, amplification, labeling, or any combination thereof. One or more results from the method can be input to a processor 204. One or more input parameters, such as sample identification, subject identification, sample type, standards, or other information, can be input to the processor 204. One or more metrics from the assay can be input to the processor 204 so that the processor can generate a result, such as a recommendation for the diagnosis or treatment of a neuromuscular disease. The processor can transmit the result, the input parameters, the metrics, the standards, or any combination thereof to a display 205, such as a visual display or a graphical user interface. The processor 204 may (i) send results, input parameters, metrics, or any combination thereof to the server 207, (ii) receive results, input parameters, metrics, or any combination thereof from the server 207, or (iii) or any combination thereof.

[0139] The methods and systems of the present disclosure can be implemented in the form of one or more algorithms. The algorithms can be implemented in the form of software when executed by the central processing unit 105. The algorithms can determine optimized constructs, for example, via supervised learning, to optimize therapeutic efficacy, stability, or other attributes of one or more constructs. EXAMPLES

[0140] DUX4 sequences from skeletal muscle samples Analysis was performed from RNA-seq data of a total of 95 skeletal muscle samples, of which 70 were from FSHD patients and 25 from healthy individuals. The samples used were from three publicly available datasets: Yao et al. 2014 (28), Wong et al. 2020 (17), and Wang et al. 2019 (29). The results of this analysis are shown in Figure 3. However, these approaches did not succeed in generating enough data to predict RNA sequence variants in patients from the majority of the DUX4 coding sequence due to the fact that DUX4 is expressed at such low levels that only one or two reads were identified per sequenced sample. To confidently predict sequence variants between individuals, 50–100× the number of reads per sample is usually required. Only a small region of DUX4 located in exon 1 met this criterion. EXAMPLES

[0141] DUX4 sequences from testis samples We decided to test this negative hypothesis and analyzed RNA-seq data of testis samples from 206 individuals (30). Unexpectedly, this dataset was sufficient to predict variability across exons 1, 2, and 3 of muscle-specific transcripts with an average coverage across the sequence of 117× (Figure 4). From this dataset, we demonstrated promise for this approach, with several regions of the DUX4 coding sequence conserved at >85%. However, several regions of DUX4, likely due to differences in splice isoforms, did not yet have sufficient coverage to accurately predict conservation. EXAMPLES

[0142] DUX4 sequences from an integrated database of muscle and testis samples To solve this problem, we took a very unique and unprecedented approach, integrating muscle RNA-seq and testis RNA-seq into one combined dataset and performed the analysis. In this final analysis, we were able to obtain 486 testis samples from GTEX and utilized 95 skeletal muscle samples from Example 1. This final analysis generated the best data resulting in >50x read coverage for over 97% of the DUX4 gene, allowing accurate prediction of DUX4 gene site and OTN pairs that are conserved in over 85% of patients (Table 4). As mentioned above, all resulting OTN sequences and paired DUX4 target site sequences, all represented in DNA form, have been submitted as xml files encompassing SEQ ID NOs: 1-41,922. This data will be useful in identifying suitable intervals of 15-25 bases within the DUX4 sequence that are conserved in the majority of FSHD patients and in the selection of OTN drug development.

[0143] [Table 4]

[0144] With reference to Table 4, the contiguous sequence encoding DUX4 on chromosome 4q35 is >85% conserved between individuals and may serve as a target site for ONT targeting DUX4 for the treatment of FSHD. The DNA sequence and listed coordinates of DUX4 match Ensemble release 101 (GRCh38.pl3).

[0145] Referring to Figure 1, this diagram illustrates the genetic recombinations that result in FSHD. In FSHD, type 1 is the result of a deletion of D4Z4 repeats on chromosome 4q35 from approximately 100 to less than 11 repeats, resulting in chromosome opening and expression of DUX4. FSHD type 2 is the result of a loss-of-function mutation in the epigenetic factor SMCHD1, resulting in demethylated D4Z4 repeats on 4q35, chromosome opening and expression of DUX4.

[0146] Referring to Figure 2, this figure shows alternatively spliced ​​DUX4 transcripts originating from the D4Z4 region. ENST00000616166.1, ENST00000569241.5, and ENST00000570263.5 are associated with FSHD when expressed in muscle tissue. Transcripts ENST00000565211.1, ENST00000563716.5, and ENST00000564366.1 are normally expressed in other tissues and are not associated with the disorder. For example, ENST00000563716.5 is expressed in the testis.

[0147] Referring to FIG. 3, this figure shows read coverage from RNA-Seq data of alternatively spliced ​​DUX4 transcripts from FSHD and healthy muscle biopsy tissue.

[0148] Referring to Figure 4, this figure shows read coverage from RNA-Seq data of alternatively spliced ​​DUX4 transcripts from testis. The alternatively spliced ​​DUX4 transcripts ENST00000616166.1, ENST00000569241.5, and ENST00000570263.5 are associated with FSHD when expressed in muscle tissue.

[0149] Referring to Figure 5, this figure shows that chemical modification of DUX4 can improve the stability of ASO against biological nucleases by targeting ASO. DUX4-targeted ASOs were incubated in 10% human serum at 37°C for the indicated length of time. The stability of ASOs at each time point was visualized by denaturing urea-PAGE. Unmodified Oligo shows an example of unmodified DNA nucleic acid with extremely low half-life, Neg Con Oligo shows ASOs that do not target DUX4 but have similar chemical modifications, while the other panels show chemically engineered ASOs that target DUX4. An exceptional example shows stability against biological nucleases for up to 7 days (168 hours).

[0150] Referring to Table 5, this table shows the calculated half-lives of chemically modified ASOs targeting DUX4 against biological nucleases. DUX4 ASOs were incubated at 37° C. in 10% human serum for various time points and visualized via urea-PAGE. Concentration measurements were performed at each time point and ASO stability at each time point was calculated according to the formula N (T) =N0(1 / 2) t / t(1 / 2) Calculated and averaged based on, where N (T) is the signal at time t, N0 is the signal at the beginning before incubation with nuclease, and t(1 / 2) is the half-life. Unmodified refers to unchemically modified RNA, while Neg Con Oligo refers to ASO that is chemically modified but does not target DUX4.

[0151] [Table 5]

[0152] Referring to FIG. 6A, this figure shows a reduction in innate immune activation for engineered DUX4 ASO. Human peripheral blood mononuclear cells (PBMCs) (approximately 2-6×10 5 Cells) were plated in round-bottom 96-well plates and transfected with the indicated ASOs at 133 nM concentration using RNAiMAX reagent for 48 hours. Levels of IFN-α (left axis) and TNF-α (right axis) in the supernatant media were quantified by ELISA for six patients. Poly(dA:dT) (Pos Con#1) and immunostimulatory oligonucleotides (Pos Con#2) were utilized as positive controls for immunostimulation. RNAiMAX without oligonucleotides was utilized as a baseline negative control (Baseline), while transfection with non-immunostimulatory RNA (Neg Con) not targeting DUX4 demonstrated low immunostimulation.

[0153] Referring to FIG. 6B, this figure further shows that engineered DUX4 ASOs reduced innate immune activation according to the Raw-Blue cell assay (Invivogen, raw-sp). Briefly, cells were plated at 100,000 cells / well in 150 μL in DMEM (Thermo, 11965092) containing 10% FBS (Thermo, 10082147) in U-bottom 96-well plates (Thermo, 163320). After 24 hours, 22.34 μL of OptiMEM (Thermo, 31985088) was mixed with 2.66 μL (per well) of 10 μM ASO and 1 μL (per well) of Lipofectamine. The Lipofectamine / ASO mixture was then added to each well of Raw Cell to give a final ASO concentration of 133 nM. Poly(dA:dT) (Invivogen, tlrl-patn) @1-10ng / mL, CpG (invivogen, tlrl-1585) @133nM were used as positive controls. Cells are incubated with the transfection / ASO mixture @37 degrees / 5% CO2 for 1 day. After incubation, plates are gently spun at 300xg for 5 minutes, then 20μL from each well is taken and added to a new flat-bottom 96-well plate (VWR, 29442-056). 180uL of QUANTI-Blue (Invivogen, rep-qbs) is added to each well of supernatant and incubated @37 degrees / 5% CO2 for 30 minutes to 6 hours. Absorbance is read at 620-655nm using Cytation5 (Biotek). Data represents the average of six replicate wells, error bars represent standard deviation.

[0154] Referring to FIG. 7, DUX4 ASO HTS assay plan. Stable human or mouse myoblasts expressing eGFP with the coding sequence of DUX4 in the 3'UTR. Constitutive expression of this construct is driven by CMV for strong and ubiquitous expression. The mRNA encoding the eGFP-UTR-DUX4 transcript is transcribed intact and the eGFP sequence is translated. Translation of the toxic DUX4 protein is prevented by a stop codon at the end of the eGFP sequence and a mutation in the start codon of DUX4. ASOs that efficiently target DUX4 can bind to the fusion transcript and induce degradation through RNAse H or RISC, thus preventing the expression of GFP protein. After treatment, assays could be performed by a plate reader or image analysis to observe a decrease in fluorescence in untreated cells and negative control transfections compared to the experimental ASOs, efficiently generating reproducible results comparing the efficacy of DUX4-targeted ASOs. Two reporter assays were developed, one in the immortalized mouse myoblast cell line C2C12 and the other in the immortalized human FSHD myoblast cell line 15Abic.

[0155] Referring to Table 6, this table displays the knockdown of stable DUX4GFP reporter screening assay. 10,000 15Abic stable cells or 1500 C2C12 stable cells were plated in respective media in black-walled, clear-bottom 96-well plates. The next day after attachment, cells were then transfected using Lipofectamie™ RNAiMAX Transfection Reagent (13778075, Thermo Fisher Scientific). To each well, 0.20 μL / well of Lipofectamine® RNAiMAX was mixed with 5 μL of Opti-MEM and incubated for 5 minutes. An equal volume of 20× ASO in Opti-MEM was then added such that the total volume of the two transfection mixtures was 10 μL / well, and the final concentration of ASO in the total well volume of 200 μL was 12.5 nM for c2c12 cells and 25 nM for 15Abic cells. The ASO-Opti-MEM mixture was incubated at room temperature for 15 min. Then, 10 μL of the resulting transfection reagent mixture was added to each experimental well. After 6 h, regular cell culture medium was added to each well, and then the plate was incubated at 37 °C for 72–96 h. The medium was then replaced with 50 μL of FluoroBrite DMEM medium (A1896701, Thermo Fisher Scientific) supplemented with L-glutamine and sodium pyruvate to 4 mM and 1 mM, respectively, for reading. The fluorescence intensity of each well was measured at 390+10 nm excitation and 510+10 nm emission in a Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments). After fluorescence measurement, serum-free FluoroBrite DMEM was removed, 100 μL of regular medium was added to each well, and cell viability / cell number was measured using WST-8. The cell viability measurement followed the manufacturer's protocol. Briefly, 10 μL of WST-8 (ab228554, Abcam) was then added to each well and the plate was rocked to evenly distribute the reagent.Plates were then returned to the incubator for 30 min to 3 h depending on cell density and cell type. To measure cell viability, absorbance at 460 nm was measured on a Cytation 5. GFP measurements for each well were normalized to WST-8 cell number. Values ​​in the table represent the average GFP expression from six replicate wells and are expressed as a fraction of treatment with negative control ASO.

[0156] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0157] Referring to FIG. 8A, this figure shows that therapeutic ASOs have a strong knockdown of DUX4 in FSHD myotubes. Immortalized FSHD myoblasts were plated at 80% confluence and transfected with 50 nM or 25 nM control or anti-DUX4 ASOs using lipofectamine RNAimax (Thermo) after 24 hours, followed by incubation for 24 hours. Cells were then differentiated into DUX4-positive FSHD myotubes for 96 hours, after which total RNA harvesting and qRT-PCR were performed. GAPDH was used as an internal control. Values ​​represent the average of two experiments with three technical replicates each, and error bars represent SEM ( * p<0.05 by Student's t-test for both doses).

[0158] See Figure 8B and Table 7, which show knockdown of DUX4 and DUX4-inducible genes ZSCAN4 and SLC34A2 in FSHD myoblasts. 15Abic or C6 cells were plated in human Myogenic Precursor Cell (hMPC) medium at a density of 150,000 cells per well of twelve 24-well cell culture plates. hMPC medium consisted of 500 mL RoosterBasal™-MSC, 10 mL RoosterBooster™-MSC (KT-001, RoosterBio), 91 mL fetal bovine serum (10082147, Thermo Fisher), and 50 mL 100 mM sodium pyruvate (11360070, Thermo Fisher). Three days after plating the cells in hMPC medium, the medium was replaced with hMPC differentiation medium consisting of 500 mL RoosterBasal™-MSC, 10 mL RoosterBooster™-MSC, 11.4 mL horse serum (16050130, Thermo Fisher), and 50 mL 100 mM sodium pyruvate. Ten days after plating the cells in hMPC medium (seven days after plating the cells in hMPC differentiation medium), cells were forward transfected using 0.875 μL Lipofectamine™ RNAiMAX Transfection Reagent (13778075, Thermo Fisher) and 3.125 nM or 6.5 nM ASO in a 1 mL well volume according to the manufacturer's instructions. Five days after transfection, the cells were dissolved in 350 μL of buffer RLT (79216, Qiagen). RNA was extracted using the Direct-zol-96RNA kit (R2056, Zymo Research) according to the manufacturer's instructions. Purified RNA concentration was determined using a NanoDrop 1000 (Thermo Fisher) according to the manufacturer's instructions.cDNA was generated by following the manufacturer's instructions for the SuperScript™ IV First-Strand Synthesis System using the ezDNase™ Enzyme kit (18091150, Thermo Fisher) with the following modifications: After digestion of genomic DNA, 1 μL of 110 μM dithiothreitol was added. 1 μL of 50 μM anchor Oligo d(T)20 was used as a primer for the reverse transcription process. After cDNA generation, qPCR was performed to quantify three target genes: DUX4-fl (DUX4-full length), SLC34A2, and ZSCAN4, and one control gene: RPL13A. Dual-quenched PrimeTime qPCR probes and primers were used to perform multiplexed, probe-based qPCR reactions (forward primer, reverse primer, and probe constitute the assay). Briefly, in each well of a 96-well plate, 10 μL of PrimeTime® Gene Expression Master Mix (1055772, Integrated DNA Technologies) was mixed with 100 ng of cDNA and 0.25 μL of each 20× assay (DUX4-fl, SLC34A2, ZSCAN4, RPL13A) with a volume of water for a total volume of 20 μL for each. Thermal cycling and plate reading were performed using a LightCycler96 (Roche Diagnostics). Cycle conditions were as follows: polymerase activation at 95° C. for 180 s, denaturation at 95° C. for 15 s, and annealing / extension at 60° C. for 60 s, with 40 cycles of denaturation and annealing / extension steps. Fluorescence was read at the end of the annealing / extension step after each cycle. Cycle thresholds were determined automatically using the LightCycler 96 software. Normalized relative expression of target genes was calculated according to the method described by Taylor et al., 2019 ("The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time").Expression of the three target genes was then summed and a bar graph was generated depicting the normalized, relative, combined knockdown of the target genes compared to the negative control ASO.

[0159] [Table 7-1] [Table 7-2]

[0160] Referring to Table 8, this table displays the LD-50 value of ASOs targeting DUX4 in HepG2 liver cells. HEPG2 cells (HB-8065, ATCC, Manassas, VA) were grown in DMEM (10-013-CV, Corning Inc.) supplemented with 10% FBS (FBS, 16000044, Thermo Fisher Scientific) and 1× penicillin-streptomycin (15140122, Thermo Fisher Scientific). Cells were grown at 37° C. with 5% CO2 in a humidified incubator. 5,000 cells were plated in 180 μL of antibiotic-free medium in a 96-well plate. Immediately after plating, cells were transfected using Lipofectamine™ RNAiMAX Transfection Reagent (13778075, Thermo Fisher Scientific). For 100 nM transfection of ASO, 0.4 μL / well of RNAiMAX was diluted in 10 μL of Opti-MEM, then combined with 10 μL of 1 μM ASO (10x final culture volume) in Opti-MEM and incubated for 15 min at room temperature. Lower doses of ASO were made by 1:2 serial dilutions of the 100 nM complex. Higher concentrations were prepared by increasing the concentration of ASO, but by maintaining 0.4 μL / well of RNAiMAX, the highest dose that could be used without causing cytotoxicity. 20 μL of the appropriate diluted ASO / RNAiMAX complex was then added to each well within 30 min of complex formation. The plate was gently rocked to evenly distribute the transfection reagent within the wells, then returned to the incubator. Cells were treated for 72–96 h at 37°C. After treatment, transfection medium was removed, 100 μL of fresh medium was added to each well, and a WST-8 assay was performed to measure cell viability / cell number. The cell viability measurement followed the manufacturer's protocol. Briefly, 10 μL of WST-8 (ab228554, Abcam) was added to each well, and the plate was shaken to distribute the reagent evenly. The plate was then returned to the incubator for 90 min.The absorbance at 460 nm was then measured on a Cytation 5. Data were analyzed by subtracting the average background cell viability measurements of cell-free wells (wells with only media and WST8 reagent in the wells) from wells containing cells. Cell viability was calculated by normalizing to wells mock transfected with Opti-mem only. Lethal dose 50 (LD50) concentration values ​​were extrapolated from the dose curves using a custom Excel macro designed for this purpose.

[0161] [Table 8]

[0162] Referring to Figure 9, this figure shows the simultaneous knockdown of DUX4 and DBET RNA transcripts in FSHD patient myoblasts by multi-targeted antisense oligonucleotides (ASOs). AS-DX-10 targets only the DUX4 transcript, whereas AS-DX-25, -37, and -55 target both DUX4 and DBET transcripts. Immortalized 15Abic myoblasts were plated in 12-well plates and transfected the next day with 50 nM of control or targeted ASOs using the transfection agent RNAiMAX. One day after plating, differentiation medium was added to induce myoblast formation and DUX4 expression. 72 hours after transfection, cells were lysed, total RNA was harvested from the wells, and RT-qPCR was performed to determine the expression of DUX4 or DBET transcripts. ASOs AS-DX-25, -37, and -55 knocked down both DUX4 and DBET transcripts, whereas AS-DX-10 knocked down only DUX4. EXAMPLES

[0163] MC-DX4 off-target analysis and validation of ASO target sequence Identifying off-target transcripts All potential reverse complement ASO sites from 15bp to 20bp within the DUX4 coding gene (ENSG00000258389.2) were generated with 1bp sliding within the reference sequence spanning the DUX4 region chr4:190,173,774-190,185,942. A modified script from GGGenome (https: / / gggenome.dbcls.jp / ) was used for rapid alignment of our oligonucleotide sequences to the human transcriptome (Human RNA Refseq release 205, March 2021). This script identified all transcripts that were partially complementary to each ASO that could potentially target DUX4. We then algorithmically analyzed these hits to identify those with higher off-target potential. These can contain up to three mismatches, gaps, or bulges (WO2021203043), but must follow a set of other principles related to structural conformation, affinity, and transcript expression. Even with these filters, there will still be many predicted off-target transcripts that are likely false positives or context-dependent and need to be validated through in vitro and in vivo experimental testing.

[0164] Filtering off-target interactions for potential positive FSHD-related targets Patient isolation and gene expression analysis are crucial parts of the analysis strategy of the disclosed data to understand disease biology. This starts with collecting available datasets from the literature reporting RNA expression patterns from muscle tissue and patient cells. We assembled a database of 10 studies with strict criteria for sample handling, transcriptome profiling by microarray and RNAseq, and key patient information. These 10 studies included: genes with upregulation in myoblasts overexpressing DUX4 (Tsumagari et al. 2011 (31), Pakula et al. 2013, (32) Geng et al. 2012 (33), and Mitsuhashi et al. 2021 (34)); microarray studies of human muscle biopsies (Winokur et al. 2003 (35), and Rahimov et al. 2012 (36)); and RNA-seq profiles (Yao et al. 2014 (28), Wong et al. 2020 (17), and Wang et al. 2019 (29)). One drawback of these studies is that they include low numbers of patients and, as a result, often lack statistical power. To overcome this problem, we created a novel dataset from the three RNA-seq studies using available data to improve statistical power and the ability to derive correlations with patient clinical attributes. FIG. 10 shows an overview of our dataset and our analysis.

[0165] First, we identified genes that were commonly upregulated in FSHD versus control muscles, either within public datasets or using our own RNA-seq analysis. We also used principal component analysis and hierarchical clustering to separate patients into groups and compare expression patterns between control samples and these groups. Interestingly, the clusters match well with clinical severity scores (i.e., mild, moderate, or severe disease). In support of this analysis, a similar analysis from a subset of samples included in a larger meta-analysis yielded similar results, as displayed in Figure 11. From this analysis, we created a database of upregulated genes in FSHD, preserving the supporting evidence for this dysregulation and any relevant clinical correlations with each gene. From this database, we then performed a pathway enrichment analysis using GO pathway analysis (37). The most upregulated pathways include inflammatory response and other immune regulatory pathways, cell proliferation, cell cycle regulation, and fibrosis.

[0166] We assembled this database of FSHD-associated genes and pathways and then filtered the potential off-target interactions we identified against this list. Potential off-target interactions that match FSHD-associated genes or co-targets are displayed in the right-most column of Table 2. For example, AS-DX-007 is predicted to target three co-targets associated with FSHD, such as DBET, MKI67, and IRF5. DBET is a non-coding RNA associated with the opening of D4Z4 repeats and the expression of DUX4 (38). MKI67 encodes the Ki-67 protein, which we detected in FSHD muscle tissue as upregulated, and may be involved in DUX4 induction of myofibroblast proliferation and injury. (Figure 12A). IRF5 (interferon regulatory factor 5) encodes a transcription factor that is upregulated by several inflammatory signals and leads to the expression of several cytokines, such as TNF, and the induction of intracellular interferon responses (Figure 12B). Our analysis also demonstrated that expression of these genes was elevated in mild and severe FSHD (Figure 13).

[0167] Filtering off-target interactions for potential negative toxicity-related interactions To identify potential off-target interactions that may be associated with toxicity that may be desirable to avoid, we used the Ingenuity knowledge database, which accumulates peer-reviewed publications, and toxicity-related gene expression datasets from Tox net and other databases to link potential toxicity to off-target genes. We also identified genes associated with muscle differentiation, development and function through Go pathway analysis. We filtered oligonucleotide sequences and identified off-target interactions for matches to IPAs Toxicity knowledge base or Go pathway. For example, NR4A1 is associated with cell death and fibrosis in the liver and kidney, and differentiation of muscle cells.

[0168] Validation of co-target interactions by qRT-PCR To validate off-target interactions, the FSHD myoblast cell line 15Abic was used. 2.5e5 15abic myoblasts were plated in a 6-well plate. After 24 hours, the replicate medium was removed and 2 mL of differentiation medium and 250 μL of the appropriate ASO RNAimax complex-containing optimized conditions were added so that the final concentration of each ASO was 50 nM. ASO treatments included fluorescent negative control ASO, AS-DX-015-1 targeting only DUX4 as a positive control, or AS-DX-007-1 or AS-DX-050-1 that can co-target DBET, IRF5, and MKI67. At the start of transfection, differentiation medium was added to the cells to induce fusion into myotubes and DUX4 expression. See Figure 14A, 48 hours after transfection, under optimized conditions, displaying nearly 100% transfection efficiency of fluorescent ASO. 96 hours after transfection, myotube fusion was observed by cell morphology and total RNA was harvested. cDNA was generated and qRT-PCR was performed for DUX4 and co-target genes. Referring to Figure 14B, the graph displays the average of triplicate biological wells and the error bars represent the standard error of the mean. * Figure 2 depicts p-values ​​<0.05 by two-tailed Student's t-test. Robust knockdown of DUX4 was observed for all ASOs, with significant knockdown of co-targets observed for AS-DX-007-1 and AS-DX-050-1.

[0169] Although preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions can be made. It should be understood that various alternatives to the disclosed embodiments described herein can be used in implementing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Claims

**Claim 1** An engineered DUX4-targeted oligonucleotide that is from about 15 to about 25 nucleotides in length and that contains at least about 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 20,962 to 42,138. **Claim 2** An engineered DUX4-targeted oligonucleotide that is from about 15 to about 25 nucleotides in length and that contains at least about 80%, 85%, 90%, or 95% sequence identity with any one of SEQ ID NOs: 42,006 to 42,138, the engineered DUX4-targeted oligonucleotide of claim 1. **Claim 3** The engineered DUX4-targeted oligonucleotide is complementary to a binding site of DUX4 RNA that is conserved at more than 85% among individuals; The engineered DUX4-targeted oligonucleotide contains DNA nucleotides and RNA nucleotides and optionally The engineered DUX4-targeted oligonucleotide is a small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (snRNA), U-spliceosomal RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small rDNA-derived RNA (srRNA), transfer RNA-derived small RNA (tsRNA), ribosomal RNA-derived small RNA (rsRNA), long non-coding RNA-derived small RNA (lncsRNA), or messenger RNA-derived small RNA (msRNA), an antisense oligonucleotide (ASO), a gapmer, a mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), an RNA activating oligonucleotide (RNAa), or an exon skipping oligonucleotide; The engineered DUX4-targeted oligonucleotide contains at least one nucleobase selected from the list consisting of locked nucleic acid nucleobases, 2′O-methyl nucleobases, or 2′-methoxyethyl nucleobases; and / or The engineered DUX4-targeted oligonucleotide has a melting temperature of from about 45 to about 65 °C An engineered DUX4-targeted oligonucleotide according to claim 1, which binds to the DUX4 coding sequence in an aqueous solution for measuring the melting temperature (Tm), wherein the aqueous solution has a pH range from about 7.2 to about 7.

6. **Claim 4** An engineered DUX4-targeted oligonucleotide according to any one of claims 1 to 3, comprising at least about 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 30,392 or SEQ ID NO: 30,365. **Claim 5** A conjugate comprising: i) an engineered DUX4-targeted oligonucleotide according to any one of claims 1 to 4; ii) an antibody, antibody fragment, single monomer variable antibody domain, naturally occurring ligand, small molecule, or peptide; and optionally, iii) a linker that links i) to ii). A conjugate comprising the above. **Claim 6** A vector comprising or encoding an engineered DUX4-targeted oligonucleotide according to claim 1 or 2, optionally including a viral vector, nanoparticle vector, liposome vector, exosome vector, extracellular vesicle vector, or a combination thereof. **Claim 7** A pharmaceutical composition comprising: an engineered DUX4-targeted oligonucleotide according to any one of claims 1 to 4, the conjugate according to claim 5, the vector according to claim 6, and a pharmaceutically acceptable excipient, diluent, carrier, or a combination thereof. A pharmaceutical composition comprising the above. **Claim 8** A kit comprising: an engineered DUX4-targeted oligonucleotide according to any one of claims 1 to 4, the conjugate according to claim 5, the vector according to claim 6, or the pharmaceutical composition according to claim 7, and a container, optionally including a jar, ampoule, syringe, bag, box, or a combination thereof. A kit comprising the above. **Claim 9** A method of treating a disease or condition in a subject, comprising: Administering a therapeutically effective amount of the pharmaceutical composition according to claim 7, optionally, the disease or condition is a DUX4-mediated disease or condition, optionally, the DUX4-mediated disease or condition is facioscapulohumeral muscular dystrophy, optionally, the administration is at a dosage of from about 0.001 mg to about 10,000 mg of the pharmaceutical preparation per kg of the subject's body weight, and the administration is oral, intranasal, rectal, topical, intraocular, intramuscular, intravenous, intraperitoneal, intracardiac, subcutaneous, intracranial, intrathecal, or any combination thereof; and / or The pharmaceutical composition comprises a liquid dosage form administered in a volume of from about 1 ml to about 5 ml, from about 5 ml to 10 ml, from about 15 ml to about 20 ml, from about 25 ml to about 30 ml, from about 30 ml to about 50 ml, from about 50 ml to about 100 ml, from about 100 ml to 150 ml, from about 150 ml to about 200 ml, from about 200 ml to about 250 ml, from about 250 ml to about 300 ml, from about 300 ml to about 350 ml, from about 350 ml to about 400 ml, from about 400 ml to about 450 ml, from about 450 ml to 500 ml, from about 500 ml to 750 ml, or from about 750 ml to 1000 ml, a method.

10. The administration includes systemic administration or topical administration, optionally, the advanced administration includes systemic administration including at least one of parenteral administration, intravenous administration, subcutaneous administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intravascular administration, injection, oral administration, inhalation administration, duodenal administration, intraocular administration, transdermal administration, rectal administration, or any combination thereof, a method.

11. The method according to claim 10, further comprising administering a combination therapy simultaneously or sequentially.

12. A method comprising: administering to a subject an engineered DUX-4 target-directed oligonucleotide according to any one of claims 1-4, after administration, the engineered DUX-4 target-directed oligonucleotide selectively hybridizes to two different endogenous disease-related RNAs, wherein one of the two different endogenous disease-related RNAs is DUX4 RNA transcribed from a first locus, and one of the two different endogenous disease-related RNAs is transcribed from a locus different from the first locus, a method.

13. The second RNA of the two different endogenous disease-related RNAs is selected from SEQ ID NOs: 42,139-42,894; and / or The engineered DUX4-targeted oligonucleotide hybridizes to an endogenous disease-associated RNA transcribed from a locus different from the first locus, such that, upon hybridization, there are four or fewer mismatches, bulges, insertions, or deletions at the binding site, and the resulting duplex contains two regions of complementarity of at least seven contiguous nucleobases in length, or one region of at least ten contiguous nucleobases in length, the method of claim 12.

14. A method of treating a disease or condition that is a DUX4-mediated disease or condition, optionally wherein the DUX4-mediated disease or condition is facioscapulohumeral muscular dystrophy, the method of claim 12 or 13.

15. Upon hybridization between the engineered DUX4-targeted oligonucleotide and the second RNA, the predicted melting temperature is from about 40 °C to about 65 °C, the method of claim 13.

16. A composition for use in treating a neuromuscular disease, comprising the engineered DUX4-targeted oligonucleotide according to any one of claims 1 to 4, the conjugate according to claim 5, the vector according to claim 6, the pharmaceutical composition according to claim 7, and a pharmaceutically acceptable excipient, diluent, or carrier A composition comprising.

17. The neuromuscular disease is facioscapulohumeral muscular dystrophy, the composition for use according to claim 16.

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