Compositions and methods comprising nuclear small RNA (snRNA) for treating DMD

Abstract

An snRNA system that targets DMA RNA sequences is disclosed herein. TIFF2026534279000058.tif52160

Description

TECHNICAL FIELD

[0001] Related Applications This application claims priority and the benefit of U.S. Provisional Application No. 63 / 585,293 filed on September 26, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (LOCN_024_001WO_SeqList_ST26.xml, size: 1,227,589 bytes, creation date: September 25, 2024) is incorporated herein by reference in its entirety.

[0003] Field of the Disclosure The present disclosure relates to molecular biology, gene therapy, and compositions and methods for modifying the expression and activity of RNA molecules. BACKGROUND ART

[0004] Background Providing effective therapies for correcting dysfunctional messenger RNA has been a long-standing unmet need in the art.

[0005] Nuclear small RNAs (snRNAs) are one of the smallest types of RNA, with an average size of approximately 150 nucleotides. snRNAs are functional non-coding RNAs. Eukaryotic genomes encode a diverse range of non-coding RNAs, including snRNAs, which are a class of abundant, nuclear-localized RNAs that play important roles in intron splicing and RNA processing. During premRNA splicing, snRNAs can form ribonucleoprotein particles (snRNPs) with other proteins. These snRNPs and additional proteins form a large, particulate complex (spliceosome) bound to the unspliced ​​premRNA transcript. In addition to splicing, snRNAs also function in nuclear maturation of nascent transcripts, gene expression regulation, as splice donors in non-standard systems, and in the 3' end processing of replication-dependent histone mRNA. U7 snRNAs can be programmed to bind to and modulate mRNA without the need for exogenous protein expression, which ultimately reduces the risk of immunogenicity observed with other protein-based gene therapy approaches. Furthermore, the small size of these programmed snRNAs creates opportunities to develop highly specific (allele-specific) single-target and multi-target gene therapy approaches using a single vector.

[0006] Duchenne muscular dystrophy is caused by mutations in the DMD gene. Monogenic diseases or disorders associated with DMD are Duchenne muscular dystrophy or Becker muscular dystrophy. DMD is the largest gene in the human genome, with 79 exons separated by introns up to 250 kb. DMD encodes the dystrophin protein, which stabilizes the cell membrane of striated muscle. Exon skipping to restore the frame (e.g., caused by an exon 50 deletion frameshift) reduces the severity of Duchenne muscular dystrophy to the level seen in patients with Becker muscular dystrophy. Therefore, exon skipping to restore nearly full-length dystrophin protein offers a promising opportunity to treat Duchenne muscular dystrophy (DMD) patients with mRNA frameshift deletions in the DMD gene; however, exon skipping is generally limited to antisense oligonucleotides, which are poorly taken up by diseased tissue. Recent efforts involving AAV9-U7 snRNA gene therapy targeting exon 2, and the use of modified U7 snRNAs to convert out-of-frame mutations to in-frame mutations (Goyenvalle et al., 2009, 17(7):1234-1240 (Non-Patent Literature 1)) are promising, but these early strategies suffer from challenges such as low titers and inconsistent transgene expression. Therefore, this disclosure provides compositions and methods comprising a novel therapeutic RNA targeting platform composed of engineered snRNAs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Goyenvalle et al.,2009,17(7):1234-1240 [Overview of the project]

[0008] overview This disclosure provides an RNA-targeting nucleic acid molecule comprising nuclear small RNA (snRNA), wherein the snRNA comprises a targeting sequence that binds to a dystrophin (DMD) RNA sequence, and the DMD RNA sequence comprises at least one splicing control sequence selected from a splice acceptor sequence, a splice donor sequence, and an exon splice enhancer sequence.

[0009] In some aspects, the DMD RNA sequence is at least one of exon 2, exon 44, exon 45, exon 51, and / or exon 53.

[0010] In some aspects, the targeting sequence contains a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence described in any one of SEQ ID NO: 59-118, 126, 206-227, and 237-1344.

[0011] In some aspects, snRNAs include a stem-loop (SL) containing a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences to one or more nucleic acid sequences described in any one of SEQ ID NO:1-SEQ ID NO:11, SEQ ID NO:144-SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:186, SEQ ID NO:190-SEQ ID NO:205, SEQ ID NO:228-SEQ ID NO:230, SEQ ID NO:235, or SEQ ID NO:236.

[0012] RNA targeting nucleic acid molecule according to claim 1. An engineered stem-loop containing nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more nucleic acid sequences described in any one of SEQ ID NO:1 to SEQ ID NO:11, and a stem-loop (eSL) containing nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between).

[0013] In some aspects, snRNAs contain an engineered stem-loop (eSL) comprising one or more nucleic acid sequences described in any one of SEQ ID NO:1 to SEQ ID NO:11.

[0014] In some aspects, DMD RNA sequences are either pre-mRNA or mRNA sequences.

[0015] In some cases, snRNAs contain two targeting sequences that target two RNAs of interest. In some cases, the two targeting sequences are a fusion sequence.

[0016] In some aspects, the snRNA contains an Sm-binding domain (SmBD) selected from the group consisting of U1, U2, U4, and U5 SmBDs. In some aspects, the SmBD contains a nucleic acid sequence described in one of SEQ ID NO:31 to SEQ ID NO:38.

[0017] In some aspects, the snRNA contains a 5' interaction stabilization domain (5'ISD) that includes a nucleotide sequence selected from one of SEQ ID NO:12 to SEQ ID NO:23.

[0018] In some aspects, the snRNA includes the nucleic acid sequence described in this disclosure.

[0019] A vector comprising one or more snRNAs of any aspect of the present disclosure. In some aspects, the vector is an AAV vector.

[0020] In some aspects, the snRNA is operably linked to a promoter. In some aspects, the snRNA is operably linked to a U7 promoter or a U1 promoter. In some aspects, the snRNA is operably linked to a downstream terminator (DT). In some aspects, the snRNA is operably linked to a U7 downstream terminator or a U1 downstream terminator.

[0021] In some aspects, the vector comprises at least 1, at least 2, at least 3, at least 4, or at least 5 snRNAs. In some aspects, the at least 1, at least 2, at least 3, at least 4, or at least 5 snRNAs each target the same target RNA sequence. In some aspects, the at least 1, at least 2, at least 3, at least 4, or at least 5 snRNAs target two or more target RNA sequences.

[0022] In some aspects, each snRNA is separated by a buffer sequence. In some aspects, the buffer sequence comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 24 to SEQ ID NO: 30.

[0023] In some aspects, the vector comprises a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in any one of SEQ ID NO: 130 to SEQ ID NO: 138.

[0024] The present disclosure provides a method of targeting one or more target RNAs of interest, and blocking, knocking down, editing, exon-skipping, or splicing the one or more target RNAs, comprising the step of contacting a snRNA of the present disclosure with a cell comprising the one or more target RNAs.

[0025] The present disclosure provides a DMD RNA-targeting nucleic acid molecule comprising a targeting sequence set forth in any one of SEQ ID NOs: 59 to 118, 206 to 227, or 237 to 333.

[0026] The present disclosure provides a method of treating a disease or disorder in a subject, comprising the step of administering the RNA-targeting nucleic acid molecule of the present disclosure or the AAV vector of the present disclosure.

[0027] In some aspects, the disease or disorder is Duchenne muscular dystrophy.

[0028] In some aspects, the administration is intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and / or parenteral administration. [Brief Description of the Drawings]

[0029] [Figure 1A]Figures 1A–1C show that fusion spacers promote a higher level of exon skipping than single spacers alone. Modified U7 snRNAs containing spacers that bind to splicing control sequences (SA = splice acceptor, SD = splice donor, and / or ESE = exon enhancer sequence) to skip exon 53 result in recovery of the DMD mRNA reading frame and dystrophin protein in the case of del52 mutations in DMD patients. Figure 1A provides a schematic diagram showing the expected PCR product sizes using primers within exon 51 and exon 54. Figure 1B shows TapeStation images of RT-PCR products after U7 snRNA treatment using single and fusion U7 synthetic RNA, showing PCR products with DMD exon 53 skipped (upper band, 365 bp) and those without (lower band, 153 bp). Untreated cells were used as negative control samples. Figure 1C provides the quantification results from Figure 1B, shown as the percentage of exon 53 skipping (skipped bands / skipped bands + non-skipped bands). [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 2] Figure 2 shows AAV9 snRNA-mediated exon 53 skipping in human del45-52 DMD myotubes, as indicated by the recovery of dystrophin protein staining. The image shows immunofluorescence of dystrophin protein one week after transduction of DMD myotubes with A05014 (a scAAV9 dual snRNA cassette targeting exon 53) in a 1E5 vector genome / cell. Untreated cells, shown on the right, serve as a negative control. DAPI staining represents the nucleus. [Figure 3]Figures 3A–3B illustrate in vitro and in vivo AAV9 snRNA-mediated exon 53 skipping. Figure 3A provides a graph showing the percentage of exon 53 skipping in human DMD myotubes 7 days after treatment with A05211 at MOIs of 1E3, 1E4, and 1E5 vg / cell. Figure 3B provides a graph showing the percentage of exon 53 skipping in multiple tissues (gastrocnemius muscle, heart, liver, tibialis anterior muscle, brain, diaphragm, and triceps brachii muscle) 4 weeks after post-orbital administration of 2.4E12 vg / animal AAV9-A05211. [Figure 4A] Figures 4A–4C illustrate DMD exon 44 targeting by synthetic fused U7 snRNA in patient myotubes with exon 45 deletion. Figure 4A illustrates the mechanism of action of snRNA that promotes DMD exon 44 skipping and frame recovery in patient mutations such as exon 45 deletion. Therefore, U7 snRNA was modified to bind to a splicing control sequence to promote exon 44 skipping. Figure 4B shows tapestation images of RT-PCR products after U7 snRNA treatment using single U7 synthetic RNA and fused U7 synthetic RNA in Δ45 Inserm myotubes, showing PCR products where DMD exon 44 was not skipped (upper band) and products where it was skipped (lower band). Non-targeting U7 (NT) and untreated cells (U) were used as negative control samples. Figure 4C shows the quantification results from 4B, expressed as the percentage of exon 44 skipping (intensity of skipped band / intensity of skipped band + intensity of non-skipped band). [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 5A]Figures 5A–5B show the increased level of exon 44 skipping using a second-generation fusion spacer targeting exon 44 in Δ45 myotubes. Figure 5A shows a Tapestation image of RT-PCR products after U7 snRNA treatment (synthetic RNA) using an optimized second-generation fusion spacer. The gel image shows PCR products where DMD exon 44 was not skipped (upper band) and products where it was skipped (lower band). Untargeted U7 (NT) and untreated cells (U) were used as negative control samples. Figure 5B shows the quantification results from Figure 5A, expressed as the percentage of exon 44 skipping (intensity of skipped band / intensity of skipped band + intensity of non-skipped band). [Figure 5B] See the explanation in Figure 5A. [Figure 6A] Figures 6A–6D show AAV9-mediated snRNA exon 44 skipping in patient myotubes. Figure 6A provides a graph showing the percentage of exon 44 skipping (intensity of skipped bands / intensity of skipped bands + intensity of non-skipped bands) in Δ45 inserm myotubes transduced with A05374 and A05375 (scAAV9 vector containing a 2×U7 snRNA cassette) at varying MOIs. Synthetic U7 sR220334 was used as a positive control, and untreated (UNT) and AAV null (empty capsid) were used as negative controls. Figure 6B shows U7 snRNA expression at varying MOIs for A05374 and A05375, expressed as snRNA copy number per nanogram of total RNA by ddPCR. Figure 6C shows Jess blots of dystrophin protein expression after treatment with either A05374 or A05375 at various MOIs. AAV null and untreated (NT) are negative controls. Lysates derived from wild-type (WT) myotubes were used to generate 0–50% standard curves for dystrophin (Dys) expression. Alpha-actinin (α-ACT) is a loading control. Figure 6D provides a graph showing quantification of dystrophin expression compared to WT levels, standardized with alpha-actinin. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 7A] Figures 7A–7C show the screening of synthetic snRNAs for targeting exon 45 skipping. U7 snRNAs were engineered to bind to splicing control sequences (SA, splice acceptor; SD, splice donor; and / or ESE, exon splice enhancer sequence) to facilitate exon 45 skipping, which results in the recovery of the DMD mRNA reading frame and dystrophin protein in DMD patient Δ44 mutations. Figure 7A provides a schematic diagram showing the expected RT-PCR product size using primers within exon 43 and exon 46 / 47 after transfecting human myotubes with 100 nM synthetic snRNA. The predicted size is 487 bp when exon 45 is included and 311 bp when exon 45 is excluded. Figure 7B shows TapeStation images of the RT-PCR products after transfecting with synthetic RNA. sR04, a synthetic snRNA that retains non-targeting sequences, and untreated cells (unt) were used as negative control samples. Figure 7C provides a graph showing the quantification in Figure 7B, which is shown as the percentage of exon 45 skipping (skipped bands / skipped bands + non-skipped bands). [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 8]Figures 8A–8B show the screening of synthetic snRNAs for identifying strong spacer sequences at lower concentrations (25 nM) of synthetic snRNA. Figure 8A shows TapeStation images of RT-PCR products after transfection with U7 snRNA synthetic RNA. sR04, a synthetic snRNA that retains a non-targeting sequence, and untreated cells (unt) were used as negative control samples. Exon 45 is predicted to be 487 bp if present and 311 bp if excluded. Figure 8B provides a graph showing the quantification of Figure 8A, expressed as the percentage of exon 45 skipping (skipped bands / skipped bands + non-skipped bands). [Figure 9] Figures 9A and 9B show AAV9-mediated exon 45 skipping in human Δ44 (exon 44 deletion) myotubes. Figure 9A provides a graph showing the percentage of exon 45 skipping 7 days after transduction with A05189 and A05190 (scAAV9 dual snRNA cassettes) with varying MOIs. Figure 9B provides a graph showing the copy number of snRNA per nanogram of total RNA assessed by ddPCR for snRNAs expressed from A05189 and A05190. [Figure 10] Figures 10A–10B show single-exon and multi-exon skipping of DMD exons 50–53. Figure 10A provides TapeStation images of RNA extracted by RT-PCR 7 days after transduction of human del52 myotubes with the AAV-based 4× construct A05178 for identifying exon skipping. A05178 holds engineered snRNAs targeting exons 50, 51, 52, and 53. Figure 10B provides a graph of the percentage of exon skipping. Exon-skipped bands were quantified by dividing the peak molar concentration of the amplicon (representing single-exon or multi-exon skipping) by the signal of the total DMD amplicon. [Figure 11]Figures 11A–11B illustrate a multi-exon skipping strategy for skipping exons 44 and 45. Figure 11A provides TapeStation images of RT-PCR products for identifying skipping 24 hours after transfecting human wild-type myotubes with U7 synthetic snRNA targeting exons 44 and 45. Figure 11B provides a graph of the percentage of exon skipping. To determine the multi-skipping percentage, the signal of the bands where exons 44 and 45 were skipped was divided by the signal of the total DMD amplicons. [Modes for carrying out the invention]

[0030] Detailed explanation This disclosure provides a gene therapy composition comprising a therapeutic RNA targeting platform comprising nuclear small RNA (snRNA) that targets a sequence of mRNA precursor (premRNA) or mRNA encoding dystrophin (DMD). The targeted premRNA or mRNA sequence may include the exon region of DMD and / or the splicing regulatory sequence of DMD.

[0031] Compositions comprising nucleic acid molecules and vectors containing DMD-targeting snRNA constructs are disclosed herein. The snRNA molecules of this disclosure may be non-natural, modified, and / or manipulated snRNAs (esnRNAs). The DMD-targeting esnRNAs of this disclosure include a mutant snRNA stem-loop. In some aspects, the DMD-targeting snRNAs of this disclosure include a native stem-loop.

[0032] Small nuclear ribonucleic acid (snRNAs) are essential components of small nuclear ribonucleoprotein complexes (snRNPs). When assembled with additional proteins, they form a large ribonucleoprotein complex known as the spliceosome, a cellular mechanism that mediates the entire mRNA maturation process. The spliceosome is responsible for mRNA precursor splicing, the process of removing introns from RNA transcripts before protein production. Individual snRNAs are generally about 250 nucleotides or less in size. For example, U1 snRNA is 164 nucleotides long and is encoded by a gene that exists in several copies in the human genome. U1 snRNA represents the ribonucleic acid component of the nuclear particle U1 snRNP. U1 snRNA has a stem-and-loop three-dimensional structure and a single-stranded sequence, generally about 9 nucleotides long, is located in the 5' region, which can bind to a splicing donor site on a premRNA molecule through complementary base pairing. (Horowitz et al., 1994, Trends Genet., 10(3):100-6). Various spliceosome snRNAs contain large amounts of uridylic acid and have therefore been named U1, U2, U4, U5, U6, U4ATAC, U6ATAC, U7, U11, and U12 (Mattaj et al., 1993, FASEB J, 15, 7:47-53).

[0033] snRNA systems can be used to treat toxic mutations. For example, in exons containing toxic mutations, antisense oligonucleotides that interfere with splice sites and regulatory elements can induce skipping of specific exons at the preRNA level. Such antisense sequences can be packaged into snRNA sequences and delivered using viral vectors that carry nucleic acid sequences to which the snRNA can be transcribed. U7 snRNAs are intrinsically involved in the 3' end processing of histone premRNAs, but small changes in the Sm / Lsm protein binding site can transform them into versatile tools for splicing modulation. In one such therapeutic strategy for treating Duchenne muscular dystrophy, modified U7 snRNAs are used to convert out-of-frame mutations into in-frame mutations, thereby producing functional dystrophin, although toxic RNA with a deleted interior is generated (Goyenvalle et al., 2009, 17(7):1234-1240).

[0034] Most U-rich snRNPs are complexes that mediate premRNA splicing. U7 snRNPs are an exception. U7 is not involved in splicing, but is a key factor in the unique 3' end processing of replication-dependent histone mRNA. By modifying the histone-binding sequence and Sm motif of U7 snRNA, U7 becomes unable to participate in histone premRNA processing and instead targets premRNA or smRNA for blockade or splicing modulation. Thus, U7 snRNA can be used as an effective gene therapy platform. The U7 snRNA platform also has the additional advantages of being compact in size, having the ability to accumulate in the nucleus without causing cytotoxicity, and possessing little to no immunoreactivity (Gadgil et al., 2021, J Gene Med, 23(4):e3321).

[0035] In several aspects, esnRNAs containing engineered stem-loops (eSLs) are disclosed herein. Compensatory modifications made to the native stem-loop sequence create engineered stem-loops (eSLs) that communicate (fold and anneal) more effectively with the snRNA interaction stabilization domain (ISD), resulting in an snRNA platform with increased stability. U7 snRNAs have been previously shown to be programmable to modulate mRNA. Improvements to programmed and engineered snRNAs usable as gene therapy tools are disclosed herein.

[0036] The snRNA systems disclosed herein are configured to bind to a target DMD RNA sequence to modulate RNA splicing, resulting in the skipping or inclusion of one or more exons of the targeted sequence of the DMD RNA. The DMD-targeting snRNAs are configured to bind to a DMD premRNA molecule at a site that regulates RNA splicing. This splicing regulatory site may include a splice acceptor sequence, a splice donor sequence, and an exon splice enhancer sequence. The snRNA sequences disclosed herein can induce exon skipping (of one or more exons) of the targeted exon sequence.

[0037] In one embodiment, these snRNAs are human snRNAs. In another embodiment, these snRNAs are mouse snRNAs. In another embodiment, the snRNAs are of any species. In another embodiment, the snRNAs are a combination of human and mouse snRNAs. In one embodiment, the U7 snRNA is either human U7 snRNA or mouse U7 snRNA. In another embodiment disclosed herein, the snRNAs include various types of snRNAs (e.g., U1-U12) by combining domains of endogenous snRNAs to fine-tune platform stabilization and / or reduce off-target effects. For example, in one embodiment, the snRNA system includes a combination of a human or mouse U7 snRNA component and a human or mouse U1 snRNA component.

[0038] Additional elements that can modulate the processing and abundance of RNA can be further incorporated into snRNA, or esnRNA containing eSL. In one embodiment, additional elements that can modulate the processing, stability, and abundance of esnRNA can be further incorporated into the 5' or 3' end of esnRNA. In another embodiment, such elements may include, but are not limited to, stem-loops, hairpins, GC clamps, kissing loops, triple helix, quadruple helix, and protein-binding sites.

[0039] The snRNA platforms and portions thereof disclosed herein may be used in any therapeutic environment and situation, provided that appropriate spacers or target sequences (TS) are included in the design of the therapeutic composition. In certain embodiments, the therapeutic snRNA composition is used to treat diseases associated with dysregulation, variant, or non-functional dystrophin. In some aspects, the disease or disorder is Duchenne muscular dystrophy.

[0040] Targeting array An snRNA system can be programmed to include a targeting sequence (TS) (also called a "spacer") that targets an RNA of interest. An snRNA system can be programmed by one or more targeting sequences that target one or more RNAs of interest. In some cases, the targeting sequence is a 5' targeting sequence (5'TS) that targets one or more RNAs of interest. This is TIFF2026534279000002.tif5128. In this regard, 5' refers to the 5' end of the snRNA insert and not necessarily the entire vector structure including the snRNA insert. The TS may be located at or near the 5' end of the snRNA. In another embodiment, in particular when the snRNA construct is not a U7-based snRNA, the targeting sequence (TS) may be located at or near the 3' position within the snRNA construct, and thus generate a 3' targeting sequence (3'TS).

[0041] The targeting sequences of this disclosure, including the 5'TS and 3'TS, may be about 1 to about 200 nucleotides in length. In some aspects, the targeting sequences of this disclosure are about 10 to about 150 nucleotides in length. In some aspects, the targeting sequences of this disclosure are about 10 to about 100 nucleotides in length. In some aspects, the targeting sequences of this disclosure are about 20 to about 60 nucleotides in length. In some aspects, the targeting sequences of this disclosure are at least about 10, 20, 30, 40, 50, 60, or about 70 nucleotides in length.

[0042] The snRNA compositions of this disclosure may include multiple targeting sequences, each of which binds to a different RNA sequence. In some aspects, the esnRNAs of this disclosure include a fusion targeting sequence. In some aspects, the fusion targeting sequence is a nucleic acid sequence comprising two targeting sequences that are directly linked or linked by one or more linker nucleic acid sequences, each of which binds to a different target RNA sequence.

[0043] In one example, a U7 snRNA may be programmed by substituting its histone mRNA-binding sequence with a sequence complementary to the target of interest. In several aspects, the snRNA systems of this disclosure bind to the target mRNA or premRNA sequence of interest. The exemplary snRNA systems shown herein result in exon skipping (e.g., DMD exon skipping in the case of DMD treatment).

[0044] In some embodiments, the snRNAs of this disclosure target a premRNA or mRNA sequence encoding the DMD gene. DMD is a gene encoding the protein dystrophin. Mutations in DMD are associated with Duchenne muscular dystrophy. In some embodiments, the DMD RNA sequence targeted by the snRNA composition of this disclosure may be any exon or intron of the DMD RNA sequence. In some embodiments, the DMD RNA sequence targeted by the snRNA composition of this disclosure is exon 2, exon 44, exon 45, exon 51, or exon 53 of the DMD RNA sequence. In some embodiments, the DMD RNA sequence targeted by the snRNA composition of this disclosure is a combination of sequences from the group consisting of exons 1, 2, 3, 4, 5, 44, 45, 50, 51, 52, 53, 54, and 55 of the DMD RNA sequence, such that several exons are skipped. In some embodiments, the targeting sequence targets the RNA of interest to skip DMD exons 50, 51, 52, and 53. In some embodiments, the targeting sequence targets the RNA of interest to skip exons 44 and 45. In some embodiments, the DMD RNA sequence targeted by the snRNA composition of this disclosure is a combination of sequences from the group consisting of exon 1, exon 2, exon 3, exon 4, and exon 5. In some embodiments, the DMD RNA sequence targeted by the snRNA of this disclosure is a splicing control sequence selected from splice acceptor sequences, splice donor sequences, and / or exon splice enhancer sequences.

[0045] In some embodiments, the nucleic acid sequence encoding wild-type human DMD mRNA includes or consists of SEQ ID NO:139. In some embodiments, the nucleic acid sequence encoding wild-type human DMD mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence described in SEQ ID NO:139. In some embodiments, the nucleic acid sequence encoding wild-type human DMD pre-mRNA includes or consists of SEQ ID NO:140. In some embodiments, the nucleic acid sequence encoding wild-type human DMD pre-mRNA includes a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence described in SEQ ID NO:140. In some embodiments, the nucleic acid sequence encoding human DMD includes a deletion. In some embodiments, the deletion is a single nucleotide. In some embodiments, the deletion may be one or more nucleotides. In some embodiments, the deletion may be an exon sequence or an intron sequence. In some embodiments, the deletion may include both an intron sequence and an exon sequence. In some embodiments, the deletion may be located in a region containing one or more of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55, as well as any upstream or downstream introns between these exons. In some embodiments, the amino acid sequence encoding dystrophin may include or consist of SEQ ID NO:141. In some embodiments, the amino acid sequence of human dystrophin may include a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence described in SEQ ID NO:141.

[0046] The target sequence that binds to DMD exon 2 contains, essentially, or may contain at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) nucleic acid sequences that are identical to one or more of the following nucleotide sequences listed in Table 1 below.

[0047] (Table 1) DMD exon 2 RNA targeting sequences TIFF2026534279000003.tif96170

[0048] In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 59. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 60. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 61. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 62. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 63. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 64. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 65. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 66. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 67. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 68. In some embodiments, the target sequence that binds to human DMD exon 2 includes the sequence described in SEQ ID NO: 69. In some embodiments, the target sequence that binds to human DMD exon 2 includes one or more sequences described in any of SEQ ID NO: 334 to 674. In some embodiments, the target sequence that binds to human DMD exon 2 includes one or more sequences described in any of SEQ ID NO: 675 to 1011. In some embodiments, the target sequence that binds to human DMD exon 2 includes one or more sequences described in any of SEQ ID NO: 1012 to 1344.

[0049] The target sequence that binds to DMD exon 44 contains, essentially, or may contain, one or more nucleic acid sequences listed in Table 2 below that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences.

[0050] (Table 2) DMD exon 44 RNA targeting sequences TIFF2026534279000004.tif228153

[0051] In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:70. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:71. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:72. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:73. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:74. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:75. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:76. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:77. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:78. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:79. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:80. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:81. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:82. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:83. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:84. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:85. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:86.In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:87. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:88. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:89. In some embodiments, the target sequence that binds to human DMD exon 44 includes the sequence described in SEQ ID NO:90.

[0052] The target sequence that binds to DMD exon 45 contains, essentially, or may contain, one or more nucleic acid sequences listed in Table 3 below that are identical by at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of the following nucleotide sequences.

[0053] (Table 3) DMD exon 45 RNA targeting sequences TIFF2026534279000005.tif84170TIFF2026534279000006.tif236170

[0054] In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:91. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:92. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:93. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:94. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:95. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:96. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:97. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:98. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:99. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 100. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 101. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 102. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 103. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 104. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 105. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 106. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 107.In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 108. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 109. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 110. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 111. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 112. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 113. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 114. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 115. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO: 116. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:117. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:118. In some embodiments, the target sequence that binds to human DMD exon 45 includes the sequence described in SEQ ID NO:126.

[0055] The target sequence that binds to DMD exon 51 contains, essentially, or may contain, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of the following nucleotide sequences listed in Table 4 below.

[0056] (Table 4) DMD exon 51 RNA targeting sequence TIFF2026534279000007.tif208163

[0057] In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:206. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:207. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:208. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:209. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:210. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:211. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:212. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:213. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:214. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:215. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:216. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:217. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:218. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:219. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:220. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:221. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:222.In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:223. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:224. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:225. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:226. In some embodiments, the target sequence that binds to human DMD exon 51 includes the sequence described in SEQ ID NO:227.

[0058] The target sequence that binds to DMD exon 53 contains, essentially, or may contain, one or more nucleic acid sequences listed in Table 5 below that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences.

[0059] (Table 5) DMD exon 53 RNA targeting sequence TIFF2026534279000008.tif104170TIFF2026534279000009.tif231170TIFF2026534279000010.tif230170TIFF2026534279000011.tif23017 0TIFF2026534279000012.tif235170TIFF2026534279000013.tif239170TIFF2026534279000014.tif235170TIFF2026534279000015.tif35170

[0060] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:237. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:238. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:239. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:240. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:241. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:242. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:243. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:244. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:245. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:246. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:247. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:248. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:249.

[0061] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:250. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:251. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:252. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:253. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:254. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:255. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:256. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:257. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:258. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:259.

[0062] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:260. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:261. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:262. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:263. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:264. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:265. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:266. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:267. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:268. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:269.

[0063] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:270. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:271. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:272. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:273. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:274. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:275. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:276. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:277. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:278. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:279.

[0064] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:280. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:281. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:282. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:283. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:284. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:285. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:286. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:287. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:288. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:289.

[0065] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:290. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:291. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:292. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:293. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:294. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:295. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:296. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:297. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:298. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:299.

[0066] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:300. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:301. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:302. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:303. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:304. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:305. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:306. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:307. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:308. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:309.

[0067] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:310. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:311. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:312. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:313. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:314. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:315. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:316. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:317. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:318. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:319.

[0068] In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:320. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:321. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:322. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:323. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:324. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:325. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:326. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:327. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:328. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:329. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:330. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:331. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:332. In some embodiments, the target sequence that binds to human DMD exon 53 includes the sequence described in SEQ ID NO:333.

[0069] Manipulated stem loop The engineered snRNA (esnRNA) systems disclosed herein may include engineered stem-loops (eSLs) that include compensatory modifications to the native snRNA stem-loop. These modifications result in increased stability of the engineered nuclear small ribonucleoprotein complex (eSNRNP) compared to snRNPs containing unmodified stem-loops. The eSLs disclosed herein may be derived from any snRNP, such as U1-U12. In one embodiment, the eSL is a human or mouse U7 eSL. In one embodiment, the eSL is a human or mouse eSL. In several embodiments, the eSL is a human and mouse eSL. In several embodiments, the eSL is a non-human eSL selected from the group consisting of mouse, pig, sheep, goat, cattle, dog, cat, horse, or combinations thereof. In several embodiments, the eSL is a non-human eSL selected from the group consisting of human, mouse, pig, sheep, goat, cattle, dog, cat, horse, or combinations thereof. In some embodiments, the eSL sequence is not a native stem-loop sequence. In some embodiments, the nucleic acid sequence of the eSL is not a native stem-loop sequence by at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between). The manipulated stem-loop is described in WO2023168458, the contents of which are incorporated herein by reference in their entirety.

[0070] In some embodiments, human eSL has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000016.tif85128.

[0071] In some embodiments, the human eSL includes the sequence described in SEQ ID NO:1. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:2. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:3. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:4. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:5. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:6. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:7. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:8. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:9. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:10. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:111. In some embodiments, the human eSL includes the sequence described in SEQ ID NO:186.

[0072] In some embodiments, the mouse eSL has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000017.tif49131.

[0073] In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:164. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:146. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:147. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:148. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:228. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:229. In some embodiments, the mouse eSL contains the sequence described in SEQ ID NO:230.

[0074] In some embodiments, the human or mouse eSL is the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000018.tif20138.

[0075] In some embodiments, the human or mouse eSL includes the sequence described in SEQ ID NO:190. In some embodiments, the human or mouse eSL includes the sequence described in SEQ ID NO:191. In some embodiments, the human or mouse eSL includes the sequence described in SEQ ID NO:192.

[0076] In some embodiments, the eSL of a dog or cat is the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000019.tif5128.

[0077] In some embodiments, the canine or feline eSL includes the sequence described in SEQ ID NO:193.

[0078] In some embodiments, the eSL of a cattle, sheep, or goat is the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000020.tif12134.

[0079] In some embodiments, the eSL of a cattle, sheep, or goat includes the sequence described in SEQ ID NO:194. In some embodiments, the eSL of a cattle, sheep, or goat includes the sequence described in SEQ ID NO:195.

[0080] In some embodiments, porcine eSL has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000021.tif34141.

[0081] In some embodiments, the porcine eSL contains the sequence described in SEQ ID NO:198. In some embodiments, the porcine eSL contains the sequence described in SEQ ID NO:199. In some embodiments, the porcine eSL contains the sequence described in SEQ ID NO:200.

[0082] In some embodiments, the horse eSL has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000022.tif12136.

[0083] In some embodiments, the equine eSL includes the sequence described in SEQ ID NO:201. In some embodiments, the equine eSL includes the sequence described in SEQ ID NO:202.

[0084] In some embodiments, the sheep eSL has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical with one or more of TIFF2026534279000023.tif19144.

[0085] In some embodiments, the sheep eSL includes the sequence described in SEQ ID NO:203. In some embodiments, the sheep eSL includes the sequence described in SEQ ID NO:204. In some embodiments, the sheep eSL includes the sequence described in SEQ ID NO:205.

[0086] In some embodiments, the manipulated stem-loop provides enhanced snRNA stability compared to snRNA containing a native stem-loop. In some embodiments, the native snRNA stem-loop has the following nucleotide sequence: It contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to one or more of TIFF2026534279000024.tif41134.

[0087] In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:145. In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:144. In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:235. In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:236. In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:142. In some embodiments, the native snRNA stem-loop includes the sequence described in SEQ ID NO:143.

[0088] 5' interaction stabilization domain The eSLs disclosed herein possess more effective folding and annealing properties due to the 5' interaction stabilization domain (5'ISD), which results in increased esnRNA stability compared to unmanipulated snRNA. The 5'ISD has nucleotides complementary to the nucleotides in the manipulated SL, and although we do not wish to be constrained by theory, the interaction between the 5'ISD and the eSL is predicted to form a secondary structure that protects the 5' end of the snRNA. In some aspects, the 5'ISD anneals and / or hybridizes with the eSLs of this disclosure. In some aspects, the 5'ISD is a sequence that is complementary and / or reverse complementary to the sequence present in the eSLs of this disclosure. In some aspects, the 5'ISD disclosed herein has the following nucleotide sequence: It could be one of the 5'ISDs selected from TIFF2026534279000025.tif85128.

[0089] Sm-joint domain The snRNA systems disclosed herein utilize an Sm-binding domain (SmBD). The Sm protein ring assembled around the Sm-binding domain (SmBD) to form snRNPs includes SmB / B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. The U7 Sm-binding site recruits an endogenous RNA-binding factor and can be substituted for a non-U7 snRNA to make the esnRNA more stable. In one embodiment, the SmBD is selected from the group consisting of U1, U2, U4, and U5 snRNAs. In another embodiment, the SmBD is derived from a pseudo-snRNA. In another embodiment, the SmBD is a nucleotide sequence containing SEQ ID NO:31(ATTTTT). In another embodiment, the SmBD is It contains a nucleotide sequence selected from the group consisting of TIFF2026534279000026.tif34170.

[0090] Promoter array The snRNA systems disclosed herein include an snRNA promoter derived from one of U1 to U12. In one embodiment, the snRNA promoter is the U7 promoter. In another embodiment, the U7 promoter is the human U7 promoter (hU7) or the mouse U7 promoter (mU7). In yet another embodiment, the U7 promoter is SEQ ID NO:39 TIFF2026534279000027.tif41159 is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical endogenous human U7 promoter.

[0091] In one embodiment, the snRNA promoter is the U1 promoter. In another embodiment, the U1 promoter is the human U1 promoter or the mouse U1 promoter.

[0092] In another embodiment, the same snRNA promoter drives the expression of each copy of the snRNA insert. In another embodiment, each copy of the snRNA insert is identical. In another embodiment, different snRNA promoters drive each copy of the snRNA insert. In one embodiment, 2×snRNA includes a mouse U7 promoter that drives one copy of the snRNA insert and a mouse U1 promoter that drives the other copy of the snRNA insert.

[0093] In other contexts, the snRNA promoter is either a Pol II promoter or a Pol III promoter. In other contexts, the snRNA promoter contains, is essentially, or consists of nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the promoters and / or promoter sequences listed in Table 6 below.

[0094] (Table 6) Table of exemplary promoters TIFF2026534279000028.tif251160TIFF2026534279000029.tif242160TIFF2026534279000030.tif226160

[0095] In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:40. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:41. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:42. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:43. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:44. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:45. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:46. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:47. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:48. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:151. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:39. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:152. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:153. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:154. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:155. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:165. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:166. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:167. In some embodiments, the snRNA promoter includes the sequence described in SEQ ID NO:168.

[0096] Terminator array The snRNA systems disclosed herein include an snRNA downstream terminator (DT). The downstream terminator defines the end of a transcription unit such as esnRNA or snRNA. In another embodiment, the snRNA DT is This is a U7 DT file containing TIFF2026534279000031.tif12160.

[0097] In one embodiment, the esnRNA comprises an eSL, one or more promoters, a TS targeting the DMD, a SmBD, a 5'ISD, and a DT. In one aspect, combinations of promoters and DTs are mixed and matched. In another embodiment, the snRNA comprises a native stem loop, one or more promoters, a TS targeting the DMD, a SmBD, and a DT.

[0098] In some embodiments, DT contains, essentially consists of, or comprises nucleic acid sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the DT sequences listed in Table 7 below.

[0099] (Table 7) Exemplary DT table TIFF2026534279000032.tif214160TIFF2026534279000033.tif121160

[0100] In some embodiments, DT includes the sequence described in SEQ ID NO: 50. In some embodiments, DT includes the sequence described in SEQ ID NO: 51. In some embodiments, DT includes the sequence described in SEQ ID NO: 52. In some embodiments, DT includes the sequence described in SEQ ID NO: 53. In some embodiments, DT includes the sequence described in SEQ ID NO: 54. In some embodiments, DT includes the sequence described in SEQ ID NO: 55. In some embodiments, DT includes the sequence described in SEQ ID NO: 56. In some embodiments, DT includes the sequence described in SEQ ID NO: 57. In some embodiments, DT includes the sequence described in SEQ ID NO: 58. In some embodiments, DT includes the sequence described in SEQ ID NO: 156. In some embodiments, DT includes the sequence described in SEQ ID NO: 49. In some embodiments, DT includes the sequence described in SEQ ID NO: 234. In some embodiments, DT includes the sequence described in SEQ ID NO: 157. In some embodiments, DT includes the sequence described in SEQ ID NO: 158. In some embodiments, DT includes the sequence described in SEQ ID NO: 169. In some embodiments, DT includes the sequence described in SEQ ID NO:170. In some embodiments, DT includes the sequence described in SEQ ID NO:171. In some embodiments, DT includes the sequence described in SEQ ID NO:172. In some embodiments, DT includes the sequence described in SEQ ID NO:TTTTTT.

[0101] In one embodiment, snRNA is delivered via a vector.

[0102] In one embodiment, snRNA is delivered via an AAV vector.

[0103] In some embodiments, the AAV vector contains multiple copies of snRNA. In some embodiments, the multiple copies of snRNA are 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of snRNA (2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×). In some embodiments, the multiple copies of snRNA are 4 or more copies of snRNA.

[0104] In some embodiments, each of the multiple copies of an snRNA is separated by a nucleic acid buffer sequence derived from a human non-coding genome sequence downstream of the snRNA. In one embodiment, the buffer sequence is derived from a human genome sequence downstream of U7.

[0105] In one embodiment, the buffer sequence is the following nucleic acid sequence: Selected from the group consisting of TIFF2026534279000034.tif26153 and combinations thereof.

[0106] In another embodiment, the buffer sequence is the following nucleic acid sequence: Selected from the group consisting of TIFF2026534279000035.tif49160 and combinations thereof.

[0107] In another embodiment, the buffer sequence is the following nucleic acid sequence: Selected from the group consisting of TIFF2026534279000036.tif158161 and / or combinations thereof.

[0108] The 100bp and 500bp buffer 1 sequences are derived from a sequence starting 100bp downstream of the mouse (mus musculus) U7 pseudogene 8 (location, chromosome 14: 4,409,359-4,409,421, reverse strand, GRCm39:CM001007.3). The 100bp and 500bp buffer 2 sequences are derived from a sequence starting 130bp downstream of the human U7 pseudogene 5 (chromosome X: 140,451,148-140,451,208, forward strand, GRCh38:CM000685.2). Each 100bp buffer is the first 100bp of the corresponding 500bp buffer. 30bp buffers 1, 2, and 3 are consecutive 30bp sequences within "100bp buffer 1" downstream of the mouse U7 pseudogene 8. These downstream sequences were selected because they do not contain any known regulatory sites or genes within or near them (using Gencode / Ensembl), they do not contain any repetitive sequences, the GC content of the entire buffer is 40-60%, the GC content of the 20bp regions at both ends of the buffer is 40-60%, and they have minimal sequence complexity.

[0109] snRNA sequence The exemplary snRNA sequences in this disclosure may include any combination of the characteristics of the esnRNA or snRNA described herein.

[0110] vector Vectors containing snRNAs that target DMD (e.g., recombinant expression vectors) are also provided herein.

[0111] In some embodiments of the compositions and methods of this disclosure, the vector comprises a DMD-targeting snRNA system provided herein. In some embodiments, the vector is a single vector or a unitary vector.

[0112] In some embodiments, an snRNA system can target one or more DMD RNA sequences. In some aspects, the DMD RNA sequence is a DMD premRNA sequence. In some aspects, the snRNA system can target multiple (i.e., two or more) RNAs of interest. In some embodiments, the two or more RNAs of interest may be the same premRNA molecule, but may be different sequences within the premRNA molecule.

[0113] With respect to recombinant expression vectors, the term "functionally linked" means, for example, that the hybrid promoter is linked to the nucleotide sequence of interest (NOI) in a manner that enables the expression of the nucleotide sequence when the vector is introduced (or brought into contact) with the host cell.

[0114] In some embodiments of the compositions and methods of this disclosure, the vector comprises a DMD-targeting snRNA. In some embodiments, the therapeutic snRNA is contained in a single vector or an integrated vector.

[0115] In some embodiments of the compositions and methods of this disclosure, an RNA-binding snRNA system can target a DMD RNA sequence. In some embodiments of the compositions and methods of this disclosure, an RNA targeting system can target one or more DMD RNA sequences. In some aspects, the DMD RNA sequence is a DMD premRNA sequence. In some aspects, the snRNA system can target multiple (i.e., two or more) RNAs of interest. In some embodiments, the two or more RNAs of interest may be the same premRNA molecule, but may be different sequences within the premRNA molecule.

[0116] One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, which contains a DNA or RNA sequence derived from a virus for packaging into the virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses). Viral vectors also contain polynucleotides held by the virus for transfection into host cells. In some embodiments, the vector is a lentiviral vector (e.g., an integration-deficient lentiviral vector) or an adeno-associated virus (AAV) vector. Vectors such as bacterial vectors with bacterial origins of replication and episomatic mammalian vectors are capable of autonomous replication in the host cell into which they are introduced, while other vectors, such as non-episomatic mammalian vectors, are integrated into the host cell's genome when introduced into the host cell and thereby replicate together with the host genome.

[0117] In some embodiments, vectors, such as expression vectors, can direct the expression of genes to which they are functionally linked. Common expression vectors often take the form of plasmids. In some embodiments, recombinant expression vectors contain nucleic acids provided herein, such as esnRNA, in a form suitable for the expression of RNA molecules in host cells. Recombinant expression vectors include one or more regulatory elements, which may be selected based on the host cell used for expression and are functionally linked to the nucleic acid sequence to be expressed. In recombinant expression vectors, "functionally linked" means that the nucleotide sequence of interest is linked to the regulatory element in a manner that enables the expression of the nucleotide sequence, for example, in an in vitro transcription / translation system or when the vector is introduced into a host cell. Certain embodiments of a vector depend on factors such as the selection of the host cell to be transformed and the desired expression level. By introducing a vector into a host cell, it is possible to produce transcripts, proteins, or peptides (including fusion proteins or fusion peptides), CRISPR transcripts, proteins, enzymes, their variants, their fusion proteins, etc., encoded by nucleic acids described herein, such as snRNA.

[0118] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, expression regulatory elements. “Expression regulatory element” means any sequence that controls the expression of a coding sequence, such as a gene, as used herein. Exemplary expression regulatory elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression regulatory elements may be, for example, constitutive, inducible, repressive, or tissue-specific. “Promoter” is a regulatory sequence that is a region of polynucleotide sequence on which the initiation and rate of transcription are regulated. It may contain gene elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. “Enhancer” is a region of DNA to which an activating protein that increases the likelihood or frequency of transcription can bind.

[0119] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, vector elements such as buffer sequences derived from human genome sequences downstream of the snRNA, and thus have the ability to encode multiple snRNAs from a single construct.

[0120] In some embodiments, the snRNA constructs disclosed herein include a bidirectional snRNA promoter for expressing the snRNA.

[0121] In another embodiment, the vector configuration may include a linker, a signal sequence, and / or tags.

[0122] Viral vector In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector. In some embodiments, the vector is a retrovirus vector, an adenovirus / retrovirus chimeric vector, a herpes simplex virus type I or type II vector, a parvovirus vector, a retinal endotheliopathy virus vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector, or a hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors.

[0123] In some embodiments of the compositions and methods of this disclosure, the vectors of this disclosure are viral vectors. In some embodiments, the viral vectors include sequences isolated from or derived from retroviruses. In some embodiments, the viral vectors include sequences isolated from or derived from lentiviruses. In some embodiments, the viral vectors include sequences isolated from or derived from adenoviruses. In some embodiments, the viral vectors include sequences isolated from or derived from adeno-associated viruses (AAVs). In some embodiments, the viral vectors are non-replicable. In some embodiments, the viral vectors are isolated or recombinant. In some embodiments, the viral vectors are self-complementary.

[0124] In some embodiments, the vector further comprises one or more expression regulatory elements functionally linked to a polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the vector has low toxicity. In some embodiments, the vector is not integrated into the host genome and therefore has a low probability of causing insertional mutagenesis.

[0125] Adeno-associated virus vector "AAV vector" means a vector comprising, essentially consisting of, or comprising one or more nucleic acid molecules and one or more AAV inverted terminal repeat sequences (ITRs), as used herein. In some cases, the nucleic acid molecules encode the esnRNA of this disclosure. Such an AAV vector may be replicated and packaged into infectious viral particles when present in a host cell that provides functionality for the gene products of rep and cap, for example, by transfection of the host cell. In some cases, the AAV vector contains a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or an enhancer and / or a terminator within adjacent ITRs, and is packaged into infectious AAV particles. The nucleic acid portion encapsulated in the capsid may be called the AAV vector genome. Plasmids containing an AAV vector may also contain elements for the purpose of production, such as antibiotic resistance genes, origin of replication sequences, etc., but these are not encapsulated in the capsid and therefore do not form part of the AAV particle.

[0126] In some aspects, an AAV vector may contain at least one nucleic acid encoding the composition of the snRNA or esnRNA of this disclosure. In some aspects, an AAV vector may contain at least one control sequence. In some aspects, an AAV vector may contain at least one AAV inverse end (ITR) sequence. In some aspects, an AAV vector may contain a first ITR sequence and a second ITR sequence. In some aspects, an AAV vector may contain at least one promoter sequence. In some aspects, an AAV vector may contain at least one enhancer sequence. In some aspects, an AAV vector may contain at least one terminator sequence. In some aspects, an AAV vector may contain at least one poly(A) sequence. In some aspects, an AAV vector may contain at least one linker sequence. In some aspects, an AAV vector may contain at least one buffer sequence. In some aspects, an AAV vector of this disclosure may contain at least one nuclear localization signal or / or nuclear export signal.

[0127] In some cases, the AAV vector may include a first AAV ITR sequence, a promoter sequence, an snRNA sequence and / or an esnRNA sequence, a terminator sequence, and a second AAV ITR sequence. In some cases, the AAV vector may include, from 5' to 3', a first AAV ITR sequence, a promoter sequence, an esnRNA sequence, a terminator sequence, and a second AAV ITR sequence. In some cases, the AAV vector may include, from 5' to 3', a first AAV ITR sequence, a promoter sequence, an snRNA sequence, a terminator sequence, and a second AAV ITR sequence.

[0128] In some cases, an AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, a termination sequence, a second promoter sequence, a second snRNA sequence, a second termination sequence, and a second AAV ITR sequence. In some cases, an AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, a termination sequence, a second promoter sequence, a second snRNA sequence, a second termination sequence, a third promoter sequence, a third snRNA sequence, a third termination sequence, and a second AAV ITR sequence. In some cases, an AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, termination, a second promoter sequence, a second snRNA sequence, a second termination sequence, and a second AAV ITR sequence. In some aspects, the AAV vector may include a first AAV ITR sequence, a first promoter sequence, a first snRNA sequence, termination, a second promoter sequence, a second snRNA sequence, a second termination sequence, a third promoter sequence, a third snRNA sequence, a third termination sequence, a fourth promoter sequence, a fourth snRNA sequence, a fourth termination sequence, and a second AAV ITR sequence.

[0129] The terms “adeno-associated virus” or “AAV,” as used herein, refer to a class of viruses belonging to the genus Dependoparvovirus in the family Parvoviridae, in relation to this name. Adeno-associated viruses are single-stranded DNA viruses that replicate in cells, with certain functions provided by co-infecting helper viruses. General information and an overview of AAVs can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228 and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). Since it is well known that various serotypes are extremely closely related structurally, functionally, and even at the genetic level, it is quite expected that the same principles described in these overviews also apply to additional AAV serotypes characterized since the publication date of these overviews (see, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to exhibit extremely similar replication characteristics mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. Furthermore, the degree of relatedness is suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes throughout the entire genome and the presence of similar self-annealing segments corresponding to "inverted terminal repeats" (ITRs) at the ends. Similar infection patterns also suggest that the replication function of each serotype is under similar regulatory control. Several serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from various tissue types.

[0130] AAV possesses unique properties that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cultured cells is non-cellular, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells and target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted into plasmids as cloned DNA, enabling the construction of recombinant genomes. Furthermore, since signals directing AAV replication and capsid encapsulation of the genome are contained within the ITR of the AAV genome, an AAV vector can be generated by replacing part or all of approximately 4.3 kb inside the genome (encoding the replication protein and structural capsid protein rep-cap) with foreign DNA. The rep and cap proteins can be supplied in trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. Since AAV readily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), chilling of AAV is not particularly important. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0131] The recombinant AAV (rAAV) genome of the present invention comprises, essentially consists of, or comprises a nucleic acid molecule encoding at least one esnRNA and one or more AAV ITRs adjacent to that nucleic acid molecule. The construction of pseudotype rAAV is disclosed, for example, in WO2001083692. Other types of rAAV variants, e.g., rAAVs containing capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Nucleotide sequences of genomes of various AAV serotypes are known in the art.

[0132] In some aspects of the compositions and methods of this disclosure, the viral vector comprises a sequence isolated from or derived from adeno-associated virus (AAV). In some aspects, the viral vector comprises an inverted terminal repeat sequence or capsid sequence isolated from or derived from AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRh10, AAV11, or AAV12. In some aspects, the AAV serotype is AAVrh.74. In one aspect, the AAV vector comprises a modified capsid. In one aspect, the AAV vector is an AAV2-Tyr mutant vector. In one embodiment, the AAV vector comprises a capsid having a non-tyrosine amino acid at a position corresponding to the surface-exposed tyrosine residue at the positions of Tyr252, Tyr272, Tyr275, Tyr281, Tyr508, Tyr612, Tyr704, Tyr720, Tyr730, or Tyr673 of wild-type AAV2. See also WO2008 / 124724, which is incorporated herein in its entirety. In several embodiments, the AAV vector comprises an engineered capsid. AAV vectors comprising an engineered capsid include, but are not limited to, AAV2.7m8, AAV9.7m8, AAV2 2tYF, and AAV8 Y733F. In several embodiments, the capsid is a ubiquitination-resistant capsid. In another embodiment, the ubiquitinated capsid is an AAV2 capsid containing tyrosine (Y) and serine (S) mutations. In another embodiment, the AAV2 capsid contains Y, S, and threonine (T) mutations. In another embodiment, the AAV2 capsid includes, non-limitingly, AAV2 capsid variants such as T455V, T491V, T550V, T659V, Y444+500+730F, and Y444+500+730F+T491V. In some embodiments, the viral vector is non-replicable. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV). In some embodiments, the viral vector is single-stranded (ssAAV).

[0133] In some embodiments, the snRNAs provided herein are contained in single-stranded AAV (ssAAV). In some embodiments, the snRNAs provided herein are contained in self-complementary AAV (scAAV). Due to the single-stranded nature of the parvovirus genome, gene expression requires the use of a cellular mechanism that provides a complementary strand. This cellular recruitment activity is considered the rate-limiting factor in the efficiency of transduction and gene expression in parvoviruses and parvovirus particles. Compared to ssAAV, the use of scAAV solves this well-known problem by packaging both strands as a single double-stranded DNA molecule (or inverted repeat genome) that can be folded into dsDNA as a result of a self-complementary viral genome sequence. In this regard, the need for DNA synthesis or base pairing between multiple viral genomes is eliminated.

[0134] AAV ITR array In some embodiments of the compositions and methods of the present disclosure, the AAV inverted terminal repeat sequence may include any AAV ITR sequence known in the art. In some aspects, the AAV ITR sequence may include or consist of the AAV1 ITR sequence, AAV2 ITR sequence, AAV3 ITR sequence, AAV4 ITR sequence, AAV5 ITR sequence, AAV6 ITR sequence, AAV7 ITR sequence, AAV8 ITR sequence, AAV9 ITR sequence, AAV10 ITR sequence, AAVrh10 ITR sequence, AAV11 ITR sequence, AAV12 ITR sequence, AAV13 ITR sequence, or AAVrh74 ITR sequence.

[0135] In some aspects, the ITR sequence may contain a modified AAV ITR sequence.

[0136] In some aspects, the AAV ITR sequence contains, essentially, or may contain, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:119 or SEQ ID NO:166.

[0137] In some aspects, the first AAV ITR sequence contains, essentially consists of, or may consist of, a nucleic acid sequence identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of, SEQ ID NO:119 or SEQ ID NO:166, and the second AAV ITR sequence contains, essentially consists of, or may consist of, a nucleic acid sequence identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) of, SEQ ID NO:119 or SEQ ID NO:166. In some aspects, the first AAV ITR sequence is located at 5' of the AAV vector. In some cases, the second AAV ITR sequence is located at 3' of the AAV vector.

[0138] In some embodiments of the compositions and methods of this disclosure, the viral vector comprises a sequence isolated from or derived from adeno-associated virus (AAV). In some embodiments of the compositions and methods of this disclosure, the vector is a non-viral vector. In some embodiments, the vector comprises or consists of nanoparticles, micelles, liposomes or lipoplexes, polymerosomes, polyplexes or dendrimers. In some embodiments, the vector is an expression vector or a recombinant expression system. As used herein, the term “recombinant expression system” refers to a gene construct for the expression of a particular genetic material formed by recombinant.

[0139] lentiviral vectors In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector can encode a total polynucleotide in the range of 8kb to 10kb. In some embodiments, exemplary lentiviral vectors that may be used in any of the compositions, systems, methods, and kits described herein include human immunodeficiency virus (HIV) 1 vector, modified human immunodeficiency virus (HIV) 1 vector, human immunodeficiency virus (HIV) 2 vector, modified human immunodeficiency virus (HIV) 2 vector, and Sooty Mangabei. This may include mangabey simian immunodeficiency virus (SIVSM) vectors, modified sooty mangabey simian immunodeficiency virus (SIVSM) vectors, African green monkey immunodeficiency virus (SIVAGM) vectors, modified African green monkey immunodeficiency virus (SIVAGM) vectors, equine infectious anemia virus (EIAV) vectors, modified equine infectious anemia virus (EIAV) vectors, feline immunodeficiency virus (FIV) vectors, modified feline immunodeficiency virus (FIV) vectors, visna / maedi virus (VNV / VMV) vectors, modified visna / maedi virus (VNV / VMV) vectors, canine arthritis encephalitis virus (CAEV) vectors, modified canine arthritis encephalitis virus (CAEV) vectors, bovine immunodeficiency virus (BIV), or modified bovine immunodeficiency virus (BIV), and any combination or equivalent thereof. In some embodiments, the lentiviral vector is an integrase-possessing lentiviral vector (ICLV). In some embodiments, a lentiviral vector may refer to a transgene plasmid vector, a transgene plasmid vector combined with an associated plasmid (e.g., a packaging plasmid, a rev expression plasmid, or an envelope plasmid), or a lentiviral-based particle capable of introducing exogenous nucleic acids into cells via a viral or viral-like entry mechanism. In some embodiments, the viral vector may contain a sequence isolated from or derived from a retrovirus.In some embodiments, the viral vector comprises a sequence isolated from or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated from or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is non-replicable. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0140] Lentiviral vectors are well known in the art (see, for example, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg and Durand et al. (2011) Viruses 3(2):132-159 doi:10.3390 / v3020132).

[0141] The lentiviral vectors described herein comprise, essentially consist of, or may consist of, one or more nucleic acid molecules and one or more lentiviral LTRs. In some aspects, the nucleic acid molecules encode the snRNA and / or esnRNA of this disclosure. Such lentiviral vectors can be replicated and packaged into infectious viral particles when present in a host cell providing functionality for the rep and cap gene products, for example, by transfection of the host cell. In some aspects, the lentiviral vector comprises, within adjacent LTRs, a promoter, at least one nucleic acid capable of encoding at least one protein or RNA, and / or an enhancer and / or a terminator, and is packaged into an infectious lentiviral particle. The nucleic acid portion encapsulated in the capsid may be called the lentiviral vector genome. Plasmids containing lentiviral vectors may also contain elements for the purpose of production, such as antibiotic resistance genes, origin sequences, etc., but these are not encapsulated in the capsid and therefore do not form part of the lentiviral particle.

[0142] In some aspects, a lentiviral vector may include at least one nucleic acid encoding the snRNA and / or esnRNA of the Disclosure. In some aspects, a lentiviral vector may include at least one regulatory sequence. In some aspects, a lentiviral vector may include at least one lentiviral long-terminal repeat (LTR) sequence. In some aspects, a lentiviral vector may include a first LTR sequence and a second LTR sequence. In some aspects, a lentiviral vector may include at least one promoter sequence. In some aspects, a lentiviral vector may include at least one enhancer sequence. In some aspects, a lentiviral vector may include at least one terminator sequence. In some aspects, a lentiviral vector may include at least one poly(A) sequence. In some aspects, a lentiviral vector may include at least one linker sequence. In some aspects, a lentiviral vector may include at least one buffer sequence. In some aspects, the lentiviral vector of the Disclosure may include at least one nuclear localization signal or / or nuclear export signal.

[0143] In some aspects, a lentiviral vector may include a first lentiviral LTR sequence, a promoter sequence, an snRNA and / or esnRNA sequence, a terminator sequence, and a second lentiviral LTR sequence. In some aspects, a lentiviral vector may include, from 5' to 3', a first lentiviral LTR sequence, a promoter sequence, an snRNA sequence, a terminator sequence, and a second lentiviral LTR sequence. In some aspects, a lentiviral vector may include, from 5' to 3', a first lentiviral LTR sequence, a promoter sequence, an esnRNA sequence, a terminator sequence, and a second lentiviral LTR sequence.

[0144] In some aspects, a lentiviral vector may include a first lentiviral LTR sequence, a first promoter sequence, a first snRNA sequence, a termination sequence, a second promoter sequence, a second snRNA sequence, a second termination sequence, and a second lentiviral LTR sequence. In some aspects, a lentiviral vector may include a first lentiviral LTR sequence, a first promoter sequence, a first snRNA sequence, a termination sequence, a second promoter sequence, a second snRNA sequence, a second termination sequence, a third promoter sequence, a third snRNA sequence, a third termination sequence, and a second lentiviral LTR sequence. In some aspects, a lentiviral vector may include a first lentiviral LTR sequence, a first promoter sequence, a first snRNA sequence, termination, a second promoter sequence, a second snRNA sequence, a second termination sequence, a third promoter sequence, a third snRNA sequence, a third termination sequence, a fourth promoter sequence, a fourth snRNA sequence, a fourth termination sequence, and a second lentiviral LTR sequence.

[0145] Lentivirus LTR sequences In some aspects of the compositions and methods of this disclosure, the lentivirus long terminal repeat sequence may include any lentivirus LTR sequence known in the art. In some aspects, the lentivirus LTR sequence may include human immunodeficiency virus (HIV) 1 LTR sequence, modified human immunodeficiency virus (HIV) 1 LTR sequence, human immunodeficiency virus (HIV) 2 LTR sequence, modified human immunodeficiency virus (HIV) 2 LTR sequence, sutimangabasal immunodeficiency virus (SIV) SM )LTR sequence, modified Sutimanga Vasali Immunodeficiency Virus (SIV) SM )LTR sequence, African green monkey immunodeficiency virus (SIV) AGM )LTR sequence, modified African green monkey immunodeficiency virus (SIV) AGM)LTR sequences, equine infectious anemia virus (EIAV) LTR sequences, modified equine infectious anemia virus (EIAV) LTR sequences, feline immunodeficiency virus (FIV) LTR sequences, modified feline immunodeficiency virus (FIV) LTR sequences, Visna / Maedi (VNV / VMV) LTR sequences, modified Visna / Maedi virus (VNV / VMV) LTR sequences, capine arthritis encephalitis virus (CAEV) LTR sequences, modified capine arthritis encephalitis virus (CAEV) LTR sequences, bovine immunodeficiency virus (BIV) LTR sequences, or modified bovine immunodeficiency virus (BIV) LTR sequences may be included or comprised of such sequences.

[0146] In some aspects, the LTR sequence may contain a modified lentiviral LTR sequence.

[0147] In some aspects, the lentiviral LTR sequence contains, essentially, or may contain, a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:170 or SEQ ID NO:171.

[0148] In some embodiments, the lentiviral vectors provided herein include first and second lentiviral LTR sequences. In some aspects, the first lentiviral LTR sequence includes, essentially, or may include, a nucleic acid sequence identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) of, SEQ ID NO:170 or SEQ ID NO:171, and the second lentiviral LTR sequence includes, essentially, or may include, a nucleic acid sequence identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) of, SEQ ID NO:170 or SEQ ID NO:171. In some cases, the first lentiviral LTR sequence is located at 5' of the lentiviral vector. In some cases, the second lentiviral LTR sequence is located at 3' of the lentiviral vector.

[0149] In some aspects, the first lentiviral LTR sequence includes the sequence described in SEQ ID NO:173 or SEQ ID NO:174. In some aspects, the second lentiviral LTR sequence includes the sequence described in SEQ ID NO:173 or SEQ ID NO:174. In some embodiments, the lentiviral vector provided herein includes a first lentiviral LTR sequence containing the sequence described in SEQ ID NO:173 and a second lentiviral LTR sequence containing the sequence described in SEQ ID NO:174. In some aspects, the first lentiviral LTR sequence is located at 5' of the lentiviral vector. In some aspects, the second lentiviral LTR sequence is located at 3' of the lentiviral vector.

[0150] In some embodiments of the compositions and methods of this disclosure, the viral vector comprises a sequence isolated from or derived from a lentivirus.

[0151] In some embodiments of the compositions and methods of this disclosure, the vectors of this disclosure are nonviral vectors. In some embodiments, the vectors comprise or consist of nanoparticles, micelles, liposomes or lipoplexes, polymerosomes, polyplexes or dendrimers. In some embodiments, the vectors are expression vectors or recombinant expression systems. As used herein, the term “recombinant expression system” means a gene construct for the expression of a particular genetic material formed by recombinant.

[0152] snRNA vector construct Vector constructs targeting DMD, including the snRNA constructs described herein, are also provided herein.

[0153] An exemplary AAV vector of this disclosure is A05014, which targets DMD exon 53. The elements of A05014 are listed in Table 8. In some respects, the nucleic acid sequence encoding the AAV vector A05014 encoding the DMD exon 53 snRNA sequence contains, is essentially derived from, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:133.

[0154] (Table 8) Nucleotide sequences of plasmid elements of A05014 in 5' to 3' order TIFF2026534279000037.tif216160

[0155] The nucleotide sequence of A05014 (the entire transgene from ITR to ITR): TIFF2026534279000038.tif173161 An exemplary AAV vector in this disclosure is A05211, which targets DMD exon 53. The elements of A05211 are listed in Table 9. In some aspects, the nucleic acid sequence encoding the AAV vector A05211 encoding the DMD exon 53 snRNA sequence contains, is essentially derived from, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:134.

[0156] (Table 9) A05211: Nucleotide sequences of plasmid elements in 5' to 3' order TIFF2026534279000039.tif194160

[0157] The nucleotide sequence of A05211 (the entire transgene from ITR to ITR): TIFF2026534279000040.tif172170

[0158] An exemplary AAV vector of this disclosure is A05374, which targets DMD exon 44. The elements of A05374 are listed in Table 10. In some aspects, the nucleic acid sequence encoding the AAV vector A05374, which encodes an snRNA sequence targeting DMD exon 44, contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:135.

[0159] (Table 10) A05374: Nucleotide sequences of plasmid elements in 5' to 3' order TIFF2026534279000041.tif227160

[0160] Sequence from ITR to ITR for A05374 TIFF2026534279000042.tif172162

[0161] An exemplary AAV vector of this disclosure is A05375, which targets DMD exon 44. The elements of A05375 are listed in Table 11. In some aspects, the nucleic acid sequence encoding the AAV vector A05375, which encodes an snRNA sequence targeting DMD exon 44, contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:136.

[0162] (Table 11) A05375: Nucleotide sequences of plasmid elements in 5' to 3' order TIFF2026534279000043.tif228160

[0163] Sequence from ITR to ITR of A05375 TIFF2026534279000044.tif174162

[0164] An exemplary AAV vector of this disclosure is A05190, which targets DMD exon 45. The elements of A05190 are listed in Table 12. In some aspects, the nucleic acid sequence encoding the AAV vector A05190, which encodes an snRNA sequence that targets DMD exon 45, contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:137.

[0165] (Table 12) A05190: Stuffer-scAAV-2x_mU7 promoter-Z45-8 / 10-mU7 terminator-mU1 promoter-Z45-8 / 10-mU1 terminator; mouse loop including 5'ISD and eSL; derivative of A04376 TIFF2026534279000045.tif225160

[0166] The nucleotide sequence of A05190 (the entire transgene from ITR to ITR): TIFF2026534279000046.tif173161

[0167] An exemplary AAV vector of this disclosure is A05189, which targets DMD exon 45. The elements of A05189 are listed in Table 13. In some aspects, the nucleic acid sequence encoding the AAV vector A05189, which encodes an snRNA sequence that targets DMD exon 45, contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:138.

[0168] (Table 13) A05189: Stuffer-scAAV-2x_mU7 promoter-Z45-8 / 9-mU7 terminator_mU1 promoter-Z45-8 / 9-mU1 terminator; mouth loop including 5'ISD and eSL; derivative of A04376 TIFF2026534279000047.tif206160

[0169] The nucleotide sequence of A05189 (the entire transgene from ITR to ITR): TIFF2026534279000048.tif173161

[0170] An exemplary AAV vector in this disclosure is A05178, designed for multi-exon skipping from exon 50 to 53, containing snRNA sequences targeting DMD exons 50, 51, 52, and 53. The elements of A05178 are listed in Table 14. In some aspects, the nucleic acid sequence encoding the AAV vector A05178 contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 130.

[0171] (Table 14) Multi-exome targeting vector A05178 containing snRNA sequences targeting exons 50, 51, 52, and 53 TIFF2026534279000049.tif21166TIFF2026534279000050.tif238166TIFF2026534279000051.tif159166

[0172] The nucleotide sequence of A05178 (the entire transgene from ITR to ITR): TIFF2026534279000052.tif70161TIFF2026534279000053.tif232161

[0173] An exemplary AAV vector in this disclosure is A06107, designed for multi-exon skipping of exons 44 and 45, and containing snRNA sequences targeting DMD exons 44 and 45. The elements of A06107 are listed in Table 15. In some aspects, the nucleic acid sequence encoding the AAV vector A06107 contains, is essentially, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:131.

[0174] (Table 15) Nucleotide sequences of plasmid elements of A06107 in 5' to 3' order TIFF2026534279000054.tif230166

[0175] The nucleotide sequence of A06107 (the entire transgene from ITR to ITR): TIFF2026534279000055.tif172161

[0176] An exemplary AAV vector in this disclosure is A06108, designed for multi-exon skipping of exons 44 and 45, and containing snRNA sequences targeting DMD exons 44 and 45. The elements of A06108 are listed in Table 16. In some aspects, the nucleic acid sequence encoding the AAV vector A06108 contains, is essentially derived from, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:132.

[0177] (Table 16) Nucleotide sequences of plasmid elements of A06108 in 5' to 3' order TIFF2026534279000056.tif230166

[0178] The nucleotide sequence of A06108 (the entire transgene from ITR to ITR): TIFF2026534279000057.tif172162

[0179] Promoter array The gene therapies and RNA-targeting snRNA gene therapy compositions of this disclosure include promoter sequences derived from snRNA.

[0180] A "promoter" is a regulatory sequence, which is a region of polynucleotide sequence that regulates the initiation and rate of transcription. It may contain gene elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind.

[0181] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, expression regulatory elements. “Expression regulatory element” means any sequence that controls the expression of a coding sequence, such as a gene, as used herein. Exemplary expression regulatory elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression regulatory elements may be, for example, constitutive, inducible, repressive, or tissue-specific. “Promoter” is a regulatory sequence that is a region of polynucleotide sequence on which the initiation and rate of transcription are regulated. It may contain gene elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. “Enhancer” is a region of DNA to which an activating protein that increases the likelihood or frequency of transcription can bind.

[0182] In some embodiments of the compositions and methods of this disclosure, the expression vectors, viral vectors, or nonviral vectors provided herein include, but are not limited to, vector elements such as buffer sequences derived from human genome sequences downstream of the snRNA, and thus have the ability to encode multiple snRNAs from a single construct.

[0183] In some embodiments, the snRNA constructs disclosed herein include a bidirectional snRNA promoter for expressing the snRNA.

[0184] In another embodiment, the vector configuration may include a linker, a signal sequence, and / or a tag. In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector.

[0185] nucleic acid The NOI (nucleotide sequence of interest) includes, non-limitingly, any nucleotide sequence or transgene that can be delivered by a vector. The NOI may be synthetic, derived from naturally occurring DNA or RNA, may be codon-optimized, and may be recombinant RNA / DNA, cDNA, partial genomic DNA, and / or combinations thereof. The NOI may be a coding region or a partial coding region, but does not have to be a coding region. The NOI may be sense-directed or antisense-directed RNA / DNA. The NOI may be snRNA. In this specification, the NOI is also non-limitingly referred to as a transgene, heterologous sequence, gene, or therapeutic gene. The NOI may encode RNA (ribonucleoprotein complex), POI (protein of interest), partial POI, a variant version or variant of POI. The POI may be similar to or equivalent to a wild-type protein. The POI may be a fusion protein or a ribonucleoprotein complex such as snRNP. In some respects, the RNA sequences disclosed herein may also be represented as DNA sequences, and deriving RNA sequences from DNA sequences is within the capabilities of those skilled in the art. For example, the spacer sequences of this disclosure may represent uracil bases as either U or T. It will be readily apparent to those skilled in the art that RNA sequences may use T or U interchangeably to represent uracil.

[0186] Codon optimization In some embodiments, NOIs, transgenes, or GOIs, e.g., the nucleic acid sequences of this disclosure, are codon-optimized nucleic acid sequences. In some embodiments, the codon-optimized sequences exhibit at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased transcription or translation in human subjects compared to wild-type or non-codon-optimized nucleic acid sequences.

[0187] In some aspects, codon-optimized nucleic acid sequences exhibit increased stability. In some aspects, codon-optimized nucleic acid sequences exhibit increased stability due to increased resistance to hydrolysis. In some embodiments, codon-optimized sequences exhibit at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased stability compared to wild-type or non-codon-optimized nucleic acid sequences. In some embodiments, codon-optimized sequences exhibit at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased resistance to hydrolysis in human subjects compared to wild-type or non-codon-optimized nucleic acid sequences.

[0188] In some cases, a codon-optimized nucleic acid sequence may not contain donor splice sites. In some cases, a codon-optimized nucleic acid sequence may contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 donor splice sites. In some cases, a codon-optimized nucleic acid sequence contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer donor splice sites compared to a non-codon-optimized nucleic acid sequence.

[0189] While we do not wish to be constrained by theory, the removal of donor splice sites in codon-optimized nucleic acid sequences prevents latent splicing, which can unexpectedly and unpredictably increase the expression of proteins of interest in vivo. Furthermore, latent splicing can vary between different subjects; that is, the expression levels of proteins containing donor splice sites can vary unpredictably between subjects. Such unpredictability is unacceptable in human therapeutics. Therefore, codon-optimized nucleic acid sequences lacking donor splice sites unexpectedly and surprisingly enable increased protein expression in human subjects and homogenize protein expression across different human subjects.

[0190] In some aspects, codon-optimized nucleic acid sequences may have a different GC content than non-codon-optimized nucleic acid sequences. In some aspects, the GC content of codon-optimized nucleic acid sequences is more uniformly distributed throughout the nucleic acid sequence compared to non-codon-optimized nucleic acid sequences.

[0191] While we do not wish to be constrained by theory, codon-optimized nucleic acid sequences exhibit a more uniform melting temperature ("Tm") throughout the entire transcript due to a more uniform distribution of GC content across the entire nucleic acid sequence. This uniformity in melting temperature unexpectedly leads to increased expression of codon-optimized nucleic acids in human subjects, as less polymerase and / or ribosome stalling occurs, and transcription and / or translation of the nucleic acid sequence takes place.

[0192] In some aspects, codon-optimized nucleic acid sequences may have fewer repressive microRNA target binding sites compared to non-codon-optimized nucleic acid sequences. In some aspects, codon-optimized nucleic acid sequences may have at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, fewer repressive microRNA target binding sites compared to non-codon-optimized nucleic acid sequences.

[0193] While we do not wish to be constrained by theory, codon-optimized nucleic acid sequences unexpectedly show increased expression in human subjects due to having fewer repressive microRNA target binding sites.

[0194] Nucleic acid sequences encoding gene therapy compositions for use in the gene transfer and expression techniques described herein are provided herein. It should be understood, though not necessarily explicitly stated, that the sequences provided herein may also be used to provide expression products, or to provide substantially identical sequences encoding RNA having the same biological properties, or expressing and producing proteins having the same biological properties. These “biologically equivalent,” “biologically active,” or “equivalent” polypeptides are encoded by equivalent polynucleotides described herein. They may possess primary amino acid sequences that are at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to the reference polypeptide when compared using a sequence identity method performed under default conditions. Specific polypeptide sequences are provided as examples of specific embodiments. Modification of amino acids in a sequence to other amino acids having similar charges. Furthermore, an equivalent polynucleotide is one that hybridizes to a reference polynucleotide under stringent conditions, or its complementary chain, or, in the case of a polypeptide, a polynucleotide that hybridizes to a reference coding polynucleotide under stringent conditions, or a polypeptide encoded by its complementary chain. Alternatively, an equivalent polypeptide or protein is one expressed from an equivalent polynucleotide.

[0195] NOIs or nucleic acid sequences (e.g., polynucleotide sequences) disclosed herein can be subjected to codon optimization, a well-known technique in the art. Codon optimization refers to the fact that the frequency of use of certain codons differs among cells. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in a given cell type. It is possible to increase expression by modifying codons in a sequence to match the relative abundance of the corresponding tRNA. It is also possible to decrease expression by intentionally selecting codons for which the corresponding tRNA is known to be rare in a given cell type. Codon frequency tables are well-known in the art for mammalian cells as well as for a variety of other organisms. Based on the genetic code, nucleic acid sequences can be generated. In some embodiments, such sequences are optimized for expression in host cells or target cells, e.g., host cells used to express snRNA, or cells in which the disclosed method is performed (e.g., mammalian cells, e.g., human cells). Using specific species codon preferences and codon frequency tables, isolated nucleic acid molecules encoding snRNAs can be manipulated to leverage the codon preferences of that particular species. For example, the snRNAs disclosed herein may be designed to have codons that are preferredly used by a particular organism of interest. In one example, the nucleic acid sequence is optimized for expression in human cells and has, for example, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity with the corresponding wild-type or origin nucleic acid sequence. In some embodiments, the isolated nucleic acid molecule (which may be part of a vector) comprises at least one coding sequence that is codon-optimized for expression in eukaryotic cells, or at least one coding sequence that is codon-optimized for expression in human cells.In one embodiment, such a codon-optimized coding sequence has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the corresponding wild-type or origin sequence. In another embodiment, a codon-optimized nucleic acid sequence for eukaryotic cells encodes an snRNA having at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the corresponding wild-type or origin sequence. In yet another embodiment, diverse clones containing functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same sequence, can be routinely generated. Silent mutations in coding sequences are due to the degeneracy (i.e., redundancy) of the genetic code, where multiple codons encode the same amino acid residue. Therefore, for example, leucine may be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine by AAT or AAC; aspartic acid by GAT or GAC; cysteine ​​by TGT or TGC; alanine by GCT, GCC, GCA, or GCG; glutamine by CAA or CAG; tyrosine by TAT or TAC; and isoleucine by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3rd Edition, WH5, Freeman and Co., NY).

[0196] Hybridization refers to a reaction in which one or more polynucleotides react to form a stabilized complex via hydrogen bonds between the bases of nucleotide residues. Hydrogen bonding can occur in Watson-Crick base pairing, Hoogsteen bonds, or other sequence-specific ways. The complex may consist of two strands forming a double-stranded structure, three or more strands forming a polychain complex, one strand self-hybridizing, or any combination thereof. Hybridization reactions may constitute a step in a broader process, such as the initiation of a polynucleotide cross-compound reaction or the enzymatic cleavage of polynucleotides by ribozymes.

[0197] Examples of stringent hybridization conditions include incubation temperatures of approximately 25°C to 37°C, hybridization buffer concentrations of 6×SSC to 10×SSC, formamide concentrations of approximately 0% to 25%, and wash solutions of approximately 4×SSC to 8×SSC. Examples of moderate hybridization conditions include incubation temperatures of approximately 40°C to 50°C, buffer concentrations of approximately 9×SSC to 2×SSC, formamide concentrations of approximately 30% to 50%, and wash solutions of approximately 5×SSC to 2×SSC. Examples of highly stringent conditions include incubation temperatures of approximately 55°C to 68°C, buffer concentrations of approximately 1×SSC to 0.1×SSC, formamide concentrations of approximately 55% to 75%, and wash solutions of approximately 1×SSC, 0.1×SSC, or deionized water. Generally, the incubation time for hybridization is 5 minutes to 24 hours, and includes one, two, or more washing steps, with the incubation time for washing being approximately 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffering systems may be used.

[0198] "Homologie," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homologie can be determined by comparing positions within each sequence, which may be aligned for comparison. Molecules are homologous at a position when the positions of the compared sequences are occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. Sequences that are "unrelated" or "non-homologous" share less than 40% identity or less than 25% identity with one of the sequences of the present invention.

[0199] cell In some embodiments of the compositions and methods of the present disclosure, the cells of the present disclosure are prokaryotic cells.

[0200] In some embodiments of the compositions and methods of the Disclosure, the cells of the Disclosure are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are cattle, mouse, cat, horse, pig, dog, monkey, or human cells. In some embodiments, the cells are non-human mammalian cells, such as non-human primate cells.

[0201] In some embodiments, the cells of the Disclosure are somatic cells. In some embodiments, the cells of the Disclosure are germline cells. In some embodiments, the germline cells of the Disclosure are not human cells.

[0202] In some embodiments of the compositions and methods of the Disclosure, the cells of the Disclosure are stem cells. In some embodiments, the cells of the Disclosure are embryonic stem cells. In some embodiments, the embryonic stem cells of the Disclosure are not human cells. In some embodiments, the cells of the Disclosure are pluripotent stem cells or pluripotent stem cells. In some embodiments, the cells of the Disclosure are adult stem cells. In some embodiments, the cells of the Disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the cells of the Disclosure are hematopoietic stem cells (HSCs).

[0203] In some embodiments of the compositions and methods disclosed herein, somatic cells are muscle cells. In some embodiments, muscle cells are myoblasts or myocytes. In some embodiments, muscle cells are cardiomyocytes, skeletal muscle cells, or smooth muscle cells. In some embodiments, muscle cells are striated cells. In one embodiment, patient cells treated with the compositions disclosed herein include, but are not limited to, skeletal muscle (developing and mature muscle fibers and satellite cells), neuromuscular junctions, cardiomyocytes, smooth muscle cells, peripheral nervous system (neurons), peripheral motor neurons, and / or sensory neurons.

[0204] In some embodiments of the compositions and methods disclosed herein, somatic cells of the disclosure are neuronal cells. In one embodiment, patient cells treated with the compositions disclosed herein include, but are not limited to, central nervous system (neurons), peripheral nervous system (neurons), peripheral motor neurons, and / or sensory neurons. In one embodiment, neuronal cells are glial cells.

[0205] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are fibroblasts or epithelial cells. In some embodiments, the epithelial cells of the present disclosure form squamous epithelium, cuboidal epithelium, columnar epithelium, stratified epithelium, pseudostratified columnar epithelium, or transitional epithelium. In some embodiments, the epithelial cells of the present disclosure form glands, including but not limited to the pineal gland, thymus, pituitary gland, thyroid gland, adrenal gland, apocrine gland, holocrine gland, merocrine gland, serous gland, mucous gland, and sebaceous gland. In some embodiments, the epithelial cells of the present disclosure are in contact with the outer surface of organs, including but not limited to the lung, spleen, stomach, pancreas, bladder, intestine, kidney, gallbladder, liver, larynx, or pharynx. In some embodiments, the epithelial cells of the present disclosure are in contact with the outer surface of blood vessels or veins.

[0206] In some aspects of the present disclosure, somatic cells are ophthalmic cells. Ophthalmic cells include, but are not limited to, corneal epithelial cells, corneal stromal cells, retinal pigment epithelial (RPE) cells, lens epithelial cells, iris pigment epithelial cells, conjunctival fibroblasts, non-pigmented ciliary epithelial cells, trabecular reticular cells, choroidal fibroblasts, and conjunctival epithelial cells. In some aspects, ophthalmic cells are retinal cells or corneal cells. In one aspect, retinal cells are photoreceptor cells or retinal pigment epithelial cells. In another aspect, retinal cells are ganglion cells, amacrine cells, bipolar cells, horizontal cells, Müller glial cells, rod cells, or cone cells. In some aspects of the compositions and methods of the present disclosure, somatic cells of the present disclosure are primary cells.

[0207] In some aspects of the compositions and methods of this disclosure, the somatic cells of this disclosure are cultured cells.

[0208] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are in vivo, in vitro, ex vivo, or in situ.

[0209] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are self or allogeneic.

[0210] How to use This disclosure provides a method for encoding RNA or expressing NOI in cells using the snRNA system disclosed herein. In one embodiment, this disclosure provides a method for modifying RNA or modifying the activity of a protein encoded by an RNA molecule, comprising contacting a target RNA molecule with a composition of this disclosure under conditions suitable for binding to the RNA molecule.

[0211] This disclosure provides a method for modifying the expression level of a protein encoded by an RNA molecule, comprising contacting the composition of this disclosure with a cell containing an RNA molecule under conditions suitable for binding to the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition of this disclosure comprises a vector containing an snRNA sequence. In some embodiments, the vector is an AAV.

[0212] This disclosure provides a method for modifying the expression level of an RNA molecule or a protein encoded by an RNA molecule, comprising contacting the RNA molecule with a composition of this disclosure under conditions suitable for knockdown, blockade, splicing, multi-targeting, frame recovery, or editing of a target RNA. In some embodiments, the composition of this disclosure comprises a vector containing an snRNA sequence. In some embodiments, the vector is an AAV.

[0213] This disclosure provides a method for modifying a target RNA or modifying the activity of a protein encoded by a target RNA molecule, comprising contacting a cell containing an RNA molecule with a composition under conditions suitable for knockdown, blockade, splicing, multi-targeting, frame recovery, or editing of the target RNA. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition comprises a vector containing an snRNA sequence disclosed herein. In some embodiments, the vector is an AAV.

[0214] This disclosure provides a method for treating a disease or disorder, comprising administering a therapeutically effective amount of the snRNA composition of this disclosure to a target.

[0215] This disclosure provides a method for treating a disease in a patient requiring such treatment, comprising administering to the patient a therapeutically effective amount of the snRNA composition of this disclosure, wherein the composition comprises a vector comprising the snRNA sequence disclosed herein, and the composition modifies, reduces, disrupts, knocks down, or eliminates the expression level of the targeted RNA (compared to the expression level of the targeted RNA treated with a non-targeting (NT) control or compared to the untreated RNA). In another embodiment, the level of reduction is 1-fold or greater. In another embodiment, the level of reduction is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In another embodiment, the level of reduction is 10-fold or greater. In another embodiment, the level of reduction is 10-fold to 20-fold. In another embodiment, the level of reduction is 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold. In another embodiment, the gene therapy compositions disclosed herein result in 20% to 100% destruction of the targeted RNA when administered to a patient. In one embodiment, the elimination percentage of the targeted RNA is any of 20-99%, 25-99%, 50-99%, 80-99%, 90-99%, or 95-99%. In one embodiment, the elimination percentage is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment, the elimination percentage is complete or 100% elimination of the targeted RNA.

[0216] This disclosure provides a method for treating a disease or disorder in a subject, comprising administering a nucleic acid molecule that targets RNA (i.e., snRNA as disclosed herein) or an AAV vector containing snRNA as disclosed herein.

[0217] In some aspects, the disease or disorder is Duchenne muscular dystrophy. In some aspects, the RNA-targeting nucleic acid molecule or AAV vector targets the RNA sequence encoding dystrophin (DMD). In some aspects, the RNA sequence encoding DMD contains intronic or exonic sequences. In some aspects, the exonic sequence contains exons 2, 44, 45, 51, 53 of DMD, combinations thereof, or their adjacent regions.

[0218] In some embodiments of the methods of this disclosure, the subject of the disclosure is diagnosed with a disease to be treated. In some embodiments, the subject of the disclosure exhibits at least one sign or symptom of a disorder or disease to be treated. In some embodiments, the subject of the disclosure exhibits at least one sign or symptom of a disease.

[0219] In some embodiments of the methods of the disclosure, the subject of the disclosure is female. In some embodiments of the methods of the disclosure, the subject of the disclosure is male. In some embodiments, the subject of the disclosure has two XX or XY chromosomes. In some embodiments, the subject of the disclosure has two XX or XY chromosomes and a third chromosome, either X or Y.

[0220] In some embodiments of the method of this disclosure, the subject matter of the disclosure is newborns, infants, children, adults, the elderly, or the very elderly. In some embodiments of the method of this disclosure, the subject matter of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days old. In some embodiments of the method of this disclosure, the subject matter of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old. In some embodiments of the methods of this disclosure, the subject of this disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 years of age, or any age in between.

[0221] In some embodiments of the methods disclosed herein, the subject matter of the disclosure is mammals. In some embodiments, the subject matter of the disclosure is non-human mammals.

[0222] In some embodiments of the methods of this disclosure, the subject matter of this disclosure is human beings.

[0223] In some embodiments of the method of the Disclosure, a therapeutically effective amount comprises a single dose of the composition of the Disclosure. In some embodiments, a therapeutically effective amount comprises at least one dose of the composition of the Disclosure. In some embodiments, a therapeutically effective amount comprises one or more doses of the composition of the Disclosure.

[0224] In some embodiments of the methods of this disclosure, a therapeutically effective amount eliminates signs or symptoms of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of signs or symptoms of a disease or disorder.

[0225] In some embodiments of the methods disclosed herein, a therapeutically effective amount eliminates a disease or disorder.

[0226] In some embodiments of the methods of this disclosure, a therapeutically effective amount prevents the onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of signs or symptoms of a disease or disorder. In some embodiments, a therapeutically effective amount improves the prognosis of the subject.

[0227] In some embodiments of the methods of the present disclosure, the composition of the present disclosure is administered to a subject via intracerebral administration. In some embodiments, the composition of the present disclosure is administered to a subject via an intrastriatal route. In some embodiments, the composition of the present disclosure is administered to a subject by stereotactic injection or infusion. In some embodiments, the composition is administered to skeletal muscle. In some embodiments, the composition is administered to the brain. In some embodiments of the methods of the present disclosure, the composition of the present disclosure is administered topically to a subject.

[0228] In some embodiments, the compositions disclosed herein are formulated as pharmaceutical compositions. Briefly, the pharmaceutical compositions for use disclosed herein may contain, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, a protein or a protein-coding polynucleotide, the polynucleotide may optionally be contained in an AAV, and the AAV may optionally be immunoorthogonal. Such compositions may include buffers, e.g., neutral buffered saline, phosphate-buffered saline; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for routes of administration such as oral, enteral, topical, transdermal, intranasal, and / or inhalation, and for routes of administration by injection or infusion such as intravenous, intramuscular, subpia, subarachnoid, intraparenchymal, subarachnoid, striatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and / or parenteral administration. In certain embodiments, the compositions of this disclosure may be formulated for intracerebral or intrastriatal administration. [Examples]

[0229] Example 1: In vitro and in vivo AAV9 snRNA-mediated exon 53 skipping material and method: Transfection of muscle tubes: Patient myoblasts were differentiated into myotubes for 4-5 days. The differentiated cells were transfected with synthetic U7 snRNA (IDT) at final doses of 25 nM, 50 nM, or 100 nM using lipofectamine 2000 (Invitrogen). 24 hours after transfection, cells were collected using 1 ml of TRIzol reagent (Invitrogen). RNA was extracted using the ZYMO Direct-zol RNA microprep kit (Zymo Research).

[0230] Transduction of myotubes: Patient myoblasts were differentiated into myotubes for 3 days. On day 3 after differentiation, the cells were transduced with AAV9 virus at the indicated MOI. The culture medium was replenished 24 hours after transduction and replaced 48 hours after transduction. Seven days after transduction, cells were collected using 1 ml of TRIzol reagent (Invitrogen). RNA was extracted using the ZYMO Direct-zol RNA microprep kit (Zymo Research).

[0231] In vivo evaluation of AAV9-A05014 and A05211: Six-week-old del52hDMD / mdx mice were administered 1E12vg of AAV9-A05014 or AAV9-A05211 retroorbitally. After 4 weeks of survival, tissue was collected, homogenized with TRIzol, and extracted using the ZYMO Direct-zol RNA microprep kit (Zymo Research).

[0232] Splicing assay: Following the manufacturer's recommendations, 50–500 ng of RNA was used for cDNA synthesis using qScript Ultra SuperMix (QuantaBio). PCR was performed on the cDNA using GoTaq Green Master Mix (Promega). The PCR products were processed using a D1000 ScreenTape (Agilent) on a 4200 TapeStation (Agilent).

[0233] Immunofluorescence: After 7 days of treatment, cells were fixed with PFA and stained with a primary antibody (mouse anti-dystrophin MANDYS106 MABT827 1:100) diluted in 1% BSA containing 0.1% Triton-X 100, and incubated overnight at 4°C. The following day, cells were washed three times with PBS for 5 minutes each, and then incubated for 1 hour at RT in the dark with secondary goat anti-mouse Alexa-Fluor 647 (1:1000) in the same carrier solution as the primary antibody. Cells were washed three times with PBS and then mounted in Prolong Gold Antifade solution containing DAPI. After curing, slides were imaged with an upright fluorescence microscope using a 20x objective lens.

[0234] See Figures 1-3. Summary: Synthetic snRNAs were screened for exon 53 skipping in patient myotubes. Selected upper fusion spacers were cloned into AAV vectors as 2× snRNA cassettes and packaged in scAAV9. Treatment of patient myotubes with scAAV9 A05014 resulted in IF-mediated dystrophin recovery 7 days after transduction. Treatment of del52hDMD / mdx mice with scAAV-A05211 resulted in robust exon 53 skipping in multiple skeletal muscle tissues 4 weeks after IV delivery (see Figures 1-3).

[0235] Example 2: Exon 44 skipping mediated by AAV9 snRNA in myotubes of deletion 45 patients material and method: Transfection of muscle tubes: Patient myoblasts were differentiated into myotubes for 4-5 days. The differentiated cells were transfected with synthetic U7 snRNA (IDT) at a final dose of 25 nM using lipofectamine 2000 (Invitrogen). 24 hours after transfection, cells were collected using 1 ml of TRIzol reagent (Invitrogen). RNA was extracted using the ZYMO Direct-zol RNA microprep kit (Zymo Research).

[0236] Transduction of myotubes: Patient myoblasts were differentiated for 3 days. On day 3 after differentiation, the cells were transduced with a virus at the indicated MOI. The culture medium was replenished 24 hours after transduction and replaced 48 hours after transduction. Seven days after transduction, cells were collected using 1 ml of TRIzol reagent (Invitrogen). RNA was extracted using the ZYMO Direct-zol RNA microprep kit (Zymo Research).

[0237] Splicing assay: Following the manufacturer's recommendations, 50–500 ng of RNA was used for cDNA synthesis using qScript Ultra SuperMix (QuantaBio). PCR was performed on the cDNA using GoTaq Green Master Mix (Promega). The PCR products were processed using a D1000 ScreenTape (Agilent) on a 4200 TapeStation (Agilent).

[0238] Protein analysis: Myotube protein lysates were processed with Jess Simple Western (ProteinSimple) according to the manufacturer's instructions.

[0239] Summary: Synthetic snRNAs were screened for exon 44 skipping in patient myotubes. Selected upper fusion spacers were cloned into AAV vectors as 2× snRNA cassettes and packaged into scAAV9. scAAV9 A05374 and A05375 showed dose-dependent exon 44 skipping and dystrophin recovery in del45 myotubes 7 days after transduction (see Figures 4–6).

[0240] Example 3: Exon 45 skipping mediated by AAV9 snRNA in myotubes of deletion 44 patients material and method: To promote exon 45 skipping, which leads to the recovery of the DMD mRNA reading frame and dystrophin protein in DMD patients with Δ44 mutations, U7 snRNA was engineered to bind to splicing regulatory sequences (SA, splice acceptor, SD splice donor, and / or ESE, exon splice enhancer sequence). After transfection of human myotubes with 100 nM synthetic snRNA, RT-PCR was performed using primers for exon 43 and exon 46 / 47 to determine the product size. The predicted size was 487 bp when exon 45 was present and 311 bp when exon 45 was excluded. See Figures 7A-C.

[0241] To identify strong spacer sequences at lower concentrations of synthetic snRNA (25 nM), we performed screening of synthetic snRNAs. After transfecting with U7 snRNA synthetic RNA, RT-PCR was performed. The predicted exon 45 length was 487 bp if exon 45 was present, and 311 bp if exon 45 was excluded. Exon 45 skipping was quantified. Exon 45 sequences were aligned from humans (top) and mice (bottom). Targeting sequences contained in the synthetic snRNAs used in the screening were mapped. See Figures 8A-8C.

[0242] AAV9-mediated exon 45 skipping in human Δ44 (exon 44 deletion) myotubes is demonstrated. Myotubes were transduced with AAV9 constructs targeting exon 45, and the rate of skipping was determined 7 days after transduction with A05189 and A05190 of varying MOI. ddPCR was performed to measure the copy number of snRNA per nanogram of total RNA expressed from A05189 and A05190.

[0243] Summary: Synthetic snRNAs were screened for exon 45 skipping in patient myotubes. Selected upper fusion spacers were cloned into AAV vectors as 2× snRNA cassettes and packaged into scAAV9. scAAV9 A05189 and A05190 exhibit dose-dependent exon 45 skipping and robust snRNA expression in del44 myotubes 7 days after transduction (see Figures 7–9).

[0244] Example 4: AAV-mediated multi-exon skipping of DMD exons 50-53 in human deletion 52 myotubes Human del52 myotubes were transduced with A05178, which carries engineered snRNAs targeting exons 50, 51, 52, and 53. After 7 days, RNA was extracted and RT-PCR was performed to identify exon skipping. Exon-skipped bands were quantified by dividing the peak molar concentration of the amplicon (representing single-exon or multi-exon skipping) by the signal of the total DMD amplicon.

[0245] Summary: Treatment of del52 myotubes with A05178, which carries 4×snRNA targeting exons 50-53, results in single-exon or multi-exon skipping of DMDs 7 days after transduction.

[0246] Example 5: Strategies for multi-exon skipping of exons 44 and 45 in human wild-type myotubes Healthy human (wild-type, WT) myotubes were co-transfected with a combination of synthetic snRNAs targeting exons 44 and 45. After 24 hours, RNA was extracted and RT-PCR was performed to identify exon skipping. To determine the multi-skipping percentage, the signal of the band skipped for each of exon 44 and exon 45 was divided by the signal of the total DMD amplicon.

[0247] Summary: Treatment of WT human myotubes with a combination of synthetic snRNAs targeting exon 44 and exon 45 results in the accumulation of DMD amplicons lacking both exon 44 and exon 45, thus suggesting that this strategy can be used for multi-exon skipping of exons 44 to 45.

[0248] Conclusion These examples describe therapeutic strategies using engineered U7 snRNAs containing unique spacers to target exon splicing enhancers in various single-exon and multi-exon strategies for DMD. This study is based on our earlier DMD exon 51 (LBIO-115) study. After high-dose administration via intravenous delivery to del52hDMD / mdx mice for 3 weeks, exon 51 skipping was over 95% in the heart and up to 73% in skeletal muscle. Skipping persisted for 12 weeks in skeletal and cardiac tissues, and DMD protein doubled from week 4 to week 12. Here, we describe AAV snRNA constructs comprising novel spacer sequences that target splicing regulatory regions for exons 53, 44, and 45. These constructs exhibited relatively high levels of full-length genome (90%+). Treated patient-derived cells showed robust dose-dependent skipping of the target exon and restoration of dystrophin protein expression 7 days after transduction with the scAAV9 dual snRNA cassette. When the scAAV9-exon 53 construct was intravenously delivered to del52hDMD / mdx mice, it also enabled exon 53 skipping, showing over 95% exon skipping in the heart and up to 75% in skeletal muscle after 4 weeks. A total of 35% of DMD patients can be treated by the exon skipping approaches for exon 51, exon 53, exon 45, and exon 44, and the utilization of this strategy to skip each disease-related exon can treat up to about 70% of DMD patients.

[0249] Incorporation by Reference All documents cited herein, including cross-referenced or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. No document cited herein constitutes prior art to any invention disclosed or embodied herein, nor does it constitute teaching, suggestion, or disclosure of such invention, either alone or in combination with other references. Furthermore, in the event of any conflict between the meaning or definition of a term herein and the meaning or definition of the same term in a document incorporated by reference, the meaning or definition provided herein shall prevail.

[0250] Other aspects While specific aspects of this disclosure have been illustrated and described, various other changes and modifications are possible without departing from the spirit and scope of this disclosure. The scope of the attached claims includes all such changes and modifications that fall within the scope of this disclosure.

Claims

1. An RNA-targeting nucleic acid molecule comprising nuclear small RNA (snRNA), wherein the snRNA comprises a targeting sequence that binds to a dystrophin (DMD) RNA sequence, and the DMD RNA sequence comprises at least one splicing control sequence selected from a splice acceptor sequence, a splice donor sequence, and an exon splice enhancer sequence.

2. The RNA targeting nucleic acid molecule according to claim 1, wherein the DMD RNA sequence is at least one of exon 2, exon 44, exon 45, exon 51, and / or exon 53.

3. An RNA targeting nucleic acid molecule according to claim 1, comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical to a nucleic acid sequence described in any one of SEQ ID NO: 59–118, 126, 206–227, and 237–1344.

4. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA comprises a stem-loop (SL) containing a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between them) identical nucleic acid sequences to one or more nucleic acid sequences listed in any one of SEQ ID NO:1 to SEQ ID NO:11, SEQ ID NO:144 to SEQ ID NO:148, SEQ ID NO:164, SEQ ID NO:186, SEQ ID NO:190 to SEQ ID NO:205, SEQ ID NO:228 to SEQ ID NO:230, SEQ ID NO:235, or SEQ ID NO:

236.

5. The RNA targeting nucleic acid molecule according to claim 1, wherein the stem-loop (eSL) is an engineered stem-loop containing at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) nucleic acid sequences described in any one of SEQ ID NO:1 to SEQ ID NO:

11.

6. The RNA targeting nucleic acid molecule according to claim 1, wherein the DMD RNA sequence is a premRNA or mRNA sequence.

7. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA comprises two targeting sequences that target two RNAs of interest.

8. The RNA targeting nucleic acid molecule according to claim 7, wherein the two targeting sequences are fusion sequences.

9. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA comprises an Sm-binding domain (SmBD) selected from the group consisting of U1, U2, U4, and U5 SmBDs.

10. The RNA targeting nucleic acid molecule according to claim 9, wherein the SmBD comprises a nucleic acid sequence described in any one of SEQ ID NO:31 to SEQ ID NO:

38.

11. The RNA targeting nucleic acid molecule according to claim 1, wherein the snRNA comprises a 5' interaction stabilization domain (5'ISD) containing a nucleotide sequence selected from any one of SEQ ID NO:12 to SEQ ID NO:

23.

12. A vector comprising one or more snRNAs according to claim 1.

13. The vector according to claim 12, which is an AAV vector.

14. The AAV vector according to claim 13, wherein the snRNA is functionally linked to a promoter.

15. The AAV vector according to claim 13, wherein the snRNA is functionally linked to a U7 promoter or a U1 promoter.

16. The AAV vector according to claim 13, wherein the snRNA is functionally linked to a downstream terminator (DT).

17. The AAV vector according to claim 13, wherein the snRNA is functionally ligated to a U7 downstream terminator or a U1 downstream terminator.

18. The AAV vector according to claim 13, comprising at least one, at least two, at least three, at least four, or at least five snRNAs.

19. The AAV vector according to claim 18, wherein at least one, at least two, at least three, at least four, or at least five snRNAs each target the same target RNA sequence.

20. The AAV vector according to claim 18, wherein at least one, at least two, at least three, at least four, or at least five snRNAs target two or more target RNA sequences.

21. The AAV vector according to claim 18, wherein each snRNA is separated by a buffer sequence.

22. The AAV vector according to claim 21, wherein the buffer sequence contains a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence described in any one of SEQ ID NO:24 to SEQ ID NO:

30.

23. The AAV vector according to claim 13, comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence described in any one of SEQ ID NO:130 to SEQ ID NO:

138.

24. A method for targeting one or more target RNAs of interest and blocking, knocking down, editing, exon skipping, or splicing the one or more target RNAs, comprising the step of contacting a cell containing the one or more target RNAs with the snRNA described in claim 1.

25. A DMD RNA targeting nucleic acid molecule containing a targeting sequence described in one of the following SEQ ID NOs: 59–118, 206–227, or 237–333.

26. A method for treating a disease or disorder in a subject, comprising the step of administering an RNA-targeting nucleic acid molecule according to claim 1 or an AAV vector according to claim 12.

27. The method according to claim 26, wherein the disease or disorder is Duchenne muscular dystrophy.

28. The method according to claim 26, wherein the administration is intravenous, intramuscular, subpia, subarachnoid, intraparenchymal, subarachnoid, striatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and / or parenteral administration.

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