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Definitions

• Certain aspects of this disclosure pertain to nn engineered DUX4-targeting oligonucleotide that is from about 15 to about 25 nucleotides in length, wherein the engineered DUX4- targeting oligonucleotide comprises at least about: 80%, 85%, 90%, or 95% sequence identity to any one of SEQ. ID. NOs: 20,962 - 42,138. Further, the engineered DUX4- targeting oligonucleotide may be about from about 15 to about 25 nucleotides in length, may comprise at least about 80%, 85%, 90%, or 95% sequence identity to any one of SEQ. ID. NOs: 42,006 - 42,138.
• the engineered DUX4-targeting oligonucleotide of wherein the engineered DUX4-targeting oligonucleotide comprises a DNA nucleotide and an RNA nucleotide. In some cases, this oligonucleotide comprises a DNA nucleotide. In some cases, the oligonucleotide comprises an RNA nucleotide.
• the oligonucleotide is 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 large non-coding RNA derived small RNA (lncsRNA), or a messenger RNA derived small RNA (msRNA).
• siRNA small interfering RNA
• miRNA MicroRNA
• snRNA small nuclear RNA
• U-RNA U spliceosomal RNA
• piRNA Piwi-interacting RNA
• rasiRNA a repeat associated small interfering
• An oligonucleotide as described above may, in certain cases, comprise at least one locked nucleic acid nucleobase.
• the DUX4-targeting oligonucleotide as described above may, bind to the DUX4 coding sequence in an aqueous solution with a predicted melting temperature (Tm) from about 45 to about 65 degrees Celsius wherein the aqueous solution has a pH ranging of from about 7.2 to about 7.6.
• Another aspect of this disclosure is a conjugate of a i) DUX4-targeting oligonucleotide as described above wherein the conjugate comprises the oligonucleotide and an antibody, an antibody fragment, a single monomeric variable antibody domain, a naturally occurring ligand, a small molecule, or a peptide; and optionally iii) a linker that links i) to ii).
• the vector may comprise a viral vector, a nanoparticle vector, a liposomal vector, an exosomal vector, an extracellular vesicle vector, or a combination thereof.
• the vector may be the liposomal vector.
• the vector may be the nanoparticle vector.
• the vector may be the exosomal vector.
• the vector may be the extracellular vector.
• Another aspect of this disclosure pertains to a pharmaceutical composition
• a pharmaceutical composition comprising the engineered DUX4-targeting oligonucleotide of described herein, the conjugate of described herein, a vector as described herein vector of any one of claims 10 to 15, and a pharmaceutically acceptable: excipient, diluent, carrier, or a combination thereof.
• the pharmaceutically acceptable excipient comprises a buffering agent, a stabilizer, an antioxidant, a diluent, or any combinations thereof.
• the pharmaceutically acceptable diluent comprises distilled water, deionized water, physiological saline, Ringer's solutions, dextrose solution, a cell growth medium, phosphate buffered saline (PBS), or any combination thereof.
• the pharmaceutical compositions described herein can be in unit dose form.
• kits comprising the engineered DUX4- targeting oligonucleotide as described herein, the conjugate as described herein, the vector as described herein, or the pharmaceutical composition as described herein and a container.
• the container may comprise ajar, an ampule, a syringe, a bag, a box, or a combination thereof.
• Another aspect of this disclosure is a method of treating a disease or condition in a subject comprising administering to the subject a therapeutically effective amount the pharmaceutical composition as 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 may be a subject is in need thereof.
• the subject may be a human subject in need thereof.
• the administering is in an amount of from about 0.001 mg to about 10,000 mg of the pharmaceutical formulation per kg of body weight of the subject.
• the administering can be oral, intranasal, rectally, topically, intraocular, intramuscular, intravenous, intraperitoneal, intracardial, subcutaneous, intracranial, intrathecal, or any combination thereof.
• the method can use the pharmaceutical composition wherein the pharmaceutical composition a liquid dosage form that is administered at a volume of: about 1 ml to about 5 ml, about 5 ml to 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 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 500 ml, about 500 ml to 750 ml, or about 750 ml to 1000 ml.
• the pharmaceutical composition is in a liquid dosage form, a solid dosage form, an inhalable dosage form, an intranasal dosage form, a liposomal formulation, in the form of a pill, in the form of a capsule, or any combinations thereof.
• the administration comprises systemic or local administration.
• the systemic may be administration, wherein the systemic administration comprises at least one of: a parenteral administration, intravenous administration, subcutaneous administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intravascular administration, infusion, oral administration, inhalation administration, intraduodenal administration, rectal administration, or any combination thereof.
• the method further comprises concurrently or consecutively administering a co-therapy.
• Another aspect of the disclosure concerns a method of administering the engineered DUX-4 targeting oligonucleotide of described herein, wherein after the administering, the engineered DUX-4 targeting oligonucleotide selectively hybridizes to two different endogenous disease related RNAs wherein one of the two different endogenous disease related RNAs is a DUX4 RNA transcribed from a first genetic loci and one of the two different endogenous disease related RNAs is transcribed from a different genetic loci than the first genetic loci.
• the engineered DUX4-targeting oligonucleotide hybridizes to the endogenous disease related RNA that is transcribed from a different genetic loci than the first genetic loci, such that at least 10 continuous oligonucleotides of the engineered DUX4-targeted oligonucleotide hybridize at least two different contiguous sections of contiguous bases that are interrupted by at least one nucleobase.
• This method can be a method of treating a disease or condition which is a DUX4 mediated disease or condition.
• the disease or condition can be facioscapulohumeral muscular dystrophy.
• the predicted thermal melting point can be about 40 degrees Celsius to about 65 degrees Celsius.
• compositions for use in treating a neuromuscular disease comprising an engineered DUX4-targeting oligonucleotide as described herein, a conjugate of as described herein, a vector as described herein, a pharmaceutical composition as described herein and a pharmaceutically acceptable: excipient, diluent, or carrier.
• the composition can be for use wherein the neuromuscular disease is facioscapulohumeral muscular dystrophy.
• FIG. 1 shows genetic modifications that lead to FSHD.
• FIG. 2 shows alternately spliced DUX4 transcripts originating from D4Z4 regions.
• FIG. 3 shows a schematic of the read coverage from RNA-Seq data of alternately spliced DUX4 transcripts from FSHD and Healthy muscle biopsy tissue.
• FIG. 4 shows a schematic of the read coverage from RNA-Seq data of alternately spliced DUX4 transcripts from the Testis.
• FIG. 5 shows the serum stability of chemically modified anti-DUX4 ASOs relative to unmodified oligos.
• FIGs. 6A-B depict reduction in innate stimulation.
• FIG. 6A depicts reductions in innate IFNa and TNFa production after exposure of PBMCs to engineered anti-DUX4 ASOs.
• FIG. 6B depicts reductions in innate immunostimulation for engineered DUX4 ASOs through the Raw-blue cell assay.
• FIG. 7 show a DUX4 ASO HTS Assay Design with stable human or mouse myoblasts expressing eGFP with the coding sequence for DUX4 in the 3 ’ UTR.
• FIGs. 8A-B shows knockdown of DUX4 mRNA.
• FIG. 8A shows therapeutic ASOs have strong knockdown of DUX4 in FSHD myotubes.
• FIG. 8B shows knockdown of DUX4 and DUX4 induced genes ZSCAN4 and SLC34A2 in FSHD myotubes.
• FIG. 9 shows simultaneous knockdown of DUX4 and DBET RNA transcripts in FSHD patient myoblasts by multi-targeted ASOs.
• FIG. 10 shows a schematic overview of data analytics to identify FSHD related genes and pathways.
• FIG. 11 shows expression of genes representing six FSHD relevant biological functions separated by horizontal gaps (top to bottom): DUX4-regulated, extracellular matrix, cell cycle, immune/inflammatory response, immunoglobulin and muscle development-related
• FIGs. 12A-B show exemplary pathways for potential effects by identified co-targets.
• FIG. 12A shows pathway regulations of Ki-67 in cellular proliferation from Xie etal. (18).
• FIG. 12B shows induction of IRF5 in inflammatory signaling from Elkon etal. (19).
• FIG. 13 shows IRF5 and MKI67 RNA expression in patient biopsy samples.
• FIGs. 14A-B shows validation of co-targeted transcripts by multi-targeting ASOs.
• FIG. 14A shows myoblasts after treatment with ASOs.
• FIG. 14B shows qRT-PCT results from RNA (DUX4, DBET, IRF5, and MKI67) obtained from the myoblasts treated with ASOs.
• FIG. 15 is a diagram showing a method and system as disclosed herein.
• FIG. 16 shows a computer control system that is programmed to analyze genetic material.
• Facioscapulohumeral muscular dystrophy is the third most common form of Muscular Dystrophy (MD) with roughly 40,000 patients presenting with symptoms in the US (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 around 100 to less than 11 (3).
• FSHD Type 2 (SHD2) is the result of a loss of function mutation in the epigenetic factor, Structural Maintenance of Chromosomes flexible Hinge Domain containing 1 (SMCHDl) (3) (FIG. 1).
• RNA oligonucleotide therapeutics have the potential to directly repress DUX4, reversing muscle pathology in vitro and in mouse models (13-16).
• conservation of the complementary binding site of DUX4 targeted oligonucleotide therapeutics is still a problem.
• Oligonucleotide therapeutics designed to treat any disorder will be most effective at regulating the targeted transcript if it is perfectly complementary to the target RNA binding site in the disease transcript.
• the targeted binding sequence should have low variance between patients with this disorder. Otherwise, patients that have a SNP or mutation in the sequence of the disease gene at the target binding site may not be perfectly complementary with the therapeutic oligonucleotide resulting in less than complete silencing of the disease gene by the ONT.
• the instant application is the first to solve the problem of determining conserved variant sequences within the DUX4 gene/exons, to identify RNA therapeutics that target clinically significant DUX4 variants, and to generate RNA therapeutics with superior structural modifications for efficacy and stability.
• DUX4-targeting oligonucleotides Normally sequence databases including 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 variance in the DUX4 gene. The challenge is to find conserved therapeutic targets of DUX4. Disclosed herein is the solution and generation and validation of DUX4-targeting oligonucleotides. Most public sequence databases utilize DNA fragment sequencing technologies to efficiently and cheaply collect sequence data from populations. This involves fragmentation of long genomic DNA into pieces a few hundred bases in length that are cloned amplified and sequenced. Individual fragments are then mapped to a larger known reference genomic sequence. This technology is known to not be effective at accurately distinguishing or mapping repetitive sequences (21).
• the coding regions of the DUX4 gene reside in each D4Z4 repeat on chromosome 4.
• DNA from a normal individual contains 11-200 copies of D4Z4 on each chromosome 4 (12).
• DUX4-containing D4Z4 repeats are found on chromosome 10.
• deletion of D4Z4 repeats on chromosome 10 are not associated with development of facioscapulohumeral muscular dystrophy (FSHD) due to lack of downstream exons 3-5 in the DUX4 coding sequence.
• FSHD facioscapulohumeral muscular dystrophy
• D4Z4 pseudogenes are also found throughout the human genome (22) and significant sequence overlap occurs between DUX4 sequences in D4Z4 and other repetitive DNA sequences encoding DUX family members DUX1-DUX5 (23). This genomic complexity leads to poor mapping of sequenced DNA fragments that overlap with D4Z4 repeats, with little confidence in which genomic loci they originate from.
• This disclosure is the first to solve the problem of determining conserved variant sequences within the DUX4 gene/exons, to identify ONT therapeutics that target clinically significant DUX4 variants, and to generate ONT therapeutics with superior structural modifications for efficacy and stability.
• RNA-seq data 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 made the surprising discovery, as shown in Example 3, that sufficient read counts could be identified in RNA-seq data by combining RNA-seq data from muscle biopsies for patients into a combined databased with RNA-seq from testis samples.
• Antisense Oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA, can and do silence many RNAs besides the intended RNA target(26, 27). These non-target RNAs are often referred to as off-target effects. For gapmer ASOs this occurs when the DNA portion of the oligonucleotide causes degradation of unintended RNA off-targets by binding to partially complementary target site and inducing RNAse H cleavage. Careful sequence analysis can identify many of these potential interactions. However, simple sequence alignment does not often accurately predict a real off-target interaction. The inventors have developed a data analysis pipeline to predict and track off-target effects for RNA therapeutics that considers structural motif and binding energy as well to improve predictions (W02021203043).
• the general practice in the field is to avoid off-target effects as much as possible in oligonucleotide design.
• the novel approach described herein is instead to take a global look at off-targets.
• the inventors first look for those that would be potentially harmful and cause toxicity by filtering predicted targets through toxicity databases such as Toxnet and Ingenuity Pathway Analysis (IP A).
• IP A Ingenuity Pathway Analysis
• the inventors also consider off-targets that may be related to disease pathways through analysis of transcriptomic profiles of muscle biopsies from FSHD patients, by looking for genes that are significantly overexpressed in subsets, or related to known disease pathways such as inflammation, muscle cell division, or cell death pathways.
• the term “about” may mean the referenced numeric indication plus or minus: 5%, 10%, 15%, or 20% of that referenced numeric indication. In some instances, “about” may mean the referenced numeric indication plus or minus 15% of that referenced numeric indication. In some instances, “about” may mean the referenced numeric indication plus or minus 20% of that referenced numeric indication. With respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the disclosure and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. Also, where ranges and/or subranges of values are provided, the ranges and/or subranges can include the endpoints of the ranges and/or subranges.
• the term “substantially” as used herein 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.
• the term “homology” can refer to a % identity of a sequence to a reference sequence. As a practical matter, 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 (which can correspond with a particular nucleic acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).
• any sequence disclosed herein also comprises a sequence 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 sequence.
• oligonucleotide can refer to a DNA, RNA, or hybrid nucleic acid sequence, whether chemically modified or not, wherein a single strand, such as for example, in typically the case of DNA, reverse complementarily binds to a target RNA sequence.
• the oligonucleotide may be single stranded such as is typically the case of miRNA, wherein the single strand reverse complementarily binds to a target RNA sequence.
• an RNA oligonucleotide may be double stranded, for example, as is typically the case with siRNA, wherein one strand reverse complimentarily binds to a target RNA sequence.
• an oligonucleotide targeting DUX4 can be a DUX4- targeting oligonucleotide or an oligonucleotide that targets DUX4. It may be a DUX4- targeting oligonucleotide.
• a targeting sequence can have reverse complementarity to a DUX4 transcript.
• a targeting sequence can have at least partial reverse complementarity to a DUX4 transcript and oner more additional genetic loci, or transcripts thereof.
• the genetic loci can be used interchangeably, for example, an oligonucleotide targeting DUX4 can be a DUX4- targeting oligonucleotide or an oligonucleotide that targets DUX4. It may be a DUX4- targeting oligonucleotide.
• a targeting sequence can have reverse complementarity to a DUX4 transcript.
• a targeting sequence can have at least partial reverse complementarity to a DUX4 transcript and oner
• fragment can be a portion of a sequence, a subset that can be shorter than a full-length sequence.
• a fragment can be a portion of a gene.
• a fragment can be a portion of a peptide or 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.
• epigenetic marker can be any covalent modification of a nucleic acid base.
• administer can refer to methods that can be used to enable delivery of compounds or compositions to the desired site of biological action.
• delivery can include direct application to the affected tissue or region of the body.
• subject refers to animals, typically mammalian animals.
• the terms “treat,” “treating” or “treatment,” as used herein, may include at least partially: alleviating, abating or ameliorating a disease or condition symptom; preventing an additional symptom; ameliorating or preventing the underlying causes of a symptom; preventing a recurrence of a symptom; inhibiting the disease or condition, e.g., at least partially arresting a development of the disease or condition; relieving a disease or condition; causing regression of a disease or condition; relieving a condition caused by the disease or condition; or stopping a symptom of the disease or condition either prophylactically, therapeutically or both.
• tissue as used herein, can be any tissue sample.
• a tissue can be a tissue suspected or confirmed of having a disease or condition.
• mammalian cell can refer to any mammalian cell, typically a human cell.
• RNAi double stranded RNA-mediated interference
• ASO antisense oligonucleotides
• RNAi may operate by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces.
• Antisense oligonucleotides may bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.
• oligonucleotide therapeutics may be designed to treat any disorder amenable to regulating a targeted transcript.
• the treatment is with one or more substantially or perfectly complementary ASOs with regard to a target RNA binding site of a disease having a transcript in need of downregulation.
• the oligonucleotide therapeutics are primarily DNA, in other cases, the oligonucleotides are primarily RNA.
• ASOs that efficiently target DUX4 can bind to the fusion transcript and induce degradation through RNAse H.
• interfering RNA such as siRNA or miRNA comprising a sequence which is complementary to a DUX4 RNA transcript may be designed to treat any disorder amenable to regulating such a targeted transcript.
• a siRNA is double stranded with one strand being complementary.
• RISC uses the guide strand of miRNA or siRNA to target complementary 3 '-untranslated regions (3'UTR) of mRNA transcripts via Watson-Crick base pairing, allowing it to regulate gene expression of the mRNA transcript in a number of ways such as mRNA degradation, thereby preventing or reducing protein expression of the selected mRNA.
• Oligonucleotides as mentioned may comprise miRNA.
• miRNA may contain one or more sequence modifications, one or more chemical modifications, or a combination thereof that can: enhance stability of the miRNA; substantially reduce or eliminate immune stimulation (such as via the innate immune response); improve pharmacological activity of the miRNA; retain poly-targeting effects of the miRNA; or any combination thereof.
• 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 such nucleic acids having modified nucleobases.
• an oligonucleotide having the nucleobase sequence "ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG” and those having some DNA bases and some RNA bases such as " AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases.
• RNA transcript with the sequence "AUCGAUCG” encompasses any corresponding DNA sequence such as “ATCGATCG”.
• Nucleic acid sequences herein also comprise sequences comprising 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 sequence.
• an oligonucleotide construct may comprise a first strand comprising the DUX4-targeting oligonucleotide and a second strand comprising a sequence complementary to at least a portion of the DUX4-targeting oligonucleotide.
• the second strand may be complementary to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the first strand.
• the second strand may be complementary to at least about: 5, 10, 15, or 20 contiguous bases of the first strand.
• An oligonucleotide may comprise an end overhang, such as a 5’ end or a 3’ end.
• the first strand, the second strand or a combination thereof may comprise one or more chemical modifications. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of bases of a first strand, a second strand, or a combination thereof may comprise a chemical modification.
• the first strand, the second strand or a combination thereof may comprise one or more sugar modifications. At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of bases of a first strand, a second strand, or a combination thereof may comprise a sugar modification.
• a sugar modification may comprise a glycosylated base. In some cases, a base of a nucleotide may be glycosylated with a glycan.
• the first strand, the second strand or a combination thereof may comprise a combination of bases having a chemical modification and a sugar modification.
• an oligonucleotide as described herein such as a DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 50 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 40 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 30 nucleotides in length. In some cases, the DUX4- targeting oligonucleotide or salt thereof may be from about 5 to about 25 nucleotides in length.
• the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 60 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 80 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 100 nucleotides in length. In some cases, the DUX4-targeting oligonucleotide or salt thereof may be from about 5 to about 200 nucleotides in length.
• an interfering RNA may be a regulatory non-coding RNA (ncRNA) comprising short non-coding RNA sequences expressed in a genome that regulates expression or function of other biomolecules in mammalian cells.
• ncRNA is generally ⁇ 200 nucleotides in length and may be single stranded or double stranded and may form non-linear secondary or tertiary structures.
• An ncRNA may comprise exogenously derived 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), large non-coding RNA derived small RNA (IncsRNA), or a messenger RNA derived small RNA (msRNA).
• siRNA exogenously derived small interfering RNA
• miRNA MicroRNA
• snRNA small nuclear RNA
• U-RNA U spliceosomal RNA
• piRNA Piwi- interacting RNA
• rasiRNA small interfering RNA
• srDNA-derived RNA small RNA
• a DUX4-targeting oligonucleotide may comprise DNA, RNA or a mixture thereof.
• a DUX4-targeting oligonucleotide may comprise a plurality of nucleotides.
• a DUX4-targeting oligonucleotide may comprise an artificial nucleic acid analogue.
• a DUX4-targeting oligonucleotide may comprise DNA, may comprise cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA.
• a DUX4-targeting oligonucleotide can comprise an shRNA, or siRNA, an ncRNA mimic, a short-harpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a mixmer, double-stranded RNAs (dsRNA), single stranded RNAi, (ssRNAi), DNA-directed RNA interference (ddRNAi), an RNA activating oligonucleotide (RNAa), or an exon skipping oligonucleotide.
• shRNA short-harpin RNA
• di-siRNA dicer-dependent siRNA
• ASO antisense oligonucleotide
• a gapmer a mixmer, double-stranded RNAs (dsRNA), single stranded RNAi, (ssRNAi), DNA-directed RNA interference (ddRNAi), an
• a DUX4- targeting oligonucleotide may comprise a completely synthetic miRNA.
• a completely synthetic miRNA is one that is not derived or based upon an ncRNA. Instead, a completely synthetic miRNA may be based upon an analysis of multiple potential target sequences or may be based upon isolated natural non-coding sequences that are not ncRNAs. Modified Oligonucleotides
• a second strand may comprise a chemically modified base of a nucleotide.
• a subset of bases of the second strand may be chemically modified, such as from about 1% to about 5% of bases, from about 1% to about 10% of bases, from about 1% to about 20% of bases, from about 1% to about 30% of bases, from about 1% to about 40% of bases, from about 1% to about 50% of bases, from about 1% to about 60% of bases, from about 1% to about 70% of bases, from about 1% to about 80% of bases, or from about 1% to about 90% of bases, or more.
• a second strand as described herein may be chemically modified in the same manner as described herein for the DUX4- targeting oligonucleotide.
• An oligonucleotide may comprise a sugar modification.
• An oligonucleotide may comprise a plurality of sugar modifications.
• a sugar modification may comprise a glucose or derivative thereof.
• a sugar modification may comprise a ribose or deoxyribose.
• a sugar modification may comprise a monosaccharide, a disaccharide, a trisaccharide or any combination thereof.
• a ribonucleotide or a deoxynucleotide may be modified, such as the base component, the sugar (ribose) component, the phosphate component forming the backbone of the DUX4-targeting oligonucleotide, or any combination thereof, by a chemical modification as described herein.
• An oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemical modification.
• An oligonucleotide may comprise a plurality of chemical modifications.
• An oligonucleotide may comprise a plurality of chemical modifications within a portion of an oligonucleotide, such as a terminal end.
• a chemical modification may comprise a methyl group, a fluoro group, a methoxy ethyl group, an ethyl group, an amide group, an ester group, more than one of any of these, or any combination thereof.
• a chemical modification may comprise a chemically modified nucleotide such as guanosine, uridine, adenosine, thymidine or cytosine including, any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytidine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., 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-chloropurineriboside, 2- aminopurineriboside, 2'-araadeno
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide such as 2-amino-6-chloropurineriboside-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’-0-methyl-inosine-5'-triphosphate, 4- thiouridine-5’ -triphosphate, 5-ami noallylcytidine-5’ -triphosphate, 5-ami noallyluridine- 5’ -triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide such as pyridin-4-one ribonucleoside, 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-2-thio-uridine, 1 -taurinomethyl-4-thio- uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l -methyl-pseu
• an oligonucleotide such as a DUX4-targeting oligonucleotide, DUX4- targeting oligonucleotide may comprise a chemically modified nucleotide 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
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide 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-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyi sopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarb
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide 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, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2-methyl-6
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide 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-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1 -methyl-adenosine, 2-amino-6-chloro
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a chemically modified nucleotide, which may be chemically modified at the 2' position.
• the chemically modified oligonucleotide may comprise a substituent at the 2' carbon atom, wherein the substituent may comprise a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group or an aminoalkoxy group, such as a 2'- hydrogen (2'-deoxy), 2’-0-methyl, 2’-0-methoxyethyl, 2'-fluoro, T Methoxyethyl, 2'- fluoro, a locked nucleic acid (LNA), or any combination thereof.
• LNA locked nucleic acid
• Another chemical modification to an oligonucleotide such as a DUX4-targeting oligonucleotide may 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) or any combination thereof.
• LNA locked nucleic acid
• ENA ethylene bridged nucleic acid
• cEt constrained ethyl
• BNA bridged nucleic acid
• a backbone modification may lock the sugar of the modified nucleotide into a preferred northern conformation.
• a presence of this type of modification in the target sequence of the DUX4-targeting oligonucleotide may allow for stronger and faster binding of the DUX4-targeting oligonucleotide sequence to the target site.
• an oligonucleotide such as DUX4-targeting oligonucleotide may comprise at least one chemically modified nucleotide, wherein the phosphate backbone, which may be incorporated into the DUX4-targeting oligonucleotide, may be modified.
• the phosphate backbone which may be incorporated into the DUX4-targeting oligonucleotide, may be modified.
• One or more phosphate groups of the backbone may be modified, for example, by replacing one or more of the oxygen atoms with a different substituent.
• the modified nucleotide may include a full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
• modified phosphate groups may include a phosphorothioate, a methylphosphonate, a phosphoroselenate, a borano phosphate, a borano phosphate ester, a hydrogen phosphonate, a phosphoroamidate, an alkyl phosphonate, an aryl phosphonate or a phosphotriester.
• the phosphate linker may also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene- phosphonates).
• an oligonucleotide such as a DUX4-targeting oligonucleotide may comprise a sugar modification.
• the sugar modification may comprise a conjugate, such as a linker.
• the DUX4-targeting oligonucleotide may comprise one or more linker groups.
• the DUX4-targeting oligonucleotide may be linked to an antibody, a protein, a lipid, an aptamer, a small molecule, a drug, or any combination thereof.
• a linker may form a covalent bond.
• the DUX4-targeting oligonucleotide may be linked to one or more oligonucleotides, such as a second DUX4-targeting oligonucleotide via a linker.
• the linker may be a cleavable linker.
• a linker may comprise an azide linker.
• the DUX4-targeting oligonucleotide may comprise a base of a nucleotide that is glycosylated with a glycan.
• the DUX4-targeting oligonucleotide may comprise an abasic site, such as a nucleotide lacking an organic base.
• the abasic nucleotide may comprise a chemical modification as described herein, such as at the 2' position of the ribose.
• the 2' C atom of the ribose may 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), T -O-methyl, 2’-0-methoxyethyl or 2'-fluoro.
• an abasic site nucleotide may comprise structures 1 A or IB: dSpacer (1A) rSpacer (1 B)
• an oligonucleotide such as a DUX4-targeting oligonucleotide may be modified by the addition of a “5'-CAP” structure.
• a 5'-cap may be an entity, such as a modified nucleotide entity, which may 'cap' the 5'-end of a mature miRNA.
• a 5'-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
• the 5'-cap may be linked to the 5'-terminus of the DUX4- targeting oligonucleotide via a 5’ -5’ -triphosphate linkage.
• a 5'-cap may be methylated, e.g. m7GpppN, wherein N may be the terminal 5' nucleotide of the nucleic acid carrying the 5'- cap, such as the 5'-end of an RNA.
• a 5'-cap structure may include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L- nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, a
• a modified 5'-CAP structure may comprise a CAPl (methylation of the ribose of the adjacent nucleotide of m7G), CAP2 (methylation of the ribose of the 2nd nucleotide downstream of the m7G), CAP3 (methylation of the ribose of the 3rd nucleotide downstream of the m7G), CAP4 (methylation of the ribose of the 4th nucleotide downstream of the m7G), ARCA (anti-reverse CAP analogue, modified ARCA (e.g.
• phosphothioate modified ARCA inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7- deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido- guanosine.
• an oligonucleotide such as a DUX4-targeting oligonucleotide, may include a covalent modification may comprise 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.
• a covalent modification may comprise changing an oxidation state of a molecule associated with a nucleic acid sequence, such as an oxygen atom, or a combination thereof.
• a covalent modification may occur at any base, such as a cytosine, a thymine, a uracil, an adenine, a guanine, or any combination thereof.
• an epigenetic modification may comprise an oxidation or a reduction.
• a nucleic acid sequence may comprise one or more epigenetically modified bases.
• An epigenetically modified base may comprise any base, such as a cytosine, a uracil, a thymine, adenine, or a guanine.
• An epigenetically modified base may comprise a methylated base, a hydroxymethylated base, a formylated base, or a carboxylic acid containing base or a salt thereof.
• An epigenetically modified base may comprise a 5-methylated base, such as a 5- methylated cytosine (5-mC).
• An epigenetically modified base may comprise a 5- hydroxymethylated base, such as a 5-hydroxymethylated cytosine (5-hmC).
• An epigenetically modified base may comprise a 5-formylated base, such as a 5-formylated cytosine (5-fC).
• An epigenetically modified base may comprise a 5-carboxylated base or a salt thereof, such as a 5-carboxylated cytosine (5-caC).
• an epigenetically modified base may comprise a methyltransferase-directed transfer of an activated group (mTAG).
• An epigenetically modified base may comprise one or more bases or a purine (such as Structure 1) or one or more bases of a pyrimidine (such as Structure 2).
• An epigenetic modification may occur at one or more of any positions.
• an epigenetic modification may occur at one or more positions of a purine, including positions 1, 2, 3, 4, 5, 6, 7, 8, 9, as shown in Structure 1.
• an epigenetic modification may occur at one or more positions of a pyrimidine, including positions 1, 2, 3, 4, 5, 6, as shown in Structure 2.
• a nucleic acid sequence may comprise an epigenetically modified base.
• a nucleic acid sequence may comprise a plurality of epigenetically modified bases.
• a nucleic acid sequence may comprise an epigenetically modified base positioned within a CG site, a CpG island, or a combination thereof.
• a nucleic acid sequence may comprise different epigenetically modified bases, such as a methylated base, a hydroxymethylated base, a formylated base, a carboxylic acid containing base or a salt thereof, a plurality of any of these, or any combination thereof.
• a DUX4-targeting oligonucleotide or salt thereof, when chemically modified may be of formula: Guide Pattern 1, Guide Pattern 2, or Guide Pattern 3 as shown in Table 1.
• N and n may be any natural or non-natural nucleotide; ⁇ N ⁇ may be an LNA; [N] may be a BNA; ⁇ N> may be a 2’-methyloxy ethyl-modified uracil, guanine, adenine, or cytosine; * may be a phosphothionate-modified backbone; mp may be a methylphosphonate-modified backbone; CAP may be 5’ -terminal methyl group (5’- OMethyl) or alkylamino group such as amino-carbon 6 chain (5’ -Amino C6); a may be from 10-26; b may be from 8-24; c may be from 4-20; d may be from 5-22; e may be from
• an oligonucleotide such as DUX4-targeting oligonucleotide may comprise a chemical modification, to a base or a sugar of the DUX4-targeting oligonucleotide, relative to a natural base or sugar.
• the DUX4-targeting oligonucleotide may comprise more than one chemical modification, such as a plurality of chemical modifications.
• a portion of bases or a portion of sugars of the DUX4-targeting oligonucleotide may comprise one or more chemical modifications.
• a DUX4-targeting oligonucleotide may be engineered or modified to increase a specificity for an RNA sequence among a plurality of RNA sequences.
• a DUX4- targeting oligonucleotide may be modified to significantly increase a specificity for an RNA sequence among a plurality of RNA sequences.
• Increased specificity may be compared to a comparable oligonucleotide that may not be engineered or may be compared to a comparable oligonucleotide that may be engineered or modified in a different way.
• a specificity may be increased 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 as compared to a comparable oligonucleotide.
• a DUX4-targeting oligonucleotide may be engineered or modified to increase a specificity for a first RNA sequence as compared to a second RNA sequence.
• a reference sequence i.e., a sequence as described herein
• a subject sequence also referred to as a global sequence alignment
• FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
• the percent identity may be corrected by calculating the number of residues of the query sequence that are lateral to the N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
• a determination of whether a residue is matched/aligned may be determined by results of the FASTDB sequence alignment. This percentage may be then 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 may be used for the purposes of this aspect. In some cases, only residues to the Isl and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes 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.
• a 90-residue subject sequence may be aligned with a 100-residue query sequence to determine percent identity.
• the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N- terminus.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 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%.
• a 90-residue subject sequence is compared with a 100-residue query sequence.
• deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
• percent identity calculated by FASTDB is not manually corrected.
• residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for.
• RNA-Seq BAM files of all the samples were merged into a single BAM file using the Pysamstats vl.1.2 tool https://github.com/alimanfoo/pysamstats and custom Python scripts were used to obtain the reference base frequencies and read depth at each genomic position in the merged BAM files.
• Mean coverage was defined as the average number of reads covering each base of the window.
• a minimum conservation score was calculated for each OTN window representing the base with the lowest conservation.
• Average melting temperature (Tm) was calculated for the resulting OTN/target RNA duplex with the Primer3 v2.4.0 R tool (39), with default parameters, using the nearest neighbor model.
• DUX4- targeting oligonucleotide or salt thereof when chemically modified or when not chemically modified, may have at least 90% sequence identity to any one of SEQ. ID. NOs: 41,923- 42,115.
• a DUX4-targeting oligonucleotide or salt thereof may comprise at least about 80% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115.
• a DUX4-targeting oligonucleotide or salt thereof may comprise at least about 90% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115.
• a DUX4-targeting oligonucleotide or salt thereof may comprise from about 80% to 100% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115. In some cases, a DUX4-targeting oligonucleotide or salt thereof may comprise from about 85% to 100% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115. In some cases, aDUX4- targeting oligonucleotide or salt thereof may comprise at least 80% sequence identity to at least about 10 contiguous bases of any one of SEQ. ID. NOs: 41,923-42,115.
• a DUX4-targeting oligonucleotide or salt thereof may comprise at least 85% sequence identity to at least about 10 contiguous bases of any one of SEQ. ID. NOs: 41,923-42,115. In some cases, the DUX4-targeting oligonucleotide may comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ. ID. NOs: 41,923-42,115, or any combinations thereof.
• certain DUX4- targeting oligonucleotides as shown in Table 2 were able to interact with multiple subsequences of the target DUX4 mRNA as shown in Table 3 also submitted in xml file.
• any of the chemically modified oligonucleotides could be synthesized with a 5’ amino-carbon 6 chain even if not displayed in the table with retention of activity. Additional targeted RNAs are only listed next to the unmodified sequence of the oligonucleotide, they are not repeated for chemically modified versions of the same sequence although they would still be targeted by that sequence.
• RNAs that are partially complementary to listed DUX4 targeted oligonucleotides are partially complementary to listed DUX4 targeted oligonucleotides, but originating from a different genetic loci.
• GGGenome https://gggenome.dbcls.jp/
• This script identified all transcripts that are partially complimentary to each possible oligonucleotide targeting DUX4, containing no more than 4 mismatches, bulges, insertions or deletions, containing two regions of complementarity at least 7 contiguous bases long, or one region at least 10 contiguous bases long.
• These interactions can also have a predicted TM of about 40 °C to about 65 °C.
• the additional targeted RNAs represent transcripts that are upregulated or otherwise associated with the disease and may be beneficial to knockdown in addition to DUX4.
• AS-DX-007 (SEQ. ID. NO. 23,789) is predicted to target three co-targets associated with FSHD, for example, DBET, MKI67, and IRF5.
• DBET is a non-coding RNA associated with opening of the D4Z4 repeats, and expression of DUX4 (38).
• MKI67 encodes the Ki-67 protein, which is upregulated FSHD muscle tissue, and may be involved in the DUX4 induction of the muscle fiber cell proliferation and damage. (FIG. 12A).
• IRF5 Interferon Regulatory Factor 5
• IRF5 Interferon Regulatory Factor 5
• TNF cytokines
• FIG. 12B Target subsequences within the transcripts of the mRNA of these genes (including those of DUX4 itself, are provided herein in Table 3 as shown in RNA form.
• a DUX4-targeting oligonucleotide or salt thereof comprising a modification when contacted with a DUX4 mRNA sequence may produce lower activity of a polypeptide encoded by the DUX4 mRNA sequence as compared to contacting an equivalent amount of an otherwise comparable DUX4-targeting oligonucleotide that lacks the modification with the DUX4 mRNA sequence.
• the lower activity may be at least about 1.2-fold lower. In some cases, the lower activity may be at least about 1.5- fold lower. In some cases, the lower activity may be at least about 1.7-fold lower. In some cases, the lower activity may be at least about 2.0-fold lower.
• the lower activity may 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 lower. In some cases, the lower activity may be from about 1.2-fold to about 2.0-fold lower. In some cases, the lower activity may be from about 1.1 -fold to about 1.5-fold lower. In some cases, the lower activity may be from about 1.1-fold to about 2.5-fold lower. In some cases, the lower activity may be from about 1.2-fold to about 3.0-fold lower. In some cases, the lower activity may be at least about 1.2-fold to about at least 10-fold lower expression. In some cases, the lower activity may be at least about 14-fold lower.
• the lower expression may be at least about 18-fold lower expression.
• the lower activity may 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.
• the lower activity may be from about 1.2-fold to about 14-fold.
• the lower activity may be from about 1.1 -fold to about 20-fold lower.
• the lower activity may be from about 1.2-fold to about 30-fold lower.
• the DUX4-targeting oligonucleotide or salt thereof when contacted with the mRNA sequence, may produce at least 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-fold lower expression of a polypeptide encoded by the mRNA sequence, as compared to contacting an equivalent amount of the otherwise comparable oligonucleotide with the mRNA sequence. Lower expression may be from about 1.2-fold to about 10-fold lower expression.
• the DUX4-targeting oligonucleotide or salt thereof when contacted with the mRNA sequence, may produce at least 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-fold lower activity of a polypeptide encoded by the mRNA sequence, as compared to contacting an equivalent amount of the otherwise comparable oligonucleotide with the mRNA sequence. Lower activity may be from about 1.2-fold to about 10-fold lower activity.
• a DUX4-targeting oligonucleotide or salt thereof may comprise at least about a predicted thermal melting temperature of 45 to 65 degrees Celsius at physiological salt and pH.
• a DUX4-targeting oligonucleotide or salt thereof may bind the 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 degrees Celsius.
• a DUX4-targeting oligonucleotide or salt thereof may bind the 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.
• a subject may comprise a mammal amenable to receive a composition as described herein comprising an engineered DUX4-targeting nucleic acid (such as in the form of an oligonucleotide) or treated by a method as described herein.
• mammals may include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
• Mammals may be any age or at any stage of development, for example a mammal may be neonatal, infant, adolescent, adult or in utero. Mammals may be male or female. In some cases, a human may be from 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 old to about 110 years old or about 100 years to about 130 years old.
• a subject may not have been previously diagnosed with a disease or condition. In some cases, a subject may have been diagnosed with a disease or condition. In some cases, a subject may not have received a definitive diagnosis of a disease or condition. A subject may be at risk of developing a disease or condition (such as based at least in part on a genetic variant). A subject may have received a diagnostic test.
• a diagnostic test may include an imaging procedure, a blood count analysis, a tissue pathology analysis, a biomarker analysis, or any combination thereof.
• the subject may be a patient, such as a patient being treated for a condition or a disease such as a neuromuscular disease.
• the subject may be predisposed to a risk of developing a condition or a disease such as neuromuscular disorder.
• the subject may be in remission from a condition or a disease, such as a neuromuscular disorder.
• the subject may be healthy.
• a subject may be a subject in need thereof.
• a subject may have a disease such as treatment of facioscapulohumeral muscular dystrophy (FSHD) may include, for example, relieving the muscle weakness experienced by a mammal suffering from facioscapulohumeral muscular dystrophy (FSHD), and/or causing the regression or disappearance of muscle weakness.
• FSHD facioscapulohumeral muscular dystrophy
• DUX4-targeting oligonucleotides disclosed herein may be used to treat subjects such that the treatment results in: reduced malaise, an increase in energy, an increase in weight, a decrease in weight, an increase in muscle mass, an increase in, an increase in body flexibility, an increase in posture, an increase in range of movement, cessation of myotonia, abatement of muscle pain, or any combination thereof.
• a subject in need thereof may be treated for a disease or condition.
• a treatment may be a pre-treatment, a prophylactic treatment, or a preventive treatment.
• Treatment may include administration to the subject in need thereof the DUX4-targeting oligonucleotide, a nucleic acid construct, a vector, or a pharmaceutical composition as described herein.
• Treating may include administering an engineered DUX-4-targeted oligonucleotide highly conserved among patients and selected from SEQ. ID. NOs: 20,962-41,922 in the XML Sequence listing file submitted at the time of filing, and/or SEQ. ID. Nos: 41,923- 42,115 as shown in Table 2, or any combination thereof.
• Delivery may include direct application to the affected tissue or region of the body. Delivery may include a parenchymal injection, an intrathecal injection, an intraventricular injection, or an intraci sternal injection.
• a composition provided herein may be administered by any method. A method of administration may be by inhalation, intraarterial injection, intracerebroventricular injection, intraci sternal injection, intramuscular injection, intraorbital injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, or any combination thereof.
• Delivery may include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration. Delivery may include topical administration (such as a lotion, a cream, an ointment) to an external surface of a surface, such as a skin.
• a subject may administer the composition in the absence of supervision.
• a subject may administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.).
• a medical professional may administer the pharmaceutical formulation.
• the treatment of a neuromuscular disease such as facioscapulohumeral muscular dystrophy is by employing a composition which comprises a DUX4-targeting oligonucleotide, a vector comprising the oligonucleotide, or a pharmaceutical formulation as described below.
• a medicine maybe prepared using a DUX4-targeting oligonucleotide, a vector comprising the oligonucleotide, or a pharmaceutical formulation as described below. The medicine may be used for the treatment or prevention of facioscapulohumeral muscular dystrophy.
• Methods may of administration may include in vivo or in vitro delivery methods. Methods may include contacting a cell, such as a cell in vivo with the DUX4-targeting oligonucleotide, the nucleic acid construct, the vector, or the pharmaceutical composition as described herein. Methods may include contacting a cell, such as an isolated and purified cell (such as a cell in vitro) with the DUX4-targeting oligonucleotide, the nucleic acid construct, the vector, or the pharmaceutical composition as described herein.
• Methods may include contacting a tissue, such as an in vivo tissue or an isolated in vitro tissue, with the DUX4-targeting oligonucleotide, a nucleic acid construct, a vector, or a pharmaceutical composition as described herein.
• a tissue such as an in vivo tissue or an isolated in vitro tissue
• Treatment may include more than one DUX4-targeting oligonucleotide delivered in a single dose. Delivery may be concurrent delivery, such as delivery more than one DUX4- targeting oligonucleotide in a single injection or in two separate injections at the same time. Delivery may be sequential, such as delivery of a first dose and a second dose that may be separated by a period of time, such as minutes, hours, days, weeks, or months.
• a DUX4-targeting oligonucleotide human cell may be a cell of head or neck tissue, 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, sperm cell, egg cell, cancer cell, barrier cell, hormone-secreting cell, exocrine-secretory cell, epithelial cell, oral cell, sensory transducer cell, autonomic neuron cell, peripheral neuron cell, central nervous neuron cell, secretory cell, cardiac muscle cell, white blood cell, germ cell, nurse cell, kidney cell, or any combination thereof.
• a tissue may be a sample that may be substantially healthy, substantially benign, or otherwise substantially free of a disease or a condition.
• a tissue may be a tissue removed from a subject, such as a tissue biopsy, a tissue resection, an aspirate (such as a fine needle aspirate), a tissue washing, a cytology specimen, a bodily fluid, or any combination thereof.
• a tissue may comprise cancerous cells, tumor cells, non-cancerous cells, or a combination thereof.
• a tissue may comprise a blood sample (such as a cell-free DNA sample).
• a tissue may be a sample that may be genetically modified.
• Treatment may include treatment of a condition associated with a neuromuscular disease such as facioscapulohumeral muscular dystrophy. Treatment may result in reduced malaise, an increase in energy, an increase in weight, a decrease in weight, an increase in muscle mass, an increase in, an increase in body flexibility, an increase in posture, an increase in range of movement, cessation of myotonia, abatement of muscle pain, or any combination thereof.
• a neuromuscular disease such as facioscapulohumeral muscular dystrophy.
• a vector may be employed to deliver the DUX4-targeting oligonucleotide, the nucleic acid construct, or any combination thereof.
• a vector may comprise DNA, such as double stranded DNA or single stranded DNA.
• a vector may comprise RNA. In some cases, the RNA may comprise a base modification.
• the vector may comprise a recombinant vector.
• the vector may be a vector that is modified from a naturally occurring vector.
• the vector may comprise at least a portion of a non- naturally occurring vector.
• the vector may comprise a viral vector, a liposome, a nanoparticle, an exosome, an extracellular vesicle, or any combination thereof.
• a viral vector may comprise an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a retroviral vector, a portion of any of these, or any combination thereof.
• AAV adeno-associated viral vector
• a nanoparticle vector may comprise a polymeric-based nanoparticle, an aminolipid based nanoparticle, a metallic nanoparticle (such as gold-based nanoparticle), a portion of any of these, or any combination thereof.
• a vector may comprise an AAV vector.
• a vector may be modified to include a modified VP1 protein (such as an AAV vector modified to include a VP1 protein).
• An AAV may comprise a serotype - such as an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, a derivative of any of these, or any combination thereof.
• delivery of an oligonucleotide intended to function as an engineered DUX4-targeting oligonucleotide is through liposomal delivery.
• the liposome may be a positively charged liposome.
• the liposome may be a negatively charged liposome.
• the delivery of engineered DUX4- targeting oligonucleotide is a polymer delivery.
• the engineered DUX4- targeting oligonucleotide delivery is a dendrimer mediated delivery.
• the delivery of an engineered DUX4-targeting oligonucleotide is via microinjection, electroporation, ultrasound, gene gun or hydrodynamic applications.
• the delivery of an engineered DUX4-targeting oligonucleotide is via conjugation to or association with a nanoparticle.
• a pharmaceutical formulation may comprise a pharmaceutically acceptable excipient, diluent, carrier, or a combination thereof.
• a carrier of a pharmaceutical formulation may be, in certain cases, a solid carrier and may comprise lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
• the carrier is a liquid carrier and may comprise phosphate buffered saline solution, syrup, oil, peanut oil, olive oil, water, emulsions, a wetting agent, a sterile solution, or any combination thereof.
• a pharmaceutical formulation may comprise a pharmaceutically acceptable diluent.
• a diluent may comprise for example, sterile distilled water, deionized water, physiological saline, Ringer's solutions, dextrose solution, a cell growth medium, phosphate buffered saline (PBS), or any combination thereof.
• PBS phosphate buffered saline
• a pharmaceutical formulation may comprise a excipient.
• the excipient may comprise a pH agent, a stabilizing agent, a buffering agent, a solubilizing agent, or any combination thereof.
• An excipient may comprise a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof.
• An excipient may comprise 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, HC1, disodium edetate, lecithin, glycerin, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof.
• An excipient may be an excipient described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).
• compositions described herein may be salts, including pharmaceutically acceptable salts, of the compositions described herein.
• compositions containing compounds that are inherently charged, such as those with quaternary nitrogen, may form a salt with an appropriate counterion, e.g., a halide such as bromide, chloride, or fluoride, particularly bromide.
• a pharmaceutical composition may comprise a first active ingredient.
• the first active ingredient may comprise a DUX4-targeting oligonucleotide as described herein.
• the pharmaceutical composition may be formulated in unit dose form.
• the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, diluent, or carrier.
• the pharmaceutical composition may comprise a second, third, or fourth active ingredient, such as a second DUX4-targeting oligonucleotide.
• an engineered DUX4-targeting oligonucleotide or salt thereof comprising a modification when stored in a closed container placed in a room for a time period will remain at least about 80% of an 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% the initial amount. In some cases, the engineered DUX4-targeting oligonucleotide will remain at least about 90% the initial amount.
• the engineered DUX4-targeting oligonucleotide will remain at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%. In some cases, the engineered DUX4-targeting oligonucleotide may be at least about 60% to about at least 80%. In some cases, engineered DUX4-targeting oligonucleotide may be at least about 80% to at least about 99%. In some cases, the time period of storage may be at least 1 month. In some cases, the time period of storage may be at least about 3 months. In some cases, the time period of storage may be at least about 1 year.
• the time period of storage may be at least about 1, 2, 4, 6, 8, 12, 18, 24, 36, 48 or 60 months. In some cases, the time period of storage may be at least about 1 month to about at least 1 year. In some cases, the time period of storage may be at least about 6 months to at least about 2 years. In some cases, the time period of storage may be at least about 1 month to at least about 5 years.
• a pharmaceutical composition may be administered to a subject at a suitable unit dose.
• the pharmaceutical composition may be in unit dose form.
• unit dose may be meant to refer to pharmaceutical drug products in the form in which they are marketed for use, with a specific mixture of active ingredients and inactive components, diluents, or excipients, in a particular configuration, and apportioned into a particular dose to be delivered.
• unit dose may also sometimes encompass non-reusable packaging, although the FDA distinguishes between unit dose “packaging” or “dispensing”. More than one unit dose may refer to distinct pharmaceutical drug products packaged together, or to a single pharmaceutical drug product containing multiple drugs and/or doses.
• unit dose may also sometimes refer to the particles comprising a pharmaceutical composition, and to any mixtures involved.
• types of unit doses may vary with the route of administration for drug delivery, and the substance(s) being delivered.
• administration may comprise intravenous, intraperitoneal, intra-arterial, intertumoral, subcutaneous, intramuscular, intranasal, topical, oral, or intradermal administration.
• administration may comprise inhalation administration.
• a dosage regimen may be determined by an attending physician and clinical factors.
• a dosage for a subject may depend upon many factors, including a subject's size, body surface area, age, sex, general health, a compound to be administered, a time and route of administration, other drugs being administered concurrently, or any combination thereof.
• a range of a dose may comprise 0.001 to 1000 pg.
• a dose may be below or above such a range.
• a regimen as a regular administration of a pharmaceutical composition may be in a range of 1 pg to 10 mg.
• a regimen as a regular administration of a pharmaceutical composition may be in a range of 10 2 units to 10 12 units per day, week or month.
• a unit may be a vector or an ASO.
• a regimen comprises a continuous infusion
• it may also be in a range of 1 pg to 10,000 mg of pharmaceutical composition or engineered polynucleotide or DNA encoding the engineered polynucleotide or vector containing or encoding the engineered polynucleotide per kilogram of body weight per minute, respectively.
• the range is from 1 mg per kilogram of body weight to 1000 mg per kilogram of body weight.
• progress may be monitored by periodic assessment.
• a pharmaceutical composition when a pharmaceutical composition is a liquid it may be administered in a liquid dose form such as about 1 ml to about 5 ml, about 5 ml to 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 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 500 ml, about 500 ml to 750 ml, or about 750 ml to 1000 ml.
• a liquid dose form such as about 1 ml to about 5 ml, about 5 ml to 10 ml, about 15 ml
• composition described herein may be administered one or more days to a subject in need thereof. In some aspects, administration may occur for about: 1, 2, 3,
• administration may occur 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 aspects, administration may occur for about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
• a pharmaceutical composition described herein may be administered on 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, a composition described herein may be administered on consecutive days or on nonconsecutive days. In some cases, a composition described herein may be administered to a subject more than one time per day. In some instances, a composition described herein may be administered to a subject: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.
• a daily oral dosage regimen may be from about 0.1 milligram per kilogram (mg/kg) to about 80 mg/kg of total body weight, from about 0.2 mg/kg to about 30 mg/kg, or from about 0.5 mg/kg to about 15 mg/kg.
• a daily parenteral dosage regimen may comprise from about 0.1 mg/kg to about 10,000 mg/kg of total body weight, 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.
• a daily topical dosage regimen may be from about 0.1 mg to about 500 mg.
• a daily dosage regimen may be from about 0.01 mg/kg to about 1,000 mg/kg per day.
• an optimal quantity and spacing of individual dosages of a composition may be determined by a nature and extent of a condition being treated, a form, route and site of administration, and a particular subject being treated, and that such optimums may preferably be determined by a method described herein.
• a number of doses of compositions given per day for a defined number of days may be ascertained by those skilled in the art using conventional course of treatment determination tests.
• a dosage regimen may be determined by an attending physician and other clinical factors. In some aspects, dosages for any one subject may depend upon many factors.
• factors affecting dosage may comprise a subject's size, body surface area, age, a particular compound to be administered, sex, time and route of administration, general health, other drugs being administered concurrently or any combination thereof.
• progress may be monitored by periodic assessment.
• a pharmaceutical formulation may be administered a daily oral dosage regimen may be from about 0.1 milligram per kilogram (mg/kg) to about 80 mg/kg of total body weight, from about 0.2 mg/kg to about 30 mg/kg, or from about 0.5 mg/kg to about 15 mg/kg.
• a daily parenteral dosage regimen may comprise from about 0.1 mg/kg to about 10,000 mg/kg of total body weight, 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.
• a daily topical dosage regimen may be from about 0.1 mg to about 500 mg.
• a daily dosage regimen may be from about 0.01 mg/kg to about 1,000 mg/kg per day.
• an optimal quantity and spacing of individual dosages of a composition may be determined by a nature and extent of a condition being treated, a form, route and site of administration, and a particular subject being treated, and that such optimums may preferably be determined by a method described herein.
• a number of doses of compositions given per day for a defined number of days may be ascertained by those skilled in the art using conventional course of treatment determination tests.
• a dosage regimen may be determined by an attending physician and other clinical factors.
• dosages for any one subject may depend upon many factors.
• factors affecting dosage may comprise a subject's size, body surface area, age, a particular compound to be administered, sex, time and route of administration, general health, other drugs being administered concurrently or any combination thereof.
• progress may be monitored by periodic assessment.
• a composition or formulation may be used herein for treating treating or preventing a neuromuscular disease comprising an engineered DUX4-targeting oligonucleotide configured to hybridize to an RNA comprising a portion of a RNA transcript, wherein the engineered DUX4-targeting oligonucleotide comprises at least 70% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115, a vector encoding or comprising said oligonucleotide, and a pharmaceutically acceptable: excipient, diluent, or carrier.
• the neuromuscular disease is facioscapulohumeral muscular dystrophy.
• an engineered DUX4-targeting oligonucleotide configured to hybridize to an RNA comprising a portion of a RNA transcript, wherein the engineered DUX4-targeting oligonucleotide comprises at least 70% sequence identity to an oligonucleotide of any one of SEQ. ID. NOs: 41,923-42,115, and a pharmaceutically acceptable: excipient, diluent, or carrier in the preparation of a medicament for the treatment and prevention of facioscapulohumeral muscular dystrophy.
• a DUX4-targeting oligonucleotide or salt thereof may be administered concurrently.
• one or more additional therapeutics may be administered consecutively.
• an co-therapy may comprise immunotherapy, hormone therapy, cryotherapy, surgical procedure or any combination thereof.
• a co-therapy may include administration of a pharmaceutical composition, such as a small molecule.
• a co-therapy may include administration of a pharmaceutical composition, such as one or more antibiotics.
• a co therapy may comprise administration of a muscle relaxant, an anti-depressant, a steroid, an opioid, a cannabis-based therapeutic, acetaminophen, a non-steroidal anti-inflammatory, a neuropathic agent, a cannabis, a progestin, a progesterone, or any combination thereof.
• a neuropathic agent may comprise gabapentin.
• a non-steroidal anti-inflammatory may comprise naproxen, ibuprofen, a COX-2 inhibitor, or any combination thereof.
• a second therapy may comprise administration of a biologic agent, cellular therapy, regenerative medicine therapy, a tissue engineering approach, a stem cell transplantation or any combination thereof.
• a co-therapy may comprise a medical procedure.
• a medical procedure may comprise an epidural injection (such as a steroid injection), acupuncture, exercise, physical therapy, an ultrasound, a surgical therapy, a chiropractic manipulation, an osteopathic manipulation, a chemonucleolysis, or any combination thereof.
• a co therapy may comprise use of a breathing assist device or a ventilator.
• a co-therapy may comprise administration of a regenerative therapy or an immunotherapy such as a protein, a stem cell, a cord blood cell, an umbilical cord tissue, a tissue, or any combination thereof.
• a second therapy may comprise an anti-inflammatory compound, or an anti-fibrosis compound such as pirfenidone, nintedanib, tocilizumab, mycophenolate mofetil/mycophenolic acid prednisone, azathioprine, or a combination thereof.
• a second therapy may comprise a biosimilar.
• a co-therapy is a pharmaceutical agent
• the pharmaceutical agent included in a pharmaceutical composition in the form of a fixed dose combination drug.
• a co-therapeutic dose regimen may be administered for a duration 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.
• a dose regimen may be administered for a duration 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.
• a dose regimen may be administered for a duration of about 1 year, about 2 years or more than about 3 years.
• a daily oral dosage regimen may be from about 0.1 milligram per kilogram (mg/kg) to about 80 mg/kg of total body weight, from about 0.2 mg/kg to about 30 mg/kg, or from about 0.5 mg/kg to about 15 mg/kg.
• a daily parenteral dosage regimen may comprise from about 0.1 mg/kg to about 10,000 mg/kg of total body weight, 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.
• a daily topical dosage regimen may be from about 0.1 mg to about 500 mg.
• a daily dosage regimen may be from about 0.01 mg/kg to about 1,000 mg/kg per day.
• an optimal quantity and spacing of individual dosages of a composition may be determined by a nature and extent of a condition being treated, a form, route and site of administration, and a particular subject being treated, and that such optimums may preferably be determined by a method described herein.
• a number of doses of compositions given per day for a defined number of days may be ascertained by those skilled in the art using conventional course of treatment determination tests.
• a dosage regimen may be determined by an attending physician and other clinical factors. In some aspects, dosages for any one subject may depend upon many factors.
• factors affecting dosage may comprise a subject's size, body surface area, age, a particular compound to be administered, sex, time and route of administration, general health, other drugs being administered concurrently or any combination thereof.
• progress may be monitored by periodic assessment.
• a co-therapy described herein may be administered one or more days to a subject in need thereof. In some aspects, administration may occur for about: 1, 2, 3,
• administration may occur 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 aspects, administration may occur for about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
• a pharmaceutical composition described herein may be administered on 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, a composition described herein may be administered on consecutive days or on nonconsecutive days. In some cases, a composition described herein may be administered to a subject more than one time per day. In some instances, a composition described herein may be administered to a subject: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.
• a regimen as a regular administration of a pharmaceutical agent may be in a range of 1 pg to 10 mg.
• a regimen as a regular administration of a pharmaceutical composition may be in a range of 10 2 units to 10 10 units per day, week or month.
• if a regimen comprises a continuous infusion it may also be in a range of 1 pg to 10,000 mg of pharmaceutical agent. In certain instances, the range is from 1 mg per kilogram of body weight to 1000 mg per kilogram of body weight. In some aspects, progress may be monitored by periodic assessment. Kits
• a kit may include the DUX4-targeting oligonucleotide in a container, the nucleic acid construct in a container, the vector in a container, the pharmaceutical composition in a container.
• a kit may include more than one DUX4-targeting oligonucleotide in a container, more than one vector in a container, more than one nucleic acid construct in a container, or more than one pharmaceutical composition in a container.
• a container may be a plastic, a glass, or a metal container.
• a container may comprise a syringe, a vial, an ampule, a bag, ajar, and the like.
• a kit may include a plurality of containers, each container comprising one or more DUX4-targeting oligonucleotides, or nucleic acid constructs, or vectors, or pharmaceutical compositions.
• a kit may include an excipient or a diluent or a buffer or a liquid or gel-like medium for storage of the DUX4-targeting oligonucleotide, the nucleic acid construct, the vector, or the pharmaceutical composition.
• a kit may include an excipient or a diluent or a buffer or a liquid or gel-like medium for in vivo delivery to a subject of the DUX4-targeting oligonucleotide, the nucleic acid construct, the vector, or the pharmaceutical composition.
• kits may include a delivery vehicle, such as a syringe or needle.
• a kit may include one or more reagents for a downstream analysis.
• the time period may be from about 1 month to about 1 year. In some cases, the time period may be from about 1 month to about 2 year. In some cases, the time period may be from about 1 month to about 6 months. In some cases, the time period may be from about 1 month to about 3 year. In some cases, the time period may be from about 1 month to about 9 months. Diagnostics
• a method may further comprise diagnosing a subject as having the disease.
• a diagnosing may comprise employing an in vitro diagnostic.
• the in vitro diagnostic may be a companion diagnostic.
• the diagnosing may comprise an in vivo diagnostic.
• a diagnostic test may comprise an imaging procedure, a blood count analysis, a tissue pathology analysis, a biomarker analysis, a biopsy, a magnetic resonance image procedure, a physical examination, a urine test, an ultrasonography procedure, a genetic test, a liver function test, a positron emission tomography procedure, a X-ray, serology, an angiography procedure, an electrocardiography procedure, an endoscopy, a diagnostic polymerase chain reaction test (PCR), a pap smear, a hematocrit test, a skin allergy test, a urine test, a colonoscopy, an enzyme-linked immunosorbent assay (ELISA), microscopy analysis, bone marrow examination, rapid diagnostic test, pregnancy test, organ function test, toxicology test, infectious disease test, bodily fluids test, or any combination thereof.
• PCR diagnostic polymerase chain reaction test
• a pap smear a hematocrit test
• a skin allergy test a urine test
• FIG. 15 shows a computer system 101 that is programmed or otherwise configured to predict or confirm efficacy of various constructs for therapeutic effect, such as in the treatment of FSHD.
• the computer system 101 may regulate various aspects of the present disclosure, such as, for example, modeling or identifying constructs for various therapeutic targets, modeling efficacy or stability of constructs, or any combination thereof.
• the computer system 101 may be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
• the electronic device may be a mobile electronic device.
• the computer system 101 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 105, which may be a single core or multi core processor, or a plurality of processors for parallel processing.
• the computer system 101 also includes memory or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage and/or electronic display adapters.
• the memory 110, storage unit 115, interface 120 and peripheral devices 125 are in communication with the CPU 105 through a communication bus (solid lines), such as a motherboard.
• the storage unit 115 may be a data storage unit (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.
• the network 130 may be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
• the network 130 in some cases is a telecommunication and/or data network.
• the network 130 may include one or more computer servers, which may enable distributed computing, such as cloud computing.
• the network 130 in some cases with the aid of the computer system 101, may implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server.
• the CPU 105 may execute a sequence of machine-readable instructions, which may be embodied in a program or software.
• the instructions may be stored in a memory location, such as the memory 110.
• the instructions may be directed to the CPU 105, which may subsequently program or otherwise configure the CPU 105 to implement methods of the present disclosure. Examples of operations performed by the CPU 105 may include fetch, decode, execute, and writeback.
• 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.
• the circuit is an application specific integrated circuit (ASIC).
• the storage unit 115 may store files, such as drivers, libraries and saved programs.
• the storage unit 115 may store user data, e.g., user preferences and user programs.
• the computer system 101 in some cases may include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet.
• the computer system 101 may communicate with one or more remote computer systems through the network 130.
• the computer system 101 may communicate with a remote computer system of a user.
• remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android- enabled device, Blackberry®), or personal digital assistants.
• the user may access the computer system 101 via the network 130.
• Methods as described herein may be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115.
• the machine executable or machine-readable code may be provided in the form of software.
• the code may be executed by the processor 105.
• the code may be retrieved from the storage unit 1115 and stored on the memory 110 for ready access by the processor 105.
• the electronic storage unit 115 may be precluded, and machine- executable instructions are stored on memory 110.
• the code may be pre-compiled and configured for use with a machine having a processer adapted to execute the code or may be compiled during runtime.
• the code may be supplied in a programming language that may be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
• aspects of the systems and methods provided herein may be embodied in programming.
• Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
• Machine-executable code may be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
• “Storage” type media may include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
• another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
• a machine readable medium such as computer-executable code
• a tangible storage medium such as computer-executable code
• Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
• Volatile storage media include dynamic memory, such as main memory of such a computer platform.
• Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
• Carrier-wave transmission media may 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.
• RF radio frequency
• IR infrared
• Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH- EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
• 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.
• the computer system 101 may include or be in communication with an electronic display 135 that comprises a user interface (E ⁇ ) 140 for providing, for example, one or more results (immediate results or archived results from a previous method), one or more user inputs, a reference value or derivative thereof from a library or database, or any combination thereof.
• UI user interface
• Examples of UI’s include, without limitation, a graphical user interface (GET) and web-based user interface.
• a sample 202 containing a genetic material may be obtained from a subject 201, such as a human subject.
• a sample 202 may be subjected to one or more methods as described herein, such as performing an assay.
• an assay may comprise sequencing (such as nanopore sequencing), genotyping, hybridization, amplification, labeling, or any combination thereof.
• One or more results from a method may be input into a processor 204.
• One or more input parameters such as a sample identification, subject identification, sample type, a reference, or other information may be input into a processor 204.
• One or more metrics from an assay may be input into a processor 204 such that the processor may produce a result, such as a diagnosis of neuromuscular disease or a recommendation for a treatment.
• a processor may send a result, an input parameter, a metric, a reference, or any combination thereof to a display 205, such as a visual display or graphical user interface.
• a processor 204 may (i) send a result, an input parameter, a metric, or any combination thereof to a server 207, (ii) receive a result, an input parameter, a metric, or any combination thereof from a server 207, (iii) or a combination thereof.
• Methods and systems of the present disclosure may be implemented by way of one or more algorithms.
• An algorithm may be implemented by way of software upon execution by the central processing unit 105.
• the algorithm can, for example, determine optimized constructs via supervised learning to optimize therapeutic efficacy, stability, or other attribute of one or more constructs.
• EXAMPLE 1 DUX4 Sequence from Skeletal Muscle Samples
• Example 3 DUX4 Sequence from combined Muscle and Testis Samples database.
• FSHD type 1 is the result of a deletion of the D4Z4 repeats on chromosome 4q35 from around 100 to less than 11 repeats leading to opening of chromatin and expression of DUX4.
• FSHD Type 2 is the result of a loss of function mutation in the epigenetic factor SMCHDl leading to demethylation D4Z4 repeats on 4q35, opening of chromatin and expression of DUX4.
• SMCHDl the epigenetic factor
• SMCHDl leading to demethylation D4Z4 repeats on 4q35, opening of chromatin and expression of DUX4.
• 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 testes.
• FIG. 3 shows read coverage from RNA-Seq data of alternately spliced DUX4 transcripts from FSHD and Healthy muscle biopsy tissue.
• FIG. 4 shows read coverage from RNA-Seq data of alternately spliced DUX4 transcripts from the Testis.
• Alternately spliced DUX4 transcripts ENST00000616166.1, ENST00000569241.5, and ENST00000570263.5 are associated with FSHD when expressed in muscle tissue.
• FIG. 5 shows that chemical modifications DUX4 targeted ASOs may improve ASO stability to biological nucleases.
• DUX4 targeted ASOs were incubated in 10% human serum at 37°C for the indicated lengths of time.
• ASO stability at each time point was visualized by denaturing Urea-PAGE Unmodified Oligo, shows an example of an unmodified DNA nucleic acid that has very low half-life
• Neg Con Oligo shows an ASO that does not target DUX4 but has similar chemical modifications while the other panels show chemically engineered ASOs that target DUX4.
• An exceptional example displays stability to biological nucleases out to 7 days (168 hours).
• this table shows the calculated half-life of chemically modified ASOs targeting DUX4 to biological nucleases.
• Unmodified refers to non-chemically modified RNA while Neg Con Oligo refers to an ASO that is chemically modified but does not target DUX4.
• FIG. 6A shows reductions in innate immunostimulation for engineered DUX4 ASOs.
• Human Peripheral Blood Mononuclear Cells (PBMCs)( ⁇ 2-6 x 10 5 cells) were plated in round bottom 96 well plates and transfected at 133 nM concentrations of the indicated ASOs for 48 hours with RNAiMAX reagent.
• Levels of IFN- a (Left axis) and TNF-a (right axis) in supernatant media were quantified by ELISA for 6 patients.
• RNAiMAX without oligonucleotide served as baseline negative control (Baseline) while transfection with a non- immunostimulatory RNA that does not target DUX4 (Neg Con) demonstrated low immunostimulation.
• Baseline baseline negative control
• a non- immunostimulatory RNA that does not target DUX4 Neg Con
• FIG. 6B this figure further shows reductions in innate immunostimulation for engineered DUX4 ASOs through the Raw-Blue cell assay (Invivogen, raw-sp).
• cells were plated at 100,000 cells/well in 150uL in a U bottom 96 well plate (Thermo, 163320) in DMEM (Thermo, 11965092) with 10% FBS (Thermo, 10082147). After 24 hours, 22.34 uL of OptiMEM (Thermo, 31985088) is mixed with 2.66uL of lOuM ASO (per well), and luL of Lipofectamine (per well). Lipofectamine/ASO mixtures are then added to each well of Raw Cells to make a final ASO concentration of 133nM.
• Poly(dA:dT) (Invivogen, tlrl-patn) @ 1-10 ng/mL, CpG (invivogen, tlrl-1585) @ 133nM are used as positive controls.
• Cells are incubated for 1 day @ 37 degrees/5% C02 with the transfection/ASO mixture. After incubation, gently spin the plate at 300 xg for 5min then collect 20uL from each well and add to a new 96 well plate, flat bottom (VWR, 29442- 056).
• Add 180uL of QUANTI-Blue (Invivogen, rep-qbs) to each well of supernatant and incubate for 30m-6 hours @ 37 degrees/5% C02. Read absorbance at 620-655nm using Cytation 5 (Biotek). Data represents the mean of six replicate wells and error bars represent standard deviation.
• DUX4 ASO HTS Assay Design. Stable human or mouse myoblasts expressing eGFP with the coding sequence for DUX4 in the 3’ UTR. Constitutive expression of this construct is driven by CMV for strong, ubiquitous expression. Unmolested mRNA encoding the eGFP-UTR-DUX4 transcript is transcribed 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 mutation of the start codon for DUX4. ASOs that efficiently target DUX4 may bind to the fusion transcript and induce degradation through RNAse H or RISC, preventing GFP protein expression.
• this table display knockdown of a stable DUX4 GFP reporter screening assay.
• black wall clear bottom 96 well plates 10,000 15 Abie stables or 1500 C2C12 stable cells are plated in their respective media. The following day after attachment, cells are then transfected using LipofectamineTM RNAiMAX Transfection Reagent (13778075, Thermo Fisher Scientific). For each well 0.20 pL/well of Lipofectamine®
• RNAiMAX was mixed with 5 pL of Opti-MEM and incubated for 5 minutes. Then an equal volume of 20x ASO in Opti-MEM is added such that the total volume of the two transfection mixtures was 10 pL/well and such that the final concentration of ASO in a total well volume of 200 pL is 12.5 nM for c2cl2 cells or 25 nM for 15Abic cells. The ASO-Opti-MEM mixture was incubated at room temperature for 15 mins. 10 pL of the resultant transfection reagent mixture was then added to each experimental well. After 6 hrs normal cell culture media is added to each well and then plates were incubated for 72 - 96 h at 37 0C.
• WST-8 (ab228554, Abeam) was subsequently added to each well and the plate was oscillated to distribute the reagent evenly. Plates were then returned to the incubator for 30 mins to 3 hours depending on the cell density and cell type. To measure cell viability absorbance at 460 nm was measured on a Cytation 5. GFP measurements for each well are normalized to the WST-8 cell count. Values in the table represent mean GFP expression from six replicate wells and are displayed as a fraction of treatment with a negative control ASO.
• FIG. 8A this figure shows therapeutic ASOs have strong knockdown of DUX4 in FSHD myotubes.
• Immortalized FSHD myoblasts were plated at 80% confluence and 24 hours later transfected with control or anti-DUX4 ASOs at 50 nM or 25nM with lipofectamine RNAimax (Thermo) followed by 24-hour incubation. Cells were then differentiated for 96 hours into DUX4 positive FSHD myotubes before total RNA collection and qRT-PCR was performed. GAPDH was used as the internal control. Values represent the mean of two experiments with three technical repeats each, and error bars represent SEM *p ⁇ 0.05 by Student’s t-test for both doses.
• hMPC media is composed of 500 mL RoosterBasalTM- MSC, 10 mL RoosterBoosterTM-MSC (KT-001, RoosterBio), 91 mL of fetal bovine serum (10082147, Thermo Fisher), and 50 mL of 100 mM sodium pyruvate (11360070, Thermo Fisher).
• hMPC Differentiation media which is composed of 500 mL RoosterBasalTM-MSC, 10 mL RoosterBoosterTM-MSC, 11.4 mL of horse serum (16050130, Thermo Fisher), and 50 mL of 100 mM sodium pyruvate.
• RNAiMAX Transfection Reagent 13778075, Thermo Fisher
• 3.125 nM or 6.5 nM ASO in a well volume of 1 mL according to the manufacturer’s directions.
• Five days after transfection cells were lysed into 350 pL of Buffer RLT (79216, Qiagen).
• RNA was extracted using Direct-zol-96 RNA Kit (R2056, Zymo Research) following manufacturer’s directions. Purified RNA concentration was determined using a NanoDrop 1000 (Thermo Fisher) following manufacturer’s directions.
• cDNA was generated by following the manufacturer’s directions for the SuperscriptTM IV First-Strand Synthesis System with ezDNaseTM Enzyme kit (18091150, Thermo Fisher) with the following modifications. 1 pL of 110 pM dithiothreitol was added after digestion of genomic DNA. 1 pL of 50 pM anchored Oligo d(T)20 was used as the primer during reverse transcription. Following cDNA generation qPCR was performed to quantify three target genes: DUX4-fl (DUX4-full length), SLC34A2, and ZSCAN4 as well as one control gene: RPL13A.
• a multiplexed, probe-based qPCR reaction was done using dual quenched PrimeTime qPCR probes and primers (a forward primer, reverse primer, and probe constitute an assay). Briefly, in each well of a 96 well plate 10 pL ofPrimeTime® Gene Expression Master Mix (1055772, Integrated DNA Technologies) was mixed with 100 ng of cDNA and 0.25 pL of each 20X assay (DUX4-fl, SLC34A2, ZSCAN4, RPL13A) along with a volume of water such that the total volume of each was 20 pL. Thermal cycling and plate reading were done using a LightCycler 96 (Roche Diagnostics).
• Cycling conditions were as follows: polymerase activation at 95 °C for 180 seconds, denaturation at 95 °C for 15 seconds, and annealing/extension at 60 °C for 60 seconds with the denaturation and annealing/extension steps repeated for 40 cycles. Fluorescence was read at the end of the annealing/extension step after each cycle. Cycle threshold was automatically determined using the LightCycler 96 software. The normalized relative expression of the target genes was calculated following 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 added together and a bar chart was produced describing the normalized, relative, composite knockdown of target genes in relation to a negative control ASO.
• this table displays LD-50 values in HepG2 Liver cells for DUX4 targeted ASOs.
• 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 IX penicillin-streptomycin (15140122, Thermo Fisher Scientific) Cells were grown at 37° C at 5% C02 in a humidified incubator.In 96 well plates 5,000 cell are plated in 180 pL of media w/o antibiotics.
• RNAiMAX Transfection Reagent 13778075, Thermo Fisher Scientific.
• RNAiMAX Transfection Reagent 13778075, Thermo Fisher Scientific.
• Opti-MEM 10 pL/well of RNAiMAX
• 10 pL of 1 pM ASO 1 pM ASO (lOx final culture volume)
• Opti-MEM 10 pL of 1 pM ASO (lOx final culture volume) in Opti-MEM and incubated at room temperature for 15 minutes.
• Lower doses of ASO are created by serial 1:2 dilution of 100 nM complexes.
• Higher concentrations are prepared by increasing the concentration of the ASO but maintaining 0.4 pL/well of RNAiMAX which is the highest dose that may be used without causing cytotoxicity.
• this figure shows simultaneous knockdown of DUX4 and DBET RNA transcripts in FSHD patient myoblasts by multi -targeted antisense oligonucleotides (ASOs).
• AS-DX-10 only targets the DUX4 transcript, while AS-DX-25, -37, and -55 target both DUX4 and DBET transcripts.
• Immortalized 15 Abie myoblast cells were plated in 12- well plates and the next day transfected with control or targeted ASOs at 50 nM using the transfection agent RNAiMAX. One day after plating differentiation media was added to induce myoblast formation and DUX4 expression.
• DUX4 or DBET transcripts were 72 hrs after transfection cells were lysed and total RNA was collected from the wells and RT-qPCR was performed to determine expression of DUX4 or DBET transcripts.
• FIG. 10 shows an overview our datasets and our analysis.
• the inventors identified the genes that were commonly upregulated in FSHD muscle vs. control muscle among published datasets or using inventors’ own RNA-seq analysis.
• the inventors also utilized principal component analysis and hierarchical clustering to segregate patients into groups and compared expression patterns between the control samples and these groups. Interestingly, the clusters align well with clinical severity scores (i.e., mild, moderate, or severe diseases). Supporting this analysis, similar results were obtained from a similar analysis from a subset of the samples included in the larger meta-analysis as displayed in FIG. 11. From this analysis the inventors created a database of upregulated genes in FSHD keeping with each gene the supporting evidence for this dysregulation, and any associated clinical correlations. From this database the inventors next performed pathway enrichment analysis utilizing GO pathway analysis (37). The top upregulated pathways include inflammatory response and other immune regulated pathways, cellular proliferation, cell cycle regulation, and fibrosis.
• the inventors utilized the Ingenuity knowledge database which accumulates peer reviewed publications, and toxicity related gene expression datasets from Tox net and other databases to associate off-target genes with potential toxicity.
• the inventors also identified genes related to muscle differentiation, development and function by go-pathway analysis.
• the inventors filtered oligonucleotide sequences identified off- target interactions for matches for IP As Toxicity knowledge base or go pathways. For example, NR4A1 is associated with liver and kidney cell death and fibrosis, and muscle cell differentiation.
• the FSHD myoblast line 15Abic was used. 2.5e5 15abic myoblasts were plated in 6-well plates. After 24 hours, replication media was removed, and 2 mL of differentiation media added and 250 pL of optimum containing appropriate ASO RNAimax complexes, so that the final concentration of each ASO was 50 nM.
• FIG. 14A displays near 100% transfection efficiency of the fluorescent ASO under optimized conditions 48 hours after trasfection. After 96 hours of the transfection, myotube fusion was observed by cell morphology, and the total RNA was collected. cDNA was created, and qRT-PCR was performed for DUX4 and the co-target genes.
• FIG. 14B the graph displays the mean of three biological replicate wells, and error bars represent standard error of the mean. * denotes a p-value of ⁇ 0.05 by 2-tailed student’s t-test. Robust knockdown of DUX4 was observed for all ASOs, and significant knockdown of co-targets was observed with AS-DX-007-1 and AS-DX-050-1.
• RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units new candidates for the pathophysiology of facioscapulohumeral dystrophy. Hum Mol Genet. 2009; 18(13):2414-30. doi: 10.1093/hmg/ddpl80. PubMed PMID: 19359275; PMCID: PMC2694690.

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