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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/32—Special delivery means, e.g. tissue-specific
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/33—Alteration of splicing

Definitions

• the present invention relates to novel exon skipping expression cassettes and gene therapy vectors, such as recombinant AAV vectors encoding the novel exon skipping expression cassettes, as well as compositions and uses thereof and methods of treatment using the same.
• DMD Duchenne muscular dystrophy
• Approved DMD therapies include exon skipping, a process by which specific exons are excluded from a mutated mRNA to restore a translational reading frame and produce a functional, nearly full-length dystrophin (Servais et al. (2022) Nucl. Acid Ther. 32, 29-39; Clemens et al. (2020) JAMA Neurol. 77, 982-991). This is achieved by weekly intravenous infusion of antisense oligonucleotides, but the efficacy of dystrophin restoration is low, reaching only a few percent of normal skeletal muscle levels. Up to 80% of DMD causing mutations are addressable through exon skipping strategies, with the “hotspot” for mutations between exons 43 and 55 representing most patients.
• the dystrophin cytoplasmic protein encoded by the DMD gene functions to link cytoskeletal actin filaments to membrane proteins, acting as a shock absorber during muscle fiber contraction by linking the actin of the contractile apparatus to the layer of connective tissue that surrounds each muscle fiber.
• Dystrophin is normally localized at the cytoplasmic face of the sarcolemma membrane of muscle tissue.
• Patients that suffer from DMD may have large deletion mutations of one or more exons (60-70%), or duplication mutations (5-10%), or single nucleotide variants (including small deletions or insertions, single-base changes, and splice site changes can also cause pathogenic dystrophin variants. In-frame deletions result in the less severe Becker muscular dystrophy (BMD), in which patients express a truncated, partially functional dystrophin.
• BMD Becker muscular dystrophy
• AAV adeno-associated viral vectors can mediate prolonged expression of exon skipping antisense RNAs appended to a small nuclear (sn) RNA such as U7 (Goyenvalle et al. (2004) Science. 306, 1796-1799; Aupy et al. (2020) Mol.
• an invention based, in part, on novel gene constructs that encode novel exon skipping expression cassettes for use in gene therapy.
• the exon skipping gene constructs and expression cassettes were engineered for improved therapy with respect to efficacy, potency and safety to the subject when expressed by a viral vector in cells, in particular muscle cells.
• the exon skipping gene therapies of the present disclosure showed increased measures of skipped exons.
• exon skipping gene constructs encode for antisense oligonucleotides (AONs) that specifically target a specific exon and/or the intron/ exon junction of the specific exon of the DMD gene and such antisense sequences were operably linked to regulatory elements including a small nuclear RNA (snRNA), for example a U7 snRNA.
• exon skipping gene constructs encode for antisense oligonucleotides (AONs) that specifically target exon 53 and/or the exon 53/intron junction of the DMD gene and such antisense sequences were operably linked to regulatory elements including a small nuclear RNA (snRNA), for example a U7 snRNA.
• the exon skipping gene constructs encode for antisense oligonucleotides (AONs) that specifically target exon 51 and/or the exon 51/intron junction of the DMD gene and such antisense sequences were operably linked to regulatory elements including a small nuclear RNA (snRNA), for example a U7 snRNA.
• AONs antisense oligonucleotides
• snRNA small nuclear RNA
• expression of the snRNA in transduced cells and exon 51 skipping were strongly correlated.
• an engineered expression cassette constructed with a multiple antisense complex strategy particularly raised exon skipping efficacy to high percentages in an exon 52-deleted human DMD mouse as well as in immortalized human DMDdel52 myoblasts and engineered cells.
• Dystrophin restoration was visualized in the membranes (sarcolemma) of muscle tissues extracted from treated dystrophin-depleted mice.
• Dystrophin-positive fibers were also quantitated at high levels in these transduced tissues. Accordingly, provided are highly effective gene therapy vectors exhibiting greater than 50%, even greater than 80% exon skipping efficacy.
• Gene therapy vectors for example, recombinant AAV vectors comprising these constructs are described and have been shown to efficiently express functional exon skipping antisense complexes in vitro and in vivo. Methods of using these gene therapy vectors in therapeutic methods and methods of making these gene therapy vectors are also described herein.
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• nucleic acid compositions comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22.
• AS antisense sequence
• snRNA 3' small nuclear ribonucleic acid
• nucleic acid compositions comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) complimentary to a pre-mRNA exon 51 or exon 53 region of dystrophin and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprises a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a premessenger RNA (pre-mRNA) exon region of dystrophin and/or a pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre-mRNA premessenger RNA
• pre-mRNA pre-mRNA exon region of dystrophin
• pre-mRNA pre-mRNA exon/intron junction of the pre-mRNA
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a premessenger RNA (pre-mRNA) exon region of dystrophin and/or a pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre-mRNA premessenger RNA
• pre-mRNA pre-mRNA exon region of dystrophin
• pre-mRNA pre-mRNA exon/intron junction of the
• the nucleic acid sequence encodes at least two transgenes comprise (1) a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin; and (2) a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin.
• the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin is a splice site of the pre-mRNA exon region.
• the splice site of the pre-mRNA exon region is an acceptor splice site or a donor splice site. In some embodiments, the splice site of the pre-mRNA exon region is an acceptor splice site. In some embodiments, the splice site of the pre-mRNA exon region is a donor splice site.
• the at least two transgenes comprise: (1) a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin; and (2) a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to an acceptor splice site of the pre-mRNA exon region of dystrophin.
• the at least two transgenes comprise: (1) a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin; and (2) a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to a donor splice site of the pre-mRNA exon region of dystrophin.
• the nucleic acid sequence encodes at least three transgenes.
• the at least three transgenes comprise: (1) a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin; (2) a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin; and (3) a third transgene comprising a third AS, wherein the third AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin or the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin.
• the third AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin. In some embodiments, the third AS nucleic acid sequence is complementary to the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin. In some embodiments, the pre-mRNA exon/intron junction of the pre- mRNA exon region of dystrophin is a splice site of the pre-mRNA exon region. In some embodiments, the splice site of the pre-mRNA exon region is an acceptor splice site or a donor splice site. In some embodiments, the splice site of the pre-mRNA exon region is an acceptor splice site.
• the splice site of the pre-mRNA exon region is a donor splice site.
• the at least three transgenes comprise (1) a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to an acceptor splice site of the pre-mRNA exon region of dystrophin; (2) a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin; and (3) a third transgene comprising a third AS, wherein the third AS nucleic acid sequence is complementary to a donor splice site of the pre-mRNA exon region of dystrophin.
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a premessenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre-mRNA premessenger RNA
• nucleic acid compositions comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre-mRNA pre-messenger RNA
• nucleic acid compositions comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• nucleic acid compositions comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) and a 3' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin.
• pre- mRNA pre-messenger RNA
• the AS nucleic acid sequence is complementary to the pre- mRNA exon 53 region, the pre-mRNA region at the junction of intron 52 and exon 53, or the pre-mRNA region at the junction of exon 53 and intron 53.
• the AS nucleic acid sequence is set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• the AS nucleic acid sequence consists of the nucleic acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a premessenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre-mRNA premessenger RNA
• pre-mRNA pre-mRNA exon 51 region of dystrophin
• pre-mRNA pre-mRNA exon 51/intron junction of dystrophin
• nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre-mRNA pre-messenger RNA
• nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) and a 3' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin.
• pre- mRNA pre-messenger RNA
• the AS nucleic acid sequence is complementary to the pre- mRNA exon 51 , the pre-mRNA region at the junction of intron 50 and exon 51 , or the pre- mRNA region at the junction of exon 51 and intron 51.
• the AS nucleic acid sequence is set forth in any one of SEQ ID NOs: 48-79.
• the AS nucleic acid sequence consists of the nucleic acid sequence set forth in any one of SEQ ID NOs: 48-79.
• the U7 promoter is a mouse U7 promoter.
• the U7 promoter comprises a sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 15.
• the nucleic acid compositions further comprise a polyadenylation (poly A) signal.
• the nucleic acid compositions further comprise a polyA signal and one or more stuffer sequences located 5' and/or 3' of the polyA signal.
• the transgene comprises a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 32, or SEQ ID NO: 33.
• the nucleic acid sequence encodes more than one transgene and each transgene is operably linked to the other and the most 3’ transgene is operably linked to polyA signal and/or a stuffer sequence.
• the transgene comprises a sequence as set forth in any one of SEQ ID NOs: 80-112.
• the nucleic acid sequence encodes more than one transgene and each transgene is operably linked to the other and the most 3’ transgene is operably linked to polyA signal and/or a stuffer sequence.
• the transgene comprises a sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO: 133.
• the nucleic acid sequence encodes more than one transgene and each transgene is operably linked to the other and the most 3’ transgene is operably linked to polyA signal and/or a stuffer sequence.
• the in vivo efficacy of the transgene disclosed herein in a subject in need lasts more than 6 months by one single introduction.
• exon skipping expression cassettes and nucleic acid constructs encoding same comprising a U7 snRNA antisense complex (AS Complex), a stuffer sequence and a polyA signal, wherein expression cassettes comprise at least two AS complexes, where in the antisense sequences of the AS complexes are different.
• exon skipping expression cassettes and nucleic acid constructs encoding same comprising a U7 snRNA antisense complex (AS Complex), a stuffer sequence and a polyA signal, wherein expression cassettes comprise at least two AS complexes, where in the antisense sequences of the AS complexes are all different from each other.
• exon skipping expression cassettes having an antisense complex comprise a U7 promoter, an antisense sequence, a U7 Sm OPT, a U7 3 ' Hairpin, U7 3 ' Flank and a polylinker sequence thereof, or which have more than one AS complex.
• the U7 Sm OPT, a U7 3 ' Hairpin, U7 3 ' Flank and a polylinker sequence composite sequence is referred to as the snRNA 3' flanking sequence or element.
• expression cassettes comprise more than one antisense complex wherein the antisense sequence within each antisense complex targets the same gene or gene region.
• expression cassettes comprise more than one antisense complex wherein the antisense sequence within each antisense complex targets the same gene all at different gene regions.
• expression cassettes comprise at least three antisense complexes wherein the antisense sequence within each antisense complex targets the same gene or gene region. In some embodiments, expression cassettes comprise at least three antisense complexes wherein the antisense sequence within each antisense complex each target a different gene region on the same gene. In these embodiments, expression cassettes having more than one antisense complex have improved exon skipping efficacy, for example 50% to 90% skipped. In some embodiments, expression cassettes comprise at least one antisense complex that targets a portion of an exon region of the human DMD gene or human DMD pre-mRNA.
• expression cassettes comprise at least one antisense complex that targets a portion of an exon 53 or exon 51 region of the human DMD gene or human DMD pre-mRNA. In some embodiments, expression cassettes comprise at least one antisense complex that targets a portion of an exon 53 region of the human DMD gene or human DMD pre-mRNA. In some embodiments, expression cassettes comprise at least one antisense complex that targets a portion of an exon 53/intron junction, such as intron 52/exon 53 and/or exon 53/intron 53 junction of the human DMD gene or human DMD pre-mRNA.
• expression cassettes comprise at least one antisense complex that targets a portion of an exon 51 region of the human DMD gene or human DMD pre-mRNA. In some embodiments, expression cassettes comprise at least one antisense complex that targets a portion of an exon 51/intron junction, such as intron 50/exon 51 and/or exon 51/intron 51 junction of the human DMD gene or human DMD pre-mRNA.
• the antisense sequence has nucleic acid sequence that is set forth in any one of SEQ ID NOs: 48- 79. In some embodiments, the antisense sequence has nucleic acid sequence that consists of the nucleic acid sequence set forth in any one of SEQ ID NOs: 48-79.
• exon skipping constructs are illustrated in FIGS. 1A-1B and 13A. Certain embodiments described herein are an exon skipping construct targeting an exon of dystrophin having from amino-terminus to the carboxy -terminus:
• a first antisense complex comprising a first antisense sequence
• a second antisense complex comprising a second antisense sequence
• a third antisense complex comprising a third antisense sequence
• at least one poly adenylation signal at least one poly adenylation signal.
• a first antisense complex comprising a first antisense sequence
• a second antisense complex comprising a second antisense sequence, and [0031] at least one polyadenylation signal.
• the antisense complex is operably linked to a stuffer sequence.
• the 3’ end of the snRNA flanking element of the 3’ antisense complex is operably linked to a downstream stuffer sequence.
• the stuffer sequence is located 5' or 3' of the polyA signal.
• the antisense complex comprises a promoter, an antisense sequence and an snRNA sequence. Antisense sequences are selected for their homology to the target gene and ability to hybridize to the target gene in need of skipping.
• the promoter is a U7 promoter.
• the exon skipping transgene comprises a nucleotide sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 13, 14, 16, 17, 18, 32, 33, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 131, 132, 133, 134 or 135.
• nucleic acids encoding exon skipping transgenes, including transgenes or gene cassettes for use in gene therapy.
• the exon skipping transgenes are encoded by a nucleic acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 13, 14, 16, 17, 18, 32, 33, 35, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 131, 132, 133, 134, or 135.
• constructs are illustrated, but not limited to those in FIG. IB, and compositions and components parts of these compositions are described (by representative sequences) in Table 1, Table 2, Table 3, and Table 4.
• the constructs may include an intron 5' of the exon skipping transgene encoding sequence.
• the presence of the more than one antisense complex e.g. more than one antisense sequence may lead to improved expression of the exon skipping transgene in cells relative to expression from nucleic acid constructs not having multiple tandem antisense complexes.
• the transgenes provided herein contain promoters that drive expression of the exon skipping transgene in appropriate context, e.g. the U7 promoter is derived from U7 snRNA.
• the size of transgene is reduced and therefore to be used in gene therapy, such as with recombinant AAV vector therapy, a stuffer was added to the genome to provide stability and efficiently package the recombinant AAV vectors.
• One or more stuffer sequences may be added, wherein the stuffer sequence is a non-coding sequence.
• exon skipping transgenes or gene cassettes in which the antisense sequence is 24 nucleotides or more for effective interaction with the U7snRNA and the target gene.
• nucleic acids comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34 which encode exemplary gene cassettes or transgenes or complexes.
• nucleic acids comprising a sequence set forth in any one of SEQ ID NOs: 80-112 which encode exemplary gene cassettes or transgenes or complexes.
• nucleic acids comprising a sequence set forth in SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO: 133 which encode exemplary gene cassettes or transgenes or complexes.
• the in vivo efficacy of the expression cassette disclosed herein in a subject in need lasts more than 6 months by one single introduction.
• the recombinant vector for delivering the transgenes described herein includes non-replicating recombinant adeno-associated virus vectors (rAAV), and may be of an AAV2, AAV.hu32, AAV8 or AAV9 serotype or any other serotype appropriate for delivery of the exon skipping transgene coding sequences to muscle cells, including both skeletal muscle and cardiac muscle, which will express the exon skipping transgene and provide additional benefit to the patient, and/or deliver to muscle cells.
• the recombinant AAV particle comprises an AAV8 capsid or a variant thereof.
• AAV vectors comprising a genome encoding more than one antisense sequence (AS) such that each sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AAV vector is produced by transfecting a cell with a cis plasmid, a trans plasmid (rep/cap) and a helper plasmid, wherein the cis plasmid comprises a nucleic acid sequence as set forth in SEQ ID NO: 114.
• AS antisense sequence
• AAV vectors comprising a genome encoding more than one antisense sequence (AS) such that each sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin.
• AS antisense sequence
• the AS nucleic acid sequence is complementary to the pre-mRNA exon 53 region, the pre-mRNA region at the junction of intron 52 and exon 53, or the pre-mRNA region at the junction of exon 53 and intron 53.
• the AS nucleic acid sequence is set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• the AS nucleic acid sequence consists of the nucleic acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• AAV vectors comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre- mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AS comprises a nucleic acid sequence as set forth in SEQ ID NO: 7, 8, 9, 19, 20, 21, or 34.
• AS antisense sequence
• AAV vectors comprising a genome encoding an antisense sequence (AS) complementary to a premessenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AS consists of a nucleic acid sequence as set forth in SEQ ID NO: 7, 8, 9, 19, 20, 21, or 34.
• AS antisense sequence
• AAV vectors comprising a genome encoding more than one antisense sequence (AS) such that each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51 /intron junction of dystrophin, wherein the recombinant AAV particle further comprises an AAV8 capsid or a variant thereof, an AAV.hu32 capsid or a variant thereof, an AAV2 capsid or a variant thereof, or an AAV9 capsid or a variant thereof.
• the recombinant AAV particle comprises an AAV8 capsid or a variant thereof.
• AAV vectors comprising a genome encoding more than one antisense sequence (AS) such that each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AAV vector is produced by transfecting a cell with a cis plasmid, a trans plasmid (rep/cap) and a helper plasmid, wherein the cis plasmid comprises a nucleic acid sequence as set forth in SEQ ID NO: 114.
• AS antisense sequence
• the AS nucleic acid sequence is complementary to the pre- mRNA exon 51 region, the pre-mRNA region at the junction of intron 50 and exon 51, or the pre-mRNA region at the junction of exon 51 and intron 51.
• the AS nucleic acid sequence is set forth in any one of SEQ ID NOs: 48-79.
• the AS nucleic acid sequence consists of the nucleotide sequences set forth in any one of SEQ ID NOs: 48-79.
• AAV vectors comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre- mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AS comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 48-79.
• AS antisense sequence
• AAV vectors comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AS consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 48-79.
• the rAAV particle comprises a capsid protein having an amino acid sequence that is at least 95% identical to SEQ ID NO: 44 (AAV.hu32 capsid), having an amino acid sequence of SEQ ID NO: 44, having an amino acid sequence that is at least 95% identical to SEQ ID NO 42 (AAV8 capsid), having an amino acid sequence of SEQ ID NO: 42, having an amino acid sequence that is at least 95% identical to SEQ ID NO: 113 (AAV2 capsid), having an amino acid sequence of SEQ ID NO: 113, having an amino acid sequence that is at least 95% identical to SEQ ID NO: 43 (AAV9 capsid), or having an amino acid sequence of SEQ ID NO: 43.
• the in vivo efficacy of the rAAV vector disclosed herein in a subject in need lasts more than 6 months by one single introduction.
• compositions comprising the recombinant vectors encoding the exon skipping transgenes provided herein, including with a pharmaceutically acceptable excipient and methods of treatment to patients suffering from any dystrophinopathy, and also have a mutation in the DMD gene that is amenable to exon skipping, such as for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD or BMD female carriers, by administration of the gene therapy vectors described herein to a subject in need thereof.
• the subject is a human.
• the pharmaceutical composition is administered in combination with another therapy for treating the dystrophinopathy.
• the in vivo efficacy of the pharmaceutical composition disclosed herein in a subject in need lasts more than 6 months by one single administration.
• a dystrophinopathy such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy by administration of an rAAV containing a transgene or gene cassette described herein, by administration to a subject in need thereof such that the exon skipping transgene is delivered to the muscle (including skeletal muscle, cardiac muscle, and/or smooth muscle).
• the rAAV is administered to the subject systemically.
• the subject is a human.
• the method further comprises providing to the subject another therapy for treating the dystropinopathy.
• the in vivo efficacy of the treatment disclosed herein in a subject in need lasts more than 6 months by one single delivery.
• the method disclosed herein results in: (i) an increase in a shortened form of dystrophin RNA in a sample muscle tissue of the subject as measured by any quantitative assay such as a polymerase chain reaction assay, including digital droplet PCR (ddPCR) and electrophoresis platforms for visualization of RNA transcripts such as Tapestation, or an equivalent assay that quantifies skipped dystrophin RNA transcript copies in the sample; (ii) an increase in a shortened form of dystrophin protein in a sample muscle tissue of the subject as measured by any quantitative assay such as Western blot, capillary electrophoresis, LC-MS, or an equivalent assay that quantifies skipped dystrophin protein concentration in the sample; (iii) a decrease in creatine kinase levels as measured in a serum or urine sample of the subject by standard quantitative assays;
• any quantitative assay such
• the method disclosed herein results in: (i) an increase in percent skipped dystrophin calculated by dividing normalized skipped dystrophin transcript copies by skipped plus unskipped dystrophin copies; or (ii) an increase in percent dystrophin as measured by quantification of skipped dystrophin protein concentration compared to a standard curve of wild type dystrophin in samples of a control subject or subjects.
• rAAV vectors disclosed herein for use in a method of treating a dystrophinopathy in a subject in need thereof, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the rAAV vector disclosed herein, wherein said administration results in delivery of the exon skipping transgene to the muscle of said subject.
• nucleic acid compositions disclosed herein for use in a method of treating a dystrophinopathy in a subject in need thereof, wherein the method comprises delivering to the circulation, muscle tissue and/or cerebrospinal fluid of said subject, a therapeutically effective amount of the nucleic acid composition disclosed herein, or an expression cassette disclosed herein.
• the dystrophinopathy is DMD, BMD, X-linked dilated cardiomyopathy or the subject is a female carrier of DMD or BMD. In some embodiments, the subject is a human. In some embodiments, the method further comprises providing to the subject another therapy for treating the dystropinopathy. In some embodiments, the in vivo efficacy of the treatment in a subject in need lasts more than 6 months by one single delivery.
• the method disclosed herein results in: (i) an increase in a shortened form of dystrophin RNA in a sample muscle tissue of the subject as measured by any quantitative assay such as a polymerase chain reaction assay, including digital droplet PCR (ddPCR) and electrophoresis platforms for visualization of RNA transcripts such as Tapestation, or an equivalent assay that quantifies skipped dystrophin RNA transcript copies in the sample; (ii) an increase in a shortened form of dystrophin protein in a sample muscle tissue of the subject as measured by any quantitative assay such as Western blot, capillary electrophoresis, LC-MS, or an equivalent assay that quantifies skipped dystrophin protein concentration in the sample; (iii) a decrease in creatine kinase levels as measured in a serum or urine sample of the subject by standard quantitative assays; and/or (iv) a decrease in fibrosis as measured by histopathological tissue staining in a sample muscle tissue of the subject.
• the method disclosed herein results in: (i) an increase in percent skipped dystrophin calculated by dividing normalized skipped dystrophin transcript copies by skipped plus unskipped dystrophin copies; or (ii) an increase in percent dystrophin as measured by quantification of skipped dystrophin protein concentration compared to a standard curve of wild type dystrophin in samples of a control subject or subjects.
• rAAV vectors disclosed herein in the manufacture of a medicament for the treatment of a dystrophinopathy in a subject in need thereof, wherein the method comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the rAAV vector disclosed herein, wherein said administration results in delivery of the exon skipping transgene to the muscle of said subject.
• nucleic acid compositions disclosed herein in the manufacture of a medicament for the treatment of a dystrophinopathy in a subject in need thereof, wherein the method comprises delivering to the circulation, muscle tissue and/or cerebrospinal fluid of said subject, a therapeutically effective amount of the nucleic acid composition disclosed herein, or an expression cassette disclosed herein.
• the dystrophinopathy is DMD, BMD, X-linked dilated cardiomyopathy or the subject is a female carrier of DMD or BMD. In some embodiments, the subject is a human. In some embodiments, the method further comprises providing to the subject another therapy for treating the dystropinopathy. In some embodiments, the in vivo efficacy of the treatment in a subject in need lasts more than 6 months by one single delivery.
• the method disclosed herein results in: (i) an increase in a shortened form of dystrophin RNA in a sample muscle tissue of the subject as measured by any quantitative assay such as a polymerase chain reaction assay, including digital droplet PCR (ddPCR) and electrophoresis platforms for visualization of RNA transcripts such as Tapestation, or an equivalent assay that quantifies skipped dystrophin RNA transcript copies in the sample; (ii) an increase in a shortened form of dystrophin protein in a sample muscle tissue of the subject as measured by any quantitative assay such as Western blot, capillary electrophoresis, LC-MS, or an equivalent assay that quantifies skipped dystrophin protein concentration in the sample; (iii) a decrease in creatine kinase levels as measured in a serum or urine sample of the subject by standard quantitative assays; and/or (iv) a decrease in fibrosis as measured by histopathological tissue staining in a sample muscle tissue of the subject.
• the method disclosed herein results in: (i) an increase in percent skipped dystrophin calculated by dividing normalized skipped dystrophin transcript copies by skipped plus unskipped dystrophin copies; or (ii) an increase in percent dystrophin as measured by quantification of skipped dystrophin protein concentration compared to a standard curve of wild type dystrophin in samples of a control subject or subjects.
• kits for producing the viral vectors comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic exon skipping transgene operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsi
• host cells comprising an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleic acid composition disclosed herein.
• the present inventions are illustrated by way of examples infra describing the construction and making of exon skipping transgene vectors, and in vitro and in vivo assays demonstrating effectiveness.
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) complimentary to a pre-mRNA exon 51 or exon 53 region of dystrophin and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprises a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22. 5.
• AS antisense sequence
• snRNA small nuclear ribonucleic acid
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon region of dystrophin and/or a pre-mRNA exon/intron junction of the pre- mRNA exon region of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon region of dystrophin and/or a pre-mRNA exon/intron junction of the pre- mRNA exon region of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• nucleic acid composition of embodiment 5 or 6, wherein the at least two transgenes comprise:
• a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin;
• a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin.
• nucleic acid composition of embodiment 8 wherein the splice site of the pre-mRNA exon region is an acceptor splice site or a donor splice site.
• nucleic acid composition of embodiment 9 wherein the splice site of the pre-mRNA exon region is a donor splice site. 12. The nucleic acid composition of embodiment 7, wherein the at least two transgenes comprise:
• a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin;
• a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to an acceptor splice site of the pre-mRNA exon region of dystrophin.
• nucleic acid composition of embodiment 7, wherein the at least two transgenes comprise:
• a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to a donor splice site of the pre-mRNA exon region of dystrophin.
• nucleic acid composition of embodiment 14, wherein the at least three transgenes comprise:
• a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to the pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin;
• a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin;
• a third transgene comprising a third AS, wherein the third AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin or the pre- mRNA exon/intron junction of the pre-mRNA exon region of dystrophin.
• a first transgene comprising a first AS, wherein the first AS nucleic acid sequence is complementary to an acceptor splice site of the pre-mRNA exon region of dystrophin;
• a second transgene comprising a second AS, wherein the second AS nucleic acid sequence is complementary to the pre-mRNA exon region of dystrophin;
• a third transgene comprising a third AS, wherein the third AS nucleic acid sequence is complementary to a donor splice site of the pre-mRNA exon region of dystrophin.
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• nucleic acid composition of embodiment 27, wherein the AS nucleic acid sequence consists of the nucleic acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are different.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding more than one transgene, wherein the transgene comprises an antisense sequence (AS) having greater than 24 nucleotides, a U7 promoter operably linked to the AS at the 5' end, and a small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS at the 3 ' end, wherein each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre- mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the nucleic acid sequence encodes at least two transgenes, wherein the antisense sequences of the transgenes are all different from each other.
• AS antisense sequence
• pre- mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) having greater than 24 nucleotides and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• a nucleic acid composition comprising a nucleic acid sequence encoding a transgene, wherein the transgene comprises a U7 promoter operably linked to an antisense sequence (AS) and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element operably linked to the AS, said flanking element comprising a nucleic acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 22, wherein the AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin.
• pre-mRNA pre-messenger RNA
• U7 promoter is a mouse U7 promoter.
• nucleic acid composition of any one of embodiments 23-29, wherein the transgene comprises a sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 131, SEQ ID NO: 132, or SEQ ID NO: 133.
• nucleic acid composition of any one of embodiments 1-43, wherein the in vivo efficacy of the nucleic acid composition in a subject in need lasts more than 6 months by one single introduction.
• An expression cassette comprising: a. more than one antisense complex (AS complex), wherein the AS complex comprises a U7 promoter sequence, b. a stuff er and c. a polyadenylation (poly A) signal, wherein the expression cassette comprises at least two AS complexes, wherein the antisense sequences of the AS complexes are all different from each other.
• AS complex antisense complex
• poly A polyadenylation
• antisense complex comprises a 5' U7 promoter, an antisense sequence having greater than 24 nucleotides, and a 3 ' small nuclear ribonucleic acid (snRNA) flanking element.
• snRNA small nuclear ribonucleic acid
• pre-mRNA pre-messenger RNA
• pre-mRNA pre-messenger RNA
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding more than one antisense sequence (AS) such that each sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, where in the recombinant AAV particle further comprises an AAV8 capsid or a variant thereof, an AAV.hu32 capsid or a variant thereof, an AAV2 capsid or a variant thereof, or an AAV9 capsid or a variant thereof.
• AS antisense sequence
• the rAAV vector of embodiment 59, wherein the capsid comprises an AAV8 capsid or a variant thereof.
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding more than one antisense sequence (AS) such that each sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AAV vector is produced by transfecting a cell with a cis plasmid, a trans plasmid (rep/cap) and a helper plasmid, wherein the cis plasmid comprises a nucleic acid sequence as set forth in SEQ ID NO: 114.
• AS antisense sequence
• the rAAV vector of embodiment 62 wherein the AS nucleic acid sequence consists of the nucleic acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 34.
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AS comprises a nucleic acid sequence as set forth in SEQ ID NO: 7, 8, 9, 19, 20, 21, or 34.
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre-mRNA) exon 53 region of dystrophin and/or a pre-mRNA exon 53/intron junction of dystrophin, wherein the AS consists of a nucleic acid sequence as set forth in SEQ ID NO: 7, 8, 9, 19, 20, 21, or 34.
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding more than one antisense sequence (AS) such that each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, where in the recombinant AAV particle further comprises an AAV8 capsid or a variant thereof, an AAV.hu32 capsid or a variant thereof, an AAV2 capsid or a variant thereof, or an AAV9 capsid or a variant thereof.
• AAV8 capsid or a variant thereof an AAV.hu32 capsid or a variant thereof
• AAV2 capsid or a variant thereof or an AAV9 capsid or a variant thereof.
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding more than one antisense sequence (AS) such that each AS nucleic acid sequence is complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AAV vector is produced by transfecting a cell with a cis plasmid, a trans plasmid (rep/cap) and a helper plasmid, wherein the cis plasmid comprises a nucleic acid sequence as set forth in SEQ ID NO: 114.
• AS antisense sequence
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AS comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 48-79.
• AS antisense sequence
• pre-mRNA pre-messenger RNA
• a recombinant adeno-associated virus (AAV) vector comprising a genome encoding an antisense sequence (AS) complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin, wherein the AS consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 48-79.
• AS antisense sequence complementary to a pre-messenger RNA (pre-mRNA) exon 51 region of dystrophin and/or a pre-mRNA exon 51/intron junction of dystrophin
• An rAAV vector comprising the nucleic acid composition of any one of embodiments 1-39, or the expression cassette of any one of embodiments 45-58.
• An rAAV vector comprising an expression cassette comprising the nucleic acid composition of any one of embodiments 1-44.
• rAAV vector of any one of embodiments 59-77, wherein the in vivo efficacy of the rAAV vector in a subject in need lasts more than 6 months by one single introduction.
• a method of delivering a transgene to a cell comprising contacting said cell with the rAAV vector of any one of embodiments 59-78, wherein said cell is contacted with the vector.
• a pharmaceutical composition for treating a dystrophinopathy in a subject in need thereof comprising a therapeutically effective amount of an rAAV particle of any one of embodiments 59-78, optionally wherein said rAAV vector is formulated for administration to the circulation or muscle tissue, of said subject.
• a pharmaceutical composition for treatment of a dystrophinopathy in a subject comprising a therapeutically effective amount of a nucleic acid composition of any one of embodiments 1-44, or an expression cassette according to any one of embodiments 45-58 formulated for delivery to the circulation or muscle tissue of said subject.
• composition of embodiment 81 or 82, wherein the dystrophinopathy is DMD, BMD, X-linked dilated cardiomyopathy or the subject is a female carrier of DMD or BMD.
• a method of treating a dystrophinopathy in a subject in need thereof comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the rAAV vector of any one of embodiments 59-78, wherein said administration results in delivery of the exon skipping transgene to the muscle of said subject.
• a method of treating a dystrophinopathy in a subject in need thereof comprising delivering to the circulation, muscle tissue and/or cerebrospinal fluid of said subject, a therapeutically effective amount of a nucleic acid composition of any one of embodiments 1- 44, or an expression cassette according to any one of embodiments 45-58.
• a method of producing recombinant AAV vectors comprising:
• an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleic acid composition of any one of embodiments 1-44;
• trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans.
• a host cell comprising: an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleic acid composition of any one of embodiments 1-44.
• FIG. 1A is a schematic representation exemplifying the target gene (DMD exon 53) for exon skipping.
• Three non-overlapping antisense sequences (AS), AS1 : antisense sequence 1; AS2: antisense sequence 2; and AS3: antisense sequence 3 represent antisense oligonucleotides that each target (hybridize to) a specific region, for example, within either the junction between DMD intron 52 and exon 53 or DMD exon 53.
• AS antisense sequence 1
• AS2 antisense sequence 2
• AS3 antisense sequence 3
• IB illustrates exemplary vector gene expression cassettes flanked by ITRs, with one ITR (5'-) mutated to form a self-complementary AAV (scAAV) genome or with two wild-type ITRs (5'- and 3'-) to form a single-stranded AAV (ssAAV ) genome when packaged in rAAV.
• scAAV self-complementary AAV
• ssAAV single-stranded AAV genome when packaged in rAAV.
• Each AS transcriptionally controlled by a mouse U7 promoter, is placed within a U7 snRNA backbone to form an antisense complex which when placed in tandem and flaked by ITRs become part of a self-complementary AAV vector.
• the nucleic acid components are arranged in an expression cassette, such as on a cis plasmid for use in manufacturing an AAV vector for gene therapy (e.g. AAV-3x53AS or AAV-3x51AS vector).
• 1C illustrates the concatenation of exons 50 to 54 of human DMD mRNA (top), compared to an example of the DMD gene of patients with exon 52 deletions which produce an out of frame dystrophin mRNA (middle), and an example of skipping of exon 53 to produce a smaller, but in-frame dystrophin mRNA and, subsequently, protein (bottom).
• FIGS. 2A-H depict the results of in vitro assessment and selection of antisense sequences in construction of an exon 53 skipping vector.
• FIG. 2A Exemplary AAV2 vectors expressing a single anti-sense snRNA targeting Ex53 evaluated in human rhabdomyosarcoma (RD) cells for their ability to induce exon skipping, whereas RT-PCR amplifying both the skipped (upper band) and unskipped (lower band) dystrophin mRNA is illustrated.
• Three AS sequences AS1, AS2, AS3 exhibiting significant skipped mRNA transcripts were selected for further evaluation (Construct Exon53-2).
• FIG. 2B Single snRNA sc.
• FIG. 2C U7 snRNA expression in a human RD cell line increased in a dose-dependent manner (AS1 or AS2). snRNAs containing different antisense sequences express at approximately equivalent levels. snRNA expression normalized to TATA-binding protein RNA (TBP).
• TBP TATA-binding protein RNA
• FIG. 2E Treatment of RD cells with AAV2 encoding three snRNAs leads to near total skipping of DMD exon 53 (Construct Exon53-1).
• FIG. 2F Diagram depicting ddPCR methodology of calculating percent skipped (note that “wildtype” dystrophin in the diagram refers to the native dystrophin in RD cells as analyzed herein).
• FIG. 2G Diagram depicting ddPCR methodology of calculating percent skipped (note that “wildtype” dystrophin in the diagram refers to the native dystrophin in RD cells as analyzed herein).
• FIG. 2H miRNAscope showed robust nuclear expression of U7 snRNAs in RD cells treated with a high-dose (MOI) AAV2-3x53 AS vector (Construct Exon53-2).
• FIG. 3A-H shows the results of U7 snRNA AS-induced DMD exon 53 skipping in vivo in hDMDdel52/mdx (del52) mice containing exon 52-deleted human DMD on chromosome 5 (these mice do not express mouse dystrophin due to the mdx background).
• FIG. 3A U7 snRNA expression (ASl-U7snRNA/TBP measured by ddPCR) in del52 mouse skeletal and cardiac muscle tissues one month after vector administration. snRNA expression is normalized to TATA-Binding Protein (TBP) in the cells.
• TBP TATA-Binding Protein
• FIG. 3B Percentage of DMD transcripts with skipped exon 53 (measured by ddPCR) in del52 skeletal and cardiac muscle one month after vector administration in two dose cohorts compared to a scramble control vector.
• FIG. 3C Plotting %skipped exon 53 against snRNA expression reveals that percentage of skipped DMD transcripts correlates with antisense U7 snRNA expression. High-dose (closed square ⁇ ); low-dose (closed circle • ).
• FIG. 3D Dose-dependent vector biodistribution (AAV genomes per diploid cell) was assessed in skeletal muscle, heart, and liver.
• FIG. 3E U7 snRNA expression in del52 mouse skeletal and cardiac muscle tissues one and three months after vector administration.
• FIG. 3F Percentage of DMD transcripts with skipped exon 53 in del52 skeletal and cardiac muscle one and three months post dosing.
• FIG. 3H Percentage of skipped DMD transcripts correlates with antisense U7 snRNA expression of Hu32-Construct Exon53-2 vectors.
• FIGS. 4A-C show that Dystrophin protein expression was restored in del52 mouse muscle following administration of Hu32-Construct Exon53-2 to induce exon 53 skipping.
• FIG. 4B Quantification of (IF) dystrophin expression in del52 mouse skeletal muscles: gastrocnemius (Gastroc) and tibialis anterior (TA). High-dose (open square ⁇ ); low-dose (open circle o); scramble (open triangle A).
• FIG. 4B Quantification of (IF) dystrophin expression in del52 mouse skeletal muscles: gastrocnemius (Ga
• FIG. 4C Quantification of (IF) dystrophin expression in del52 mouse skeletal muscles: triceps and quadriceps (Quad). High-dose (open square ⁇ ); low-dose (open circle o); scramble low dose (open triangle A).
• FIG. 4E Vectorized U7 snRNA anti-sense sequences (Hu32-Construct Exon53-2) also induced significant restoration of dystrophin protein expression in quadriceps one and three months post dosing as determined by analysis by capillary electrophoresis (Jess, PROTEINSIMPLE). Dystrophin accumulation continues to increase up to three months.
• FIG. 41 Percent of normal dystrophin restored in treated mice based on wt mouse dystrophin standard curve.
• FIGS. 5A-C depicts rescued histopathological phenotypes in del52 mice by exon 53 skipping.
• FIG 5A Embryonic myosin heavy chain staining (eMyHC, left column of images in each panel), hematoxylin and eosin (H&E, middle column in each panel), and Sirius red (right column in each panel) show acute muscle regeneration, muscle tissue integrity/mononuclear infiltrates, and fibrosis, respectively. Representative images from four skeletal muscles are shown.
• FIG. 5C Quantification of Sirius Red-positive area as a percentage of whole muscle area.
• FIGS. 6A-G illustrate an in vitro assessment of AAVHu32.3x.U7.Ex53 vector in immortalized human DMDdel52 muscle cells.
• FIG. 6A U7 snRNA expression in differentiated human DMDdel52 muscle cells was dose dependent (AS1-, or AS2- or ASSET snRNA measured by ddPCR).
• FIG. 6B Presence of unskipped del52 dystrophin decreases with increasing multiplicity of infection (MOI) of AAVHu32.3x.U7.Ex53 vector.
• FIG. 6C Skipping of Ex53 increases with higher MOI of vector.
• FIG. 6D Percent skipping of dystrophin Ex53 rises with higher MOI.
• FIG. 6A U7 snRNA expression in differentiated human DMDdel52 muscle cells was dose dependent (AS1-, or AS2- or ASSET snRNA measured by ddPCR).
• FIG. 6B Presence of unskipped del52 dystrophin decreases with increasing multiplicity of infection (MOI)
• FIG. 6E Tapestation analysis RT-PCR products shows ratio of unskipped and skipped bands at different MOIs.
• FIG. 6F Restoration of dystrophin expression in dmdDel52 (patient) cells increases with percent skipping.
• FIG. 6G Expression of restored dystrophin continues to rise up to day 10 post transduction.
• FIGS. 7A-C depict an evaluation of genome integrity for Hu32.3x.U7.Ex53.
• FIG. 7A Diagram of Hu32.3x.U7.Ex53 genome (top) and coverage display in IGV of PacBio Hifi reads mapped to the scAAV genome (bottom). Vertical bars at either end of diagram indicate ITR flip/flop configurations (bottom).
• FIG. 7B Agarose gel of purified scAAV genomes.
• FIG. 7C Distribution of lengths for HiFi reads that align to the Hu32.3x.U7.Ex53 genome.
• FIGS. 8A-D depict an evaluation of skipping efficacy in 3 months compared to 1 month post-administration of low dose and high dose constructs in del 52 mouse study.
• FIG. 8A Table depicting the basic protocol for the 3 month study in hDMDdel52/mt& mice.
• FIG. 8B Results depicting values of unskipped del 52 dystrophin RNA (normalized to TBP) in different tissues at (from left to right): 1 month (high dose), 3 month (high dose), 1 month (low dose), and 3 month (low dose).
• FIG. 8C Results depicting values of skipped dystrophin RNA (normalized to TBP) in different tissues at (from left to right): 1 month (high dose), 3 month (high dose), 1 month (low dose), and 3 month (low dose).
• FIG. 8D Results depicting values of percent skipped dystrophin in different tissues at (from left to right): 1 month (high dose), 3 month (high dose), 1 month (low dose), and 3 month (low dose).
• FIGS. 9A-B depict an evaluation of snRNA expression 3 month compared to 1 month post-administration of low dose and high dose constructs in the skeletal muscle (GAS, Quad), heart and liver of del 52 mice.
• FIGS. 10A-H depict dystrophin protein levels in muscle at 3 month compared to 1 month post-administration of low dose and high dose constructs in the del52 mouse study.
• FIG. 11 depicts experimental design for functional study with del52 mice.
• FIGS. 12A-B depict the results of a hDMDdel52/mdx mouse 2 limb hanging test.
• FIGS. 12C-D depict the results of a hDMDdel52/mdx mouse 4 limb hanging test.
• FIGS. 12E-F depict the results of a hDMDdel52/mdx mouse forelimb grip strength test.
• FIG. 13A is a schematic representation exemplifying the dystrophin target gene region (DMD exon 51) for exon skipping.
• Three non-overlapping antisense sequences AS1, AS1 : antisense sequence 1; AS2: antisense sequence 2; and AS3: antisense sequence 3, represent antisense oligonucleotides that each target (hybridize to) a specific region within either the junction between DMD exon 51 and an upstream intron (such as the exon 51 acceptor splice site) or downstream intron (such as the exon 51 donor splice site) or within DMD exon 51.
• FIG. 14 depicts an agarose gel result of candidate antisense sequences in exon skipping as single snRNA constructs.
• FIGS. 16A-B depict exon 51 and exon 53 skipping in Aexon52 RD cells treated with triple-AS-snRNA constructs (plasmids).
• FIGS. 17A-G depict snRNA expression levels in RD-AAVR cells treated with triple-snRNA (exon 51 skipping) constructs by AAV delivery.
• FIGS. 18A-G depict robust exon 51 skipping in RD-AAVR cells treated with triple-snRNA constructs by AAV2 delivery and confirmed by ddPCR.
• FIGS. 19A-D depict robust exon 51 skipping in RD-AAVR cells treated with triple-snRNA constructs by AAV.hu32 delivery and confirmed by ddPCR.
• FIGS. 20A-I depict robust exon 51 skipping in immortalized human DMD del52 myoblasts treated with triple-snRNA constructs by AAV.hu32 delivery and confirmed by ddPCR.
• FIGS. 20G-I show exon 51 skipping and dystrophin restoration in immortalized human DMDdel52 myoblasts treated with AAV8.Ex51.3AS vector or Hu32.Ex51.3AS vector at increasing MOI.
• AAV8.Ex51.3AS represents AAV8-Exon51-12.
• Human32.Ex51.3AS represents AAVHu32-Exon51-12.
• FIGS. 20H-I depict dose-dependent U7 snRNA
• FIGS. 21A-C depict experimental design for in vivo assessment of vectors in hDMDdel52/mdx (del52) mouse model.
• FIGS. 22A-H depict results for in vivo assessment of vectors in hDMDdel52/mdx (del52) mouse model by measuring AAV genome, unskipped and skipped dystrophin mRNA and the skipping efficacy.
• FIGS. 23A-V depict ddPCR results for in vivo assessment of Ex51.3 AS vectors in hDMDdel52/mdx (del52) mouse model.
• FIGS. 24A-F depict the quantification of dystrophin expression in GAS (gastrocnemius), diaphragm, and heart (determined by Jess, in high dose, middle dose, and low dose treated del52 mice, and controls: untreated del52 or C57BL/6 mice).
• FIGS. 25A-D depict the restoration of dystrophin expression in gastrocnemius tissues following administration of AAVhu32.Ex51-12 as observed by immunofluorescence.
• FIG. 25E depicts expression of dystrophin protein in heart tissue induced by AAVhu32. Ex51-12 as observed by immunofluorescence.
• FIG. 26 illustrates the concatenation of exons 50 to 54 of human DMD mRNA (the first row), compared to an example of the DMD gene of patients with exon 52 deletions which produce an out of frame dystrophin mRNA (the second row), an example of skipping of exon 53 to produce a smaller, but in-frame dystrophin mRNA and, subsequently, protein (the third row), and an example of skipping of exon 51 to produce a smaller, but in-frame “skipped” dystrophin mRNA and, subsequently, protein (e.g. “skipped dystrophin”)( the fourth row of the figure).
• FIGS. 27A-B depict robust exon 53 skipping and dystrophin restoration in immortalized human DMDdel52 myoblasts treated with AAV8.Ex53.3AS vector or AAVHu32.Ex53.3AS vector.
• AAV8.Ex53.3AS represents AAV8-Exon53-2.
• AAVHu32.Ex53.3AS represents AAVHu32-Exon53-2.
• FIGS. 27C-D depict dosedependent U7 snRNA expression and exon 53 skipping in immortalized human DMDdel52 myoblasts treated with AAV8.Ex53.3AS vector or AAVHu32.Ex53.3AS vector and confirmed by ddPCR.
• FIG. 27E shows quantification of dystrophin expression normalized by actinin.
• FIG. 28 illustrates study design for evaluating exon 53 skipping efficacy of AAVHu32.Ex53.3AS vector in del52/mdx mouse model.
• AAVHu32.Ex53.3AS represents AAVHu32-Exon53-2.
• FIGS. 29A-J depict an evaluation of exon 53 skipping efficacy of AAVHu32.Ex53.3AS vector in del52/mdx mouse.
• FIGS. 29A-B illustrate dose-dependent biodistribution of AAV genomes (AAV genomes per diploid cell) and snRNA expression.
• FIG. 29E depicts dose-dependent reduction of creatine kinase levels in the serum of treated mice.
• FIG. 29A-B illustrate dose-dependent biodistribution of AAV genomes (AAV genomes per diploid cell) and snRNA expression.
• FIGS. 29G-I depict a quantification of immunofluorescence signal, and reductions in fibrosis/acute muscle regeneration associated with dystrophin expression.
• FIG. 29J depicts reduction of myosin light chain (MYL3) levels in the serum of AAVHu32.Ex53.3 AS-treated mice (both high dose (HD) and low dose (LD)) compared to vehicle administration.
• MYL3 myosin light chain
• FIGS. 30A-D depict a sustained or increased exon 53 skipping efficacy of AAVHu32.Ex53.3AS vector in del52/mdx mouse at 3-month time point, demonstrated by biodistribution of AAV genomes, snRNA expression, and percentage of skipped exon 53.
• FIG. 30D shows that percentage of skipped exon 53 correlated with snRNA expression.
• FIG. 31 depicts experimental design for functional study with del52 mice treated with AAVHu32.Ex53.3AS vector. “AAVHu32.Ex53” represents AAVHu32-Ex53-2.
• FIGS. 32A-C depict results of functional test with hDMDdel52/mdx (del52) mice treated with AAVHu32.Ex53.3AS vector.
• FIG. 32B depicts the results of a hDMDdel52/mdx mouse 2 limb hanging test.
• FIG. 32C depicts the results of a hDMDdel52/mdx mouse 4 limb hanging test.
• FIGS. 33A-G illustrate that administration of AAVHu32.Ex53.3AS vector induced significant exon skipping and restored dystrophin protein in heart up to 12 months post administration.
• FIG. 33A depicts biodistribution of AAV genomes, snRNA expression, and percent exon 53-skipping.
• FIGS. 33B-C depict dystrophin protein shown by capillary Western and immunofluorescence.
• FIG. 33A depicts biodistribution of AAV genomes, snRNA expression, and percent exon 53-skipping.
• FIGS. 33B-C depict dystrophin protein shown by capillary Western and immunofluorescence.
• FIGS. 33E-G depict quantification of immunofluorescence signals, and reductions in fibrosis/acute muscle regeneration associated with dystrophin expression.
• FIG. 34 depicts study design for evaluating exon 53 skipping efficacy of AAVhu32.Ex53.3AS vector in non-human primate (NHP).
• AAVhu32.Ex53 represents AAVHu32-Ex53-2.
• FIGS. 35A-H depict an evaluation of exon 53 skipping efficacy of AAVhu32.Ex53.3AS vector in non-human primate (NHP).
• “Ex53.3AS” represents AAVHu32-Ex53-2.
• FIG. 35A depicts efficient exon 53 skipping in heart, diaphragm and skeletal muscles of NHP treated with AAVhu32.Ex53.3AS vector.
• FIG. 35B shows a perfect junction between dystrophin exons 52 and 54 by sequencing of the skipped RT-PCR band.
• FIG. 35D depicts detection of nuclear-localized U7 snRNA in cardiac and skeletal muscles measured by miRNAscopeTM (bio-techne, Abingdon, UK).
• FIG. 35E depicts a reduction of dystrophin protein in heart of NHP treated by AAVhu32.Ex53.3AS vector.
• FIG. 35F depicts efficient exon 53 skipping in NHP heart - specifically the right ventricle, apex and left atrium of the heart - in NHP treated with AAVhu32.Ex53.3AS vector compared to vehicle control.
• FIGS. 35G and 35H depict total snRNA per TBP copy and percent skipped Exon 53, respectively, in the right ventricle, apex and left atrium of NHP heart.
• FIG. 36 depicts study design for evaluating exon 51 skipping efficacy of AAVhu32.Ex51.3AS vector in hDMDdel52/mdx model.
• AAVhu32.Ex51 represents AAVHu32-Ex51-12
• AAVhu32.Ex51-l represents AAVHu32-Ex51-12
• AAVhu32.Ex51-2 represents AAVhu32.Ex51-3
• FIGS. 37A-N depict an evaluation of exon 51 skipping efficacy of AAVhu32.Ex51.3AS vector in hDMDdel52/mdx mouse and in myoblasts.
• FIG. 37A depicts restoration of dystrophin protein in del52 myoblasts in vitro.
• FIGS. 37B-C depict snRNA expression and exon 51-skipping in hDMDdel52/mdx mice.
• FIG. 37D depicts reduced histopathological phenotypes in del52 mice. Immunofluorescence of dystrophin positive fibers (top panel) and sirius red (bottom panel) show dystrophin and fibrosis, respectively.
• FIGS. 37H-M depict the relationship between percent skipping and dystrophin levels in skeletal muscle (gastrocnemius and diaphragm) and heart.
• FIG. 37N depicts the comparison of Ex53.3AS and Ex51.3AS biodistribution, total snRNA expression (per TBP) and percent skipped dystrophin at various timepoints in gastrocnemius (GAS), tibialis anterior (TA), heart and diaphragm (DIA).
• FIG. 38 depicts study design for evaluating potency of AAVHu32.Ex53 vectors with different ITR in hDMD/del52 mouse model.
• mITR represents a vector (AAVhu32- Ex53-4) that was made with the mITR cis plasmid having the full mITR with a restored region.
• mITRdelB represents a vector (AAVhu32-Ex53-2) with the original deletion in the mutant ITR.
• FIGS. 39A-B depict an evaluation of genome integrity of AAVHu32.Ex53 vectors with different ITR.
• PacBio sequencing showed a single major population of AAV genome at the correct size in the scAAV vectors made with the mITR cis plasmid.
• mITR represents a vector (AAVhu32-Ex53-4) that was made with the mITR cis plasmid having the full mITR with a restored region.
• mITRdelB represents a vector (AAVhu32-Ex53-2) with the original deletion in the mutant ITR.
• FIGS. 40A-G depict an evaluation of potency of AAVHu32.Ex53 vectors with different ITR in hDMD/del52 mouse, demonstrated by levels of AAV genome biodistribution, snRNA expression, percent skipping, and dystrophin restoration.
• FIGS. 41A-B depict the NGS analysis strategy (FIG. 41A) and Sashimi plot (FIG. 4 IB) used to evaluate RNA transcripts from gastrocnemius tissue of treated (Ex53.3AS) and untreated hDMDdel52/mdx mice.
• exon skipping transgenes for example, as shown in FIG. IB and nucleic acid compositions and rAAV vectors encoding the same as well as pharmaceutical compositions and treatment methods related thereto.
• AAV or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses.
• the AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene.
• An example of the latter includes a rAAV having a capsid protein having a modified sequence and/or a peptide insertion into the amino acid sequence of the naturally-occurring capsid.
• rAAV refers to a “recombinant AAV.”
• a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.
• rep-cap helper plasmid refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.
• cap gene refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus.
• the capsid protein may be VP1, VP2, or VP3.
• replica gene refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.
• nucleic acids and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules.
• Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases.
• Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
• nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.
• Amino acid residues as disclosed herein can be modified by conservative substitutions to maintain, or substantially maintain, overall polypeptide structure and/or function.
• “conservative amino acid substitution” indicates that: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Vai, lie, and Leu) can be substituted with other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (i.e., Phe, Tyr, and Trp) can be substituted with other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (i.e., Arg, His, and Lys) can be substituted with other amino acids with positively charged side chains; amino acids with negatively charged side chains (i.e., Asp and Glu) can be substituted with other amino acids with negatively charged side chains; and amino acids with polar uncharged side chains (i.e., Ser, Thr, Asn, and Gin) can be substituted with other amino acids with polar uncharged side chains.
• subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.
• a non-primate e.g., cows, pigs, horses, cats, dogs, rats etc.
• a primate e.g., monkey and human
• Dystrophin is a cytoplasmic protein encoded by the DMD gene, which is the largest known human gene [NCBI human dystrophin Gene ID: 1756], DMD transcript Dp427m encodes the main dystrophin protein found in muscle and this large full-length dystrophin (427 kDa) protein comprises a number of subdomains that contribute to its function including interaction with other proteins in the dystrophin-associated protein complex (DAPC) at the sarcolemma (Constatin, B. Biochim Biophys Acta 2014, 1838(2):635- 42. doi: 10.1016/j.bbamem.2013.08.023. Epub 2013 Sep 7).
• DMD gene which is the largest known human gene
• DMD transcript Dp427m encodes the main dystrophin protein found in muscle and this large full-length dystrophin (427 kDa) protein comprises a number of subdomains that contribute to its function including interaction with other proteins in the dystrophin-associated protein complex (DAPC) at the sarcole
• DMD Duchenne muscular dystrophy
• BMD Becker muscular dystrophy
• patients express a truncated, partially functional dystrophin.
• the therapy should induce expression of a shortened form of dystrophin RNA and subsequently a translated functional protein, e.g. “skipped dystrophin”.
• skipping over the gene region containing the mutation/deletion in the dystrophin gene, means that a nucleotide, usually an antisense oligonucleotide or RNA, binds to the mutation site in a pre-mRNA such that when the mRNA is translated into protein it skips over the site with the mutation to restore the reading frame and thus create a shortened form of the protein.
• a nucleotide usually an antisense oligonucleotide or RNA
• binds to the mutation site in a pre-mRNA such that when the mRNA is translated into protein it skips over the site with the mutation to restore the reading frame and thus create a shortened form of the protein.
• skipped dystrophin means that skipping of exon on dystrophin gene pre-mRNA results in production of a smaller, but in-frame dystrophin mRNA and, subsequently, a shorter form of dystrophin protein.
• the skipped mRNA of dystrophin gene is smaller compared to a wildtype dystrophin mRNA, or an out-of-frame dystrophin mRNA in a patient who has mutation/deletion in the dystrophin gene.
• skipping of exon 53 can produce a smaller, but in-frame “skipped” dystrophin mRNA and, subsequently, a shorter form of dystrophin protein.
• skipping of exon 51 can result in a smaller, but in-frame “skipped” dystrophin mRNA and, subsequently, a shortened dystrophin protein.
• the DMD gene of patients with exon 52 deletions leads to an out of frame dystrophin mRNA, which does not produce a functional dystrophin protein.
• terapéuticaally functional exon skipping transgene or “therapeutically functional gene therapy” means that the exon skipping gene therapy exhibits therapeutic efficacy in one or more of the assays for therapeutic utility described in the Examples or in assessment of methods of treatment described in the Examples.
• percent skipped dystrophin is a measure of the function of the gene therapy, and thus the efficacy, and the test can be performed by assessing unskipped and skipped dystrophin by ddPCR (using specific primer/probes) or equivalent quantitative assay in a muscle sample of a treated subject. Percent skipped was calculated by dividing normalized skipped dystrophin copies by skipped plus unskipped dystrophin copies.
• Skipped dystrophin protein and percent dystrophin as measured by quantitative protein assays also provide assessments of the methods of treatment. Assessments of muscle strength, motor skills and tissue histopathology would, for example, be other assessments following treatment with a gene therapy.
• operably linked refers to that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous. Where necessary, operably linked may refer to joining a coding region and a non-coding region, or two protein coding regions in a contiguous manner, e.g. in reading frame. In some instances, for example enhancers which may function when separated from the promoter by several kilobases, also intronic sequences and stuffer sequences may be of variable lengths and may be operably linked while not directly contiguous with a downstream or upstream promoter and heterologous gene.
• Embodiments described herein comprise an exon skipping transgene comprising from amino-terminus to the carboxy -terminus: 5' U7 promoter (e.g. SEQ ID NO: 6 or 15), an antisense sequence, and a 3' small nuclear ribonucleic acid (snRNA) flanking sequence (e.g. SEQ ID NO: 10 or 22).
• 5' U7 promoter e.g. SEQ ID NO: 6 or 15
• an antisense sequence e.g. SEQ ID NO: 6 or 15
• snRNA 3' small nuclear ribonucleic acid flanking sequence
• the combination of U7 promoter, antisense sequence and snRNA flanking is referred to as the “antisense complex”.
• Multiple antisense complexes may be arranged in tandem within the transgene.
• An aspect of the present disclosure relates to a recombinant AAV (rAAV) vector comprising an antisense sequence (often called antisense oligonucleotides, AONs).
• the antisense sequences are assembled into snRNAs, as explained above, in order to enable exon skipping through alternative splicing by the cellular machinery.
• Antisense sequences encoded by the expression cassettes are complementary to the target gene pre-mRNA.
• Nucleic acid sequences provided herein indicate the sequence of the antisense DNA incorporated into the AAV genome (prior to transcription).
• the antisense sequence is equal to or greater than 24 nucleotides.
• the antisense sequence comprises the sequence set forth in SEQ ID NO: 7, 8, 9, 19, 20, 21, or 34.
• the antisense sequence comprises the sequence set forth in any one of SEQ ID NOs: 48-79.
• exon skipping expression cassettes having an antisense complex comprise a U7 promoter, an antisense sequence, a U7 Sm OPT, a U7 3' Hairpin, U7 3 ' Flank and a polylinker sequence thereof, or which have more than one AS complex.
• the U7 Sm OPT, a U7 3' Hairpin, U7 3' Flank and a polylinker sequence composite sequence is referred to as the snRNA 3 ' flanking sequence or element.
• expression cassettes comprise more than one antisense complex wherein the antisense sequence within each antisense complex targets the same gene or gene region.
• expression cassettes having more than one antisense complex have improved exon skipping efficacy, for example 50% to 90% skipped.
• the in vivo efficacy of the expression cassette disclosed herein in a subject in need lasts more than 6 months by one single introduction.
• constructs encode one or more antisense complex(es). In some embodiments, constructs encode one, two, three, four, or more antisense complex(es). In some embodiments, constructs encode at least one antisense complex. In some embodiments, constructs encode at least two antisense complexes. In some embodiments, constructs encode at least three antisense complexes. In some embodiments, constructs encode at least four antisense complexes. In some embodiments, constructs encode one antisense complex. In some embodiments, constructs encode two antisense complexes. In some embodiments, constructs encode three antisense complexes. In some embodiments, constructs encode four antisense complexes. In some embodiments, constructs encode one antisense complex. In some embodiments, constructs encode two antisense complexes. In some embodiments, constructs encode three antisense complexes. In some embodiments, constructs encode four antisense complexes.
• the exon skipping constructs of the present disclosure were shown to reduce pathogenesis of muscle tissues in mouse models.
• the constructs of the disclosure may further prevent progressive fibrosis, including ventricular fibrosis, as measured by the reduction in myocardial macrophage concentrations, the reduction of the expression of adhesion molecules, and/or normalized electrocardiogram (ECG) readouts, for example end systolic volume (left ventricle), end diastolic volume, stroke volume, ejection fraction, heart rate, or cardiac output, following administration of the constructs.
• ECG electrocardiogram
• End systolic volume and other cardiac readouts can also be measured using MRI (magnetic resonance tomography), cardiac CT (computed tomography) or SPECT (single photon emission computed tomography). Cardiac function improvements following administration of the constructs of the invention may also be tested in a DBA/2J-mdx mouse model.
• exon skipping constructs and other components not specifically described can have the sequences as provided in Table 1, Table 2, Table 3, and Table 4 below.
• the amino acid sequences for the exons (and introns) of dystrophin provided herein correspond to the dystrophin isoform of UniProtKB-Pl 1532 (DMD HUMAN), which is herein incorporated by reference.
• Other embodiments can comprise the sequences from naturally-occurring functional dystrophin isoforms known in the art, such as UniProtKB-A0A075B6G3 (A0A075B6G3_HUMAN) (incorporated by reference herein).
• the present disclosure also contemplates variants of these sequences so long as the function of each component and the composite sequence is substantially maintained and/or the therapeutic efficacy of exon skipping comprising such variants is substantially maintained.
• Functional activity includes (1) hybridizing to the target gene; (2) improved muscle function in an animal model (for example, in the hDMDdel52/mt& mouse model described herein) or in human subjects; and/or (3) improvement in dystrophin expression or percent skipped exon(s) in animal models, immortalized human DMD exon-deleted myoblast cells, or human patients.
• exon skipping transgene comprises a sequence set forth in SEQ ID NO: 1 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1; or comprise a sequence set forth in SEQ ID NO: 2 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2; or comprise a sequence set forth in SEQ ID NO: 13 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; or comprise a sequence set forth in SEQ ID NO: 14 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least
• the exon skipping transgene comprises a sequence set forth in any one of SEQ ID NOs: 48-79 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises one or more sequence(s) set forth in any one of SEQ ID NOs: 48-79 or one or more sequence(s) with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises at least one sequence set forth in any one of SEQ ID NOs: 48-79 or at least one sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises at least two sequences set forth in any one of SEQ ID NOs: 48-79 or at least two sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises at least three sequences set forth in any one of SEQ ID NOs: 48-79 or at least three sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises at least four sequences set forth in any one of SEQ ID NOs: 48-79 or at least four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises one, two, three, or four sequences set forth in any one of SEQ ID NOs: 48-79 or one, two, three, or four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises one sequence set forth in any one of SEQ ID NOs: 48-79 or one sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises two sequences set forth in any one of SEQ ID NOs: 48-79 or two sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises three sequences set forth in any one of SEQ ID NOs: 48-79 or three sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises four sequences set forth in any one of SEQ ID NOs: 48-79 or four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 48-79.
• the exon skipping transgene described herein comprises one or more sequence(s) set forth in Table 2 or one or more sequence(s) with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises at least one, two, three, or four sequences set forth in Table 2 or at least one, two, three, or four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises at least one sequence set forth in Table 2 or at least one sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises at least two sequences set forth in Table 2 or at least two sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises at least three sequences set forth in Table 2 or at least three sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises at least four sequences set forth in Table 2 or at least four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one, two, three, or four sequences set forth in Table 2 or one, two, three, or four sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one sequence set forth in Table 2 or one sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one or more sequence(s) complementary to a pre-mRNA exon/intron junction of the pre- mRNA exon region of dystrophin set forth in Table 2 or one or more sequence(s) complementary to a pre-mRNA exon/intron junction of the pre-mRNA exon region of dystrophin with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one or more sequence(s) complementary to a donor splice site of the pre-mRNA exon region of dystrophin set forth in Table 2 or one or more sequence(s) complementary to a donor splice site of the pre-mRNA exon region of dystrophin with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one or more sequence(s) complementary to a pre-mRNA exon region of dystrophin set forth in Table 2 or one or more sequence(s) complementary to a pre-mRNA exon region of dystrophin with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 2.
• the exon skipping transgene described herein comprises one or more sequence(s) complementary to a pre-mRNA exon region of dystrophin as depicted in Table 2.
• the exon skipping transgene comprises a sequence set forth in any one of SEQ ID NOs: 80-112 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 80-112.
• the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 84 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 84.
• the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 85 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 85. In some embodiments, the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 106 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 106.
• the exon skipping transgene comprises a sequence set forth in Table 3 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence set forth in Table 3.
• the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 131 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 131.
• the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 132 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 132.
• the exon skipping transgene comprises a sequence set forth in SEQ ID NO: 133 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 133.
• Table 1, Table 2, Table 3, and Table 4 provide the nucleic acid sequences of exon skipping construct embodiments in accordance with the present disclosure. It is contemplated that other embodiments include substituted variants of the exon skipping constructs therein such that the constructs substantially maintain their functional activity.
• nucleic acid expression cassettes comprising regulatory elements designed to confer or enhance expression of the antisense sequences within the exon skipping constructs.
• the invention further involves engineering regulatory elements, including promoter elements, and optionally enhancer elements and/or introns, to enhance or facilitate expression of the transgene.
• the rAAV vector also includes such regulatory control elements known to one skilled in the art to influence the expression of the RNA encoded by the nucleic acids (transgenes). Each element of the expression cassette was selected to provide the most efficient exon skipping for the particular target gene.
• the expression cassette of an AAV vector typically comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues.
• a regulatory sequence such as a promoter
• the U7 promoter and 3’ flanking sequence described herein are derived from U7 small nuclear RNA (snRNA) sequences and take advantage of endogenous functions of snRNAs to enable exon skipping.
• snRNA small nuclear RNA
• insertion of appropriate antisense sequences into the U7 snRNA eliminates its U7 histone processing function and changes the function to that of an effector of alternative splicing of the target gene pre-messenger RNA (pre-RNA) (Gorman et al, Proc Natl Acad Sci USA. 1998 Apr 28; 95(9): 4929-4934, which is hereby incorporated by reference in its entirety.)
• RNA polymerase Il-type promoters play an important role in coordinating transcription and pre-mRNA splicing.
• the exon skipping expression cassette comprises a U7 promoter.
• the antisense sequence of the cassette is operably linked to a U7 promoter.
• the antisense sequence is also linked to a Sm or Sm-like protein binding domain, or variant thereof.
• the antisense sequence is operably linked to a snRNA SmOPT sequence.
• the Sm OPT sequence comprises at least about nucleotides 1 to 11 of the sequence set forth in SEQ ID NO: 22.
• the snRNA also provides a hairpin.
• the hairpin is derived from mouse U7 snRNA, human U7 snRNA, or human U 1 snRNA.
• AAV vector comprising an antisense sequence (often called antisense oligonucleotides, AONs).
• the antisense sequences are assembled into snRNAs, as explained above, in order to enable exon skipping through alternative splicing by the cellular machinery.
• Antisense sequences encoded by the expression cassettes are complimentary to the target gene pre-mRNA.
• Nucleic acid sequences provided herein indicate the sequence of the antisense incorporated into the AAV genome.
• Antisense sequences were selected based on their homology to the target exon(s) of human DMD gene (encodes dystrophin protein) in the present examples.
• antisense sequences were selected based on their homology to exon 53 or exon 51 of human DMD gene (encodes dystrophin protein) in the present examples.
• Antisense sequences may also overlap with the junction of intron 52 and exon 53, or the junction of exon 53 and intron 53 (e.g. 5' or 3' splicing sites of exon 53), and still be effective for exon skipping.
• Antisense sequences may also overlap with the junction of intron 50 and exon 51, or the junction of exon 51 and intron 51 (e.g. 5' or 3' splicing sites of exon 51), and still be effective for exon skipping.
• antisense sequences are typically 19 nucleotides in length
• the antisense sequences described herein are equal to or greater than 24 nucleotides in length.
• one nucleotide mismatch was sufficient to significantly decrease AON efficacy according to ISo, et al. Genes (Basel). 2019 Jun; 10(6): 452)
• SRSFs serine and arginine-rich splicing factors
• SRSFs may increase or decrease effectiveness of the AON, depending on their location and proximity to each other.
• These splicing factors may be located on the target gene using an exonic splicing finder software tool, such as ESEfinder (exon.cshl.edu/ESE/) or RESCUE-ESE
• antisense selection is largely influenced by coverage of at least one ESE, avoidance of the formation of secondary structures, and size of the antisense sequence, and for dystrophin gene exon skipping, location of the target antisense region at or near the 5' or 3' splicing sites of the exon may be advantageous.
• the antisense sequence is equal to or greater than 24 nucleotides in length.
• the antisense sequence covers at least one splicing factor site on the target sequence.
• an antisense sequence(s) described herein cover at least two splicing factor sites on the target sequence.
• the antisense sequence covers an acceptor splice site and/or a donor splice site on the target sequence. In certain embodiments, an antisense sequence(s) described herein cover an acceptor splice site or a donor splice site on the target sequence. In certain embodiments, an antisense sequence(s) described herein cover an acceptor splice site and a donor splice site on the target sequence. In certain embodiments, an antisense sequence(s) described herein cover at least an acceptor splice site on the target sequence. In certain embodiments, an antisense sequence(s) described herein cover at least a donor splice site on the target sequence.
• an AAV vector comprising an intron within the regulatory cassette.
• Introns contain sequences that, in this context, may regulate or enhance transcription or translation of a gene or mRNA.
• An intron placed 5' or 3' of the (each) transgene coding sequence may enhance transcriptional activity, proper splicing and, thus, exon skipping U7snRNA expression.
• an intron is coupled to the 5' end of a sequence encoding an exon skipping nucleic acid construct.
• the intron can be linked to the antisense sequence or the snRNA.
• the intron is less than 100 nucleotides in length.
• the intron is a chimeric intron derived from human P- globin and Ig heavy chain (also known as P-globin splice donor/immunoglobulin heavy chain splice acceptor intron, or P-globin/IgG chimeric intron).
• introns well known to the skilled person may be employed, such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), P-globin splice donor/immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron.
• the intron is an intron comprises SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 39 as shown in Table 1.
• polyA polyadenylation
• Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure.
• Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit P-globin gene, the bovine growth hormone (BGH) gene, the human growth hormone (hGH) gene, and the synthetic polyA (SPA) site.
• the polyA signal comprises SEQ ID NO: 23 or SEQ ID NO: 36 as shown in Table 1.
• exon skipping transgene in accordance with the present disclosure can be included in an AAV vector for gene therapy administration to a human subject.
• recombinant AAV (rAAV) vectors can comprise an AAV viral capsid and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises an exon skipping transgene, operably linked to one or more regulatory sequences that control expression of the transgene in human cells, e.g. muscle cells to express and deliver the exon skipping transgene.
• ITRs AAV inverted terminal repeats
• the provided methods are suitable for use in the production of any isolated recombinant AAV particles for delivery of a exon skipping constructs described herein, in the production of a composition comprising any isolated recombinant AAV particles encoding an exon skipping construct, or in the method for treating a disease or disorder amenable for treatment with an exon skipping construct in a subject in need thereof comprising the administration of any isolated recombinant AAV particles encoding a exon skipping construct described herein.
• AAV particles and AAV vectors are used interchangeably (e.g. an AAV capsid encapsidating a genome).
• the rAAV can be of any serotype, variant, modification, hybrid, or derivative thereof, known in the art, or any combination thereof (collectively referred to as “serotype”).
• the AAV serotype has a tropism for muscle tissue.
• the AAV serotype also has a tropism for the liver, in which case the liver cells transduced with the AAV form a depot of exon skipping construct secreting cells, secreting the exon skipping construct into the circulation.
• rAAV particles have a capsid protein from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC
• rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
• AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.
• a population of rAAV particles can comprise two or more serotypes, e.g., comprising two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HS
• rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety.
• the rAAV particles comprise the capsid with one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety.
• rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos.
• rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAVPHP.B.
• rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
• rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
• rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
• rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
• rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
• rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
• rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446;
• rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
• a single-stranded AAV can be used.
• a self-complementary vector e.g., scAAV
• scAAV single-stranded AAV
• the rAAV particles comprise a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, and AAV.hu37.
• the rAAV particles have an AAV capsid serotype of AAV1 or a derivative, modification, or pseudotype thereof.
• the rAAV particles have an AAV capsid serotype of AAV2 or a derivative, modification, or pseudotype thereof.
• the rAAV particles have an AAV capsid serotype of AAV4 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV5 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV.hu32 or a derivative, modification, or pseudotype thereof.
• the rAAV particles have an AAV capsid serotype of AAV2 or a derivative, modification, or pseudotype thereof.
• rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV.hu32, AAV2, AAV8 or AAV9 capsid protein.
• rAAV particles comprise a capsid protein that has an AAV.hu32 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
• rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV8 capsid protein.
• the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
• the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
• rAAV particles comprise a mosaic capsid.
• Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV.
• rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.
• rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV.hu32, AAV.hu37, AAVrh.8, and AAVrh.10.
• rAAV particles comprise a pseudotyped rAAV particle.
• the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV 14, AAV15 and AAV16).
• rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV
• rAAV particles comprise a pseudotyped rAAV particle containing AAV.hu32 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle is comprised of AAV9 capsid protein. In some embodiments, the pseudotyped rAAV.hu32, rAAV8 or rAAV9 particles are rAAV2/hu32, rAAV2/8 or rAAV2/9 pseudotyped particles.
• rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes.
• the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc8O, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, rA
• the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV.hu32, AAVrh.8, and AAVrh.10.
• the rAAV particles comprise an AAV capsid protein chimeric of AAV.hu32 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC
• the rAAV particles comprise an AAV capsid protein chimeric of AAV.hu32 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.
• the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC1, AAV.HSC2,
• the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAV.hu32, AAV.hu37, AAVrh.8, and AAVrh.10.
• the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC1, AAV.HSC2,
• the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAV.hu32, AAVrh.8, and AAVrh.10.
• the rAAV particles comprises a Clade A, B, E, or F AAV capsid protein. In some embodiments, the rAAV particles comprises a Clade F AAV capsid protein. In some embodiments the rAAV particles comprises a Clade E AAV capsid protein.
• Table 1 below provides examples of amino acid sequences for an AAV2, AAV8, AAV9, AAV.hu32, AAV.hu37 and AAV.rh74 capsid proteins and the nucleic acid sequence of alternative AAV2 5’- and 3’ ITRs for the packaging of single-stranded DNA (ssDNA or sc AAV) genomes or self-complementary genomes (sc AAV).
• the provided methods are suitable for use in the production of recombinant AAV encoding a transgene.
• the transgene is an exon skipping construct as described herein.
• the rAAV genome comprises the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter/enhancers, b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the described transgene.
• constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a U7 promoter and a small poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid encoding a exon skipping transgene as described herein.
• ITRs AAV2 inverted terminal repeats
• control elements which include a U7 promoter and a small poly A signal
• transgene providing (e.g., coding for) a nucleic acid encoding a exon skipping transgene as described herein.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control or filler elements, which include a)stuffer sequence, b) a small poly A signal; and (3) a DMD exon 53 skipping cassette, which includes from the N-terminus to the C-terminus, the mouse U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control or filler elements, which include a) a stuffer sequence, b) a small poly A signal; and (3) a DMD exon 51 skipping cassette, which includes from the N- terminus to the C-terminus, the mouse U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes more than one antisense complex which comprises from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence; (3) a stuffer sequence; and (4) control elements, which include a small poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes two antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence; (3) optionally a stuffer sequence linked to the second antisense complex (coupled to the snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence; (3) optionally a stuffer sequence linked to the third antisense complex (coupled to the snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises SEQ ID NO: 19, an AS complex comprises SEQ ID NO: 20, and an AS complex comprises SEQ ID NO: 21; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• AS antisense
• the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes one, two, three, four, or more antisense complex(es), each comprising from the N- terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises one or more sequence(s) set forth in Table 2; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes two antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises one or more sequence(s) set forth in Table 2; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises one or more sequence(s) set forth in Table 2; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes four antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises one or more sequence(s) set forth in Table 2; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises a sequence set forth in Table 3; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and an antisense (AS) complex comprises SEQ ID NO: 58, an AS complex comprises SEQ ID NO: 52, and an AS complex comprises SEQ ID NO: 59; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• the U7 promoter is a mouse U7 promoter.
• the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense (AS) complexes, each comprising from the N- terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and each of the three AS complexes comprises any one of SEQ ID NO: 58, SEQ ID NO: 52, or SEQ ID NO: 59; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• the U7 promoter is a mouse U7 promoter.
• the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) a DMD exon skipping cassette, which includes three antisense (AS) complexes, each comprising from the N-terminus to the C-terminus, U7 promoter, antisense sequence, and an snRNA 3’ flanking sequence, and at least one of the three AS complexes comprises any one of SEQ ID NO: 58, SEQ ID NO: 52, or SEQ ID NO: 59; (3) a stuffer sequence linked to the third antisense complex (coupled to its snRNA flanking sequence); and (4) control elements, which include a poly A signal.
• the U7 promoter is a mouse U7 promoter.
• constructs described herein comprising AAV ITRs flanking an expression cassette can be between 900 nt and 2500 nt in length. In some embodiments, such constructs are less than 2200 nt, 2100 nt, 2000 nt, 1900 nt, 1800 nt, 1700 nt, or 1600 nt in length.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS).
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 134.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59. SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 52.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 61, and SEQ ID NO: 62.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 63, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 63, and SEQ ID NO: 64.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 65, and SEQ ID NO: 64.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 66, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 67, and SEQ ID NO: 64.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 52, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 76, SEQ ID NO: 52, and SEQ ID NO: 77.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 57, SEQ ID NO: 60, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 59.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome coding the antisense sequences comprises SEQ ID NO: 58, SEQ ID NO: 63, SEQ ID NO: 70, and SEQ ID NO: 71.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the antisense sequences are arranged in tandem in any order in the genome.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome further comprises an snRNA sequence.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome further comprises a U7 promoter.
• recombinant adeno-associated virus (AAV) vectors comprise a genome encoding three or more antisense sequences (AS), wherein the genome further comprises a U7 promoter and an snRNA 3’ flanking sequence.
• Self-complementary AAV refers a plasmid or vector having an expression cassette in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
• the scAAV genome is not subject to host-cell DNA polymerase and does not require synthesis of a complementary strand.
• the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription.
• Single-stranded AAV (ssAAV) vectors wherein the coding sequence and complementary sequence of the transgene expression cassette are on separate strands, are packaged in separate viral capsids.
• ssAAV single-stranded AAV
• scAAV self-complementary AAV
• Stuffer polynucleotide sequences are included in the AAV genome to enlarge genome size.
• Inert stuffer (or filler) sequences are non-coding sequences (e.g. cDNA depleted of any translation or “initiation” sites) typically incorporated in genomes and expression cassettes and can be particularly useful for transgenes delivered by viral vectors.
• stuffer (or filler) sequences may improve the packaging of the AAV and thus improve manufacturing, stability, and transduction.
• Exemplary stuffer sequences are provided in Table 1. See also, WO2022/235614 published on December 8, 2022, which is hereby incorporated by reference in its entirety.
• nucleic acid embodiments of the present disclosure comprise rAAV vectors encoding exon skipping constructs comprising or consisting of a nucleotide sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 13, 14, 16, 17, 18, 32, 33, 35, 132, 133, 134, or 135 provided in Table 1 herein.
• rAAV vectors encoding exon skipping constructs comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 80-112 provided in Table 3 herein.
• nucleic acid embodiments of the present disclosure comprise rAAV vectors encoding exon skipping constructs comprising or consisting of a nucleotide sequence set forth in SEQ ID NO: 131 provided in Table 4 herein.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 13, 14, 16, 17, 18, 32, 33, 35, 132, 133, 134, or 135 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: SO- 112 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 84 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 85 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 106 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 131 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 132 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective exon skipping construct that restores the target gene product, particularly restores dystrophin in muscle cells.
• the rAAV vectors or particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Provisional Application No. 63400724 filed August 24, 2022, International Patent Application No. PCT/US2023/065855 filed April 14, 2023, WO2019207132, W02020206189, WO2021072197, W02021050974, WO202 1077000, W02022076750 and W02022020616.
• AAV capsids comprising muscle-specific peptides or muscle-homing peptides are useful for targeting rAAV vectors to muscle.
• the muscle-specific peptide comprises an integrin receptor-binding domain or an integrin-binding domain, such as RGD and other recognition sequences for integrin (Ruoslahti E. Annu Rev Cell Dev Biol. 1996;12:697-715. doi: 10.1146/annurev.cellbio, 12.1.697. PMID: 8970741; e.g. RGD-peptide RGDLRVS (SEQ ID NO: 116), or peptide SLRSPPS (SEQ ID NO: 117), as described in Varadi, et al.
• the peptide insertion may be a sequence of consecutive amino acids from a domain that targets muscle, or an analog, or a conformational analog designed to mimic the three-dimensional structure of said domain.
• a muscle-homing peptide is VQVGRTS (SEQ ID NO: 123) or a portion thereof.
• a muscle-homing peptide comprises about or at least 3, 4, 5, or 6 contiguous amino acids of VQVGRTS (SEQ ID NO: 123).
• a muscle-homing peptide comprises about or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to VQVGRTS (SEQ ID NO: 123).
• a muscle-homing peptide is RRQPPRSISSHP (M12; SEQ ID NO: 124) or a portion thereof.
• a muscle-homing peptide comprises about or at least 4, 5, 6, or 7 contiguous amino acids of ASSLNIA (a muscle-specific peptide (MSP); SEQ ID NO: 125). In some embodiments, a muscle-homing peptide comprises about or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to ASSLNIA (a musclespecific peptide (MSP); SEQ ID NO: 125). In some embodiments, a muscle-homing peptide is AGSTSAGSAAGSSGDRRQPPRSISSHP (SEQ ID NO: 126) or a portion thereof.
• a muscle-homing peptide comprises about or at least 4, 5, 6, 7, or more than 7 contiguous amino acids of AGSTSAGSAAGSSGDRRQPPRSISSHP (SEQ ID NO: 126). In some embodiments, a muscle-homing peptide comprises about or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to AGSTSAGSAAGSSGDRRQPPRSISSHP (SEQ ID NO: 126). In some embodiments, a muscle-homing peptide is CYAIGSFDC (SEQ ID NO: 127) or a portion thereof.
• a muscle-homing peptide comprises about or at least 4, 5, 6, 7, or more than 7 contiguous amino acids of CYAIGSFDC (SEQ ID NO: 127). In some embodiments, a muscle-homing peptide comprises about or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to CYAIGSFDC (SEQ ID NO: 127). In some embodiments, a muscle-homing peptide is SGASAV (SEQ ID NO: 128) or a portion thereof. In some embodiments, a muscle-homing peptide comprises about or at least 4contiguous amino acids of SGASAV (SEQ ID NO: 128).
• a muscle-homing peptide comprises about or at least about 80%, 85%, 90%, 95%, or 100% sequence identity to SGASAV (SEQ ID NO: 128).
• a muscle-homing peptide comprises the motif GRSGXR (SEQ ID NO: 129; wherein X can be any naturally occurring amino acid).
• a muscle-homing peptide comprises the motif DFSGIAX (SEQ ID NO: 130; wherein X can be any naturally occurring amino acid).
• a muscle-homing peptide of the disclosure can be any known muscle-homing peptide or any predicted muscle-homing peptide, and any muscle-homing capsid can be any capsid serotype comprising a musclehoming peptide, for example an AAV2, AAV.hu32, AAV.rh74, AAV8 or AAV9 serotype or any other serotype appropriate for delivery of the exon skipping transgene coding sequences to muscle cells.
• a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein.
• capsid proteins are described herein.
• the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein and retains (or substantially retains) biological function of the capsid protein and the inserted peptide from a heterologous protein or domain thereof.
• the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein, while retaining (or substantially retaining) biological function of the AAV8 capsid protein and the inserted peptide.
• the capsid protein, coat, and rAAV particles may be produced by techniques known in the art.
• the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector.
• the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene.
• the cap and rep genes are provided by a packaging cell and not present in the viral genome.
• the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap plasmid in place of the existing capsid gene.
• this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat.
• Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging.
• cell culture-based systems are known in the art for production of rAAV particles, any of which can be used to practice a method disclosed herein.
• the cell culturebased systems include transfection, stable cell line production, and infectious hybrid virus production systems which include, but are not limited to, adenovirus-AAV hybrids, herpesvirus- AAV hybrids and baculovirus-AAV hybrids.
• rAAV production cultures for the production of rAAV virus particles require: (1) suitable host cells, including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperaturesensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) suitable media and media components (nutrients) to support cell growth/survival and rAAV production.
• suitable host cells including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines
• suitable helper virus function provided by wild type or mutant adenovirus (such as temperaturesensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions
• Nonlimiting examples of host cells include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, myoblast cells, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (e.g. in the case of baculovirus production systems).
• SF-9 insect-derived cell lines
• a method of producing rAAV particles comprising (a) providing a cell culture comprising an insect cell; (b) introducing into the cell one or more baculovirus vectors encoding at least one of: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
• the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome.
• the method comprises using a baculovirus encoding the rAAV genome and an insect cell expressing the rep and cap genes. In some embodiments, the method comprises using a baculovirus vector encoding the rep and cap genes and the rAAV genome.
• the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell comprising one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
• a method disclosed herein uses a baculovirus production system.
• the baculovirus production system uses a first baculovirus encoding the rep and cap genes and a second baculovirus encoding the rAAV genome.
• the baculovirus production system uses a baculovirus encoding the rAAV genome and a host cell expressing the rep and cap genes.
• the baculovirus production system uses a baculovirus encoding the rep and cap genes and the rAAV genome.
• the baculovirus production system uses insect cells, such as Sf-9 cells.
• a skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV.
• AAV helper genes e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene
• rAAV genomes comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)
• ITRs inverted terminal repeats
• helper viruses including adenovirus and herpes simplex virus (HSV)
• HSV herpes simplex virus
• helper viruses promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication, thus producing capsid proteins and facilitating packaging (or encapsidating) the genome.
• AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors (plasmids) encoding the AAV rep and cap genes, helper genes, and rAAV genome.
• AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.
• viral vectors for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.
• one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector.
• the rHSV vector encodes the AAV rep and cap genes.
• the rHSV vector encodes the helper genes.
• the rHSV vector encodes the rAAV genome.
• the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes.
• a method of producing rAAV particles comprising (a) providing a cell culture comprising a host cell; (b) introducing into the cell one or more rHSV vectors encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
• the rHSV vector encodes the AAV rep and cap genes.
• the rHSV vector encodes helper functions.
• the rHSV vector comprises one or more endogenous genes that encode helper functions. In some embodiments, the rHSV vector comprises one or more heterogeneous genes that encode helper functions. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions and the rAAV genome. In some embodiments, the rHSV vector encodes helper functions and the AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
• a method of producing rAAV particles comprising (a) providing a cell culture comprising a mammalian cell; (b) introducing into the cell one or more polynucleotides encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
• the helper functions are encoded by adenovirus genes.
• the mammalian cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
• AAV rep and cap genes are encoded by one plasmid vector.
• AAV helper genes e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene
• the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector.
• the El a gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector.
• one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector.
• the helper genes are stably expressed by the host cell.
• AAV rep and cap genes are encoded by one viral vector.
• AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector.
• the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector.
• the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector.
• one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one viral vector.
• the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors.
• a method disclosed herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged.
• the AAV cap gene is an AAV8 or AAV9 cap gene.
• the AAV cap gene is an AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu32, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene.
• the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, and AAV.hu37.
• the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs.
• the AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HS
• Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged.
• a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used.
• ITRs AAV inverted terminal repeats
• a second vector encoding AAV rep and cap genes a third vector encoding helper genes
• a mixture of the three vectors is co-transfected into a cell.
• a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.
• one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells.
• the cell constitutively expresses rep and/or cap genes.
• the cell constitutively expresses one or more AAV helper genes.
• the cell constitutively expresses El a.
• the cell comprises a stable transgene encoding the rAAV genome.
• AAV rep, cap, and helper genes can be of any AAV serotype.
• AAV ITRs can also be of any AAV serotype.
• AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.
• AAV cap gene is from AAV8 or AAV9 cap gene.
• an AAV cap gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6,
• AAV rep and cap genes for the production of a rAAV particle are from different serotypes.
• the rep gene is from AAV2 whereas the cap gene is from AAV8.
• the rep gene is from AAV2 whereas the cap gene is from AAV9.
• the rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HS
• the rep and the cap genes are from the same serotype. In still other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one modified protein domain or modified promoter domain. In certain embodiments, the at least one modified domain comprises a nucleotide sequence of a serotype that is different from the capsid serotype. The modified domain within the rep gene may be a hybrid nucleotide sequence consisting fragments different serotypes.
• Hybrid rep genes provide improved packaging efficacy of rAAV particles, including packaging of a viral genome comprising an exon skipping transgene greater than 4 kb, greater than 4.1 kb, greater than 4.2 kB, greater than 4.3 kb, greater than 4.4 kB, greater than 4.5 kb, or greater than 4.6 kb.
• AAV rep genes consist of nucleic acid sequences that encode the non-structural proteins needed for replication and production of virus. Transcription of the rep gene initiates from the p5 or pl9 promoters to produce two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively.
• Rep78/68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have common helicase and ATPase domains that function in genome replication and/or encapsidation (Maurer AC, 2020, DOI: 10.1089/hum.2020.069). Transcription of the cap gene initiates from a p40 promoter, which sequence is within the C-terminus of the rep gene, and it has been suggested that other elements in the rep gene may induce p40 promoter activity.
• the p40 promoter domain includes transcription factor binding elements EFl A, MLTF, and ATF, Fos/Jun binding elements (AP-1), Sp 1 -like elements (Spl and GGT), and the TATA element (Pereira and Muzyczka, Journal of Virology, June 1997, 71(6): 4300-4309).
• the rep gene comprises a modified p40 promoter.
• the p40 promoter is modified at any one or more of the EFl A binding element, MLTF binding element, ATF binding element, Fos/Jun binding elements (AP-1), Sp 1 -like elements (Spl or GGT), or the TATA element.
• the rep gene is of serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rhlO, rh20, rh39, rh.74, RHM4-1, hu32, or hu37, and the portion or element of the p40 promoter domain is modified to serotype 2.
• the rep gene is of serotype 8 or 9, and the portion or element of the p40 promoter domain is modified to serotype 2.
• ITRs contain A and A’ complimentary sequences, B and B’ complimentary sequences, and C and C’ complimentary sequences; and the D sequence is contiguous with the ssDNA genome.
• the complimentary sequences of the ITRs form hairpin structures by self-annealing (Berns KI. The Unusual Properties of the AAV Inverted Terminal Repeat. Hum Gene Ther 2020).
• the D sequence contains a Rep Binding Element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication.
• RBE Rep Binding Element
• TRS terminal resolution site
• the ITRs are also required as packaging signals for genome encapsidation following replication.
• the ITR sequences and the cap genes are from the same serotype, except that one or more of the A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence may be modified to contain sequences from a different serotype than the capsid. In some embodiments, the modified ITR sequences are from the same serotype as the rep gene.
• the ITR sequences and the cap genes are from different serotypes, except that one or more of the ITR sequences selected from A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence are from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are from the same serotype as the rep gene.
• the rep and the cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, p!9 promoter domain, Rep40 domain or p40 promoter domain.
• the rep and the cap genes are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype.
• an rAAV comprises a transgene flanked by AAV2 ITR sequences, an AAV8 cap, and a hybrid AAV2/8 rep.
• the AAV2/8 rep comprises serotype 8 rep except for the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2/8 rep comprises serotype 2 rep except for the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, more than two serotypes may be utilized to construct a hybrid replcap plasmid.
• a method disclosed herein comprises transfecting a cell using a chemical based transfection method.
• the chemical-based transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)), lipofection.
• the chemical-based transfection method uses cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)).
• the chemical-based transfection method uses polyethylenimine (PEI).
• the chemical-based transfection method uses DEAE dextran.
• the chemical-based transfection method uses calcium phosphate.
• Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation e.g., electroporation, lipofection).
• Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
• the foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
• the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below.
• Methods include both in vitro and in vivo tests in animal models as described herein or using any other methods known in the art for testing the activity and efficacy of exon skipping transgenes.
• transgene of a recombinant vector disclosed herein for example rAAV vectors.
• the transduction and subsequent expression of recombinant gene therapy vectors in muscle cells can be tested in human rhabdomyosarcoma (RD) cells as described in Example 2, herein.
• RD rhabdomyosarcoma
• Several muscle or heart cell lines may be utilized, including but not limited to C2C12 (mouse), T0034 (human), L6 (rat), MM 14 (mouse), P19 (mouse), G-7 (mouse), G-8 (mouse), QM7 (quail), H9c2(2-1) (rat), Hs 74. Ht (human), and Hs 171.
• Ht human cell lines. Immortalized human myoblasts that encode a mutated dystrophin gene may be used in various exon skipping detection assays (MYOBANK - AFM, Institut de Myologie, Paris France). Vector copy numbers and/or gene transcripts may be assessed using polymerase chain reaction techniques, also digital droplet PCR (ddPCR), and level of exon skipping transgene expression may be tested by measuring levels of exon skipping transgene mRNA in the cells.
• ddPCR digital droplet PCR
• the efficacy of a viral vector containing a transgene encoding a exon skipping transgene as described herein may be tested by administering to an animal model to restore mutated dystrophin, for example, by using an mdx background mouse and/or the golden retriever muscular dystrophy (GRMD) background model wherein such model also contains a mutation that is amenable to exon skipping.
• Animal models are utilized to assess the biodistribution, expression and therapeutic effect of the transgene expression. The therapeutic effect may be assessed, for example, by assessing change in dystrophin expression in the animal receiving the exon skipping transgene.
• compositions and methods for therapeutic administration comprising a dose of an exon skipping transgene encoding vector disclosed herein in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed here.
• the efficacy of gene therapy vectors may be assessed in murine models of DMD.
• the mdx mouse model (Yucel, N., et al, Humanizing the mdx mouse model of DMD: the long and the short of it, Regenerative Medicine volume 3, Article number: 4 (2016)), carries a nonsense mutation in exon 23, resulting in an early termination codon and a truncated protein (mdx).
• Mdx mice have 3-fold higher blood levels of pyruvate kinase activity compared to littermate controls.
• mdx skeletal muscles exhibit active myofiber necrosis, cellular infiltration, a wide range of myofiber sizes and numerous centrally nucleated regenerating myofibers. This phenotype is enhanced in the diaphragm, which undergoes progressive degeneration and myofiber loss resulting in an approximately 5- fold reduction in muscle isometric strength.
• GRMD golden retriever muscular dystrophy
• Phenotypic features in dogs include elevation of serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD, as in humans. GRMD dogs develop paradoxical muscle hypertrophy which seems to play a role in the phenotype of affected dogs, with stiffness at gait, decreased joint range of motion, and trismus being common features. Objective biomarkers to evaluate disease progression include tetanic flexion, tibiotarsal joint angle, % eccentric contraction decrement, maximum hip flexion angle, pelvis angle, cranial sartorius circumference, and quadriceps femoris weight.
• Methods of exon skipping gene therapy provided herein exhibit therapeutic efficacy in one or more of the assays for therapeutic utility described in the Examples or in assessment of methods of treatment described in the Examples.
• the treatment in a subject in need can show a long-term in vivo efficacy (e.g., more than 6 months, or up to 12 months) in the assessment described in the Examples.
• the function of the gene therapy and efficacy can be demonstrated by an increase in a shortened form of dystrophin RNA in a sample muscle tissue of the subject as measured by any quantitative assay such as a polymerase chain reaction assay, including digital droplet PCR (ddPCR) and electrophoresis platforms for visualization of RNA transcripts such as Tapestation, or an equivalent assay that quantifies skipped dystrophin RNA transcript copies in the sample.
• therapeutic efficacy can be demonstrated by an increase in a shortened form of dystrophin protein observed in a sample muscle tissue of the subject as measured by any quantitative assay such as Western blot, capillary electrophoresis, LC-MS, or an equivalent assay that quantifies skipped dystrophin protein concentration in the sample.
• therapeutic efficacy can be demonstrated by a decrease in creatine kinase levels as measured in a serum or urine sample of the subject by standard quantitative assays, and/or a decrease in fibrosis as measured by histopathological tissue staining in a sample muscle tissue of the subject.
• Skipped dystrophin protein and percent dystrophin as measured by quantitative protein assays also provide assessments of the methods of treatment.
• the function of the gene therapy and efficacy can be demonstrated by an increase in percent skipped dystrophin calculated by dividing normalized skipped dystrophin transcript copies by skipped plus unskipped dystrophin copies.
• Therapeutic efficacy can be demonstrated by an increase in percent dystrophin as measured by quantification of skipped dystrophin protein concentration compared to a standard curve of wild type dystrophin in samples of a control subject or subjects. Muscle strength, motor skills and tissue histopathology would, for example, be other assessments and demonstration of therapeutic efficacy following treatment with a gene therapy.
• DMD muscular dystrophy disease
• Step2 myotonic muscular dystrophy
• FSHD Facioscapulohumeral disease
• limb-girdle muscular dystrophy X-linked dilated cardiomyopathy
• oculopharyngeal muscular dystrophy provided that mutations exist and are amenable to exon skipping therapy.
• exon skipping transgene of the present disclosure may be any exon skipping transgene described herein, including those that provide one or more antisense sequences that when expressed will hybridize to the target gene.
• the exon skipping transgene has an antisense complex sequence set forth in SEQ ID NOs: 3, 4, 5, 16, 17, 18, 35, or 135, or a combination of any one or more of the antisense complex sequences set forth in SEQ ID NOs: 3, 4, 5, 16, 17, 18, 35, and 135.
• the vectors encoding the exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 1, 2, 13, 14, 32, 33, 131, 132, or 133, in certain embodiments, operably linked to regulatory elements for constitutive, muscle-specific (including skeletal, smooth muscle and cardiac muscle-specific) expression, and other regulatory elements such as poly A sites.
• the vectors encoding the exon skipping transgene include those having a nucleic acid sequence of any one of SEQ ID NOs: 80-112, in certain embodiments, operably linked to regulatory elements for constitutive, muscle-specific (including skeletal, smooth muscle and cardiac muscle-specific) expression, and other regulatory elements such as poly A sites.
• the vectors encoding the exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 131, in certain embodiments, operably linked to regulatory elements for constitutive, muscle-specific (including skeletal, smooth muscle and cardiac muscle-specific) expression, and other regulatory elements such as poly A sites.
• a vector described herein encoding an exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 84.
• a vector described herein encoding an exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 85.
• a vector described herein encoding an exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 106. In some embodiments, a vector described herein encoding an exon skipping transgene include those having a nucleic acid sequence of SEQ ID NO: 131. Such nucleic acids may be in the context of an rAAV genome, for example, flanked by ITR sequences, particularly, AAV2 ITR sequences. In certain embodiments, the methods and compositions comprising administering to a subject in need thereof, an rAAV comprising a genome having a nucleic acid sequence of SEQ ID NO: 2, 14 or 33.
• the methods and compositions comprising administering to a subject in need thereof, an rAAV comprising a genome having a nucleic acid sequence of SEQ ID NO: 131.
• the patient has been diagnosed with and/or has symptom(s) associated with DMD.
• the patient has a confirmed mutation of the DMD gene that is amenable to exon 51 or exon 53 skipping. Recombinant vectors used for delivering the transgene encoding the exon skipping constructs are described herein.
• Such vectors should have a tropism for human muscle cells (including skeletal muscle, smooth mucle and/or cardiac muscle) and can include non-replicating rAAV, particularly those bearing an AAV2 capsid, AAV8 capsid, AAV9 capsid, AAV.hu32 capsid, or AAV.rh74 capsid.
• the recombinant vectors can be administered in any manner such that the recombinant vector enters the muscle tissue, such as by introducing the recombinant vector systemically, for example intravenously.
• Subjects to whom such gene therapy is administered can be those responsive to gene therapy mediated delivery of an exon skipping transgene to muscles.
• the methods encompass treating patients who have been diagnosed with DMD or other muscular dystrophy disease, and identified as responsive to treatment with an exon skipping gene therapy, or considered a good candidate for therapy with gene mediated delivery of an exon skipping transgene.
• the patients have previously been treated with synthetic version of antisense oligonucleotides and have been found to be responsive to one or more of synthetic versions of exogenous exon skipping therapies.
• Therapeutically effective doses of any such recombinant vector or any combination of such vectors should be administered in any manner such that the recombinant vector enters the muscle (e.g., skeletal muscle or cardiac muscle), preferably by introducing the recombinant vector into the bloodstream where the vector exhibits tropism to various tissues.
• the vector is administered subcutaneously, intramuscularly or intravenously.
• Intramuscular, subcutaneous, or intravenous administration of an AAV vector in particular an AAV2, AAV9, AAV.hu32, or an AAV8, or other vector, should result in substantial expression of the transgene product in cells of the muscle (including skeletal muscle, cardiac muscle, and/or smooth muscle). The expression of the transgene product results in delivery and maintenance of the transgene product in at least the muscle.
• the actual dose amount administered to a particular subject can be determined by a clinician, considering parameters such as, but not limited to, physical and physiological factors including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject, and/or route of administration.
• Doses can range from 1 * 10 8 vector genomes per kg (vg/kg) to 1 x 10 15 vg/kg.
• Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (i.e., days, weeks, months, etc.).
• compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant viral vector comprising the exon skipping transgene in a formulation buffer comprising a physiologically compatible aqueous buffer.
• the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
• the gene therapy vectors provided herein may be administered in combination with other treatments for muscular dystrophy, including corticosteroids, beta blockers and ACE inhibitors, micro- or mini- dystrophins, synthetic antisense oligonucleotides (AONs), and muscle growth signaling molecules.
• corticosteroids including corticosteroids, beta blockers and ACE inhibitors, micro- or mini- dystrophins, synthetic antisense oligonucleotides (AONs), and muscle growth signaling molecules.
• AONs synthetic antisense oligonucleotides
• MLP A Multiplex Ligation-Dependent Probe Amplification
• PMID: 33029525; PMCID: PMC7537677) Point mutations and small deletion/insertions, which can be found in about 20% to about 35% of patients, can be detected using a high- throughput detection technique. All 79 exons and the exon-intron boundaries of the DMD gene can be studied through Multiple PCR amplification (e.g., GenSeizer DMD Panel Mix, morgene, China) and direct sequencing (e.g., Illumina MiSeq) (Han S, et. al., Biomed Res Int. 2020 Sep 27). PCR-based Sanger Sequencing can be used to further validate the nextgeneration sequencing results (Han S, et. al., Biomed Res Int. 2020 Sep 27).
• PCR-based Sanger Sequencing can be used to further validate the nextgeneration sequencing results (Han S, et. al., Biomed Res Int. 2020 Sep 27).
• Duchenne patients having the following mutations may be amenable to Exon 51 skipping therapeutics: exon(s) 3-50, 4-50, 5-50, 6-50, 9-50, 10-50, 11-50, 13-50, 14-50, 15-50, 16-50, 17-50, 19-50, 21-50, 23-50, 24-50, 25-50, 26-50, 27-50, 28-50, 29-50, 30-50, 31-50, 32-50, 33-50, 34-50, 35-50, 36-50, 37-50, 38-50, 39-50, 40-50, 41-50, 42-50, 43-50, 45-50, 47-50 48-50, 49-50, 50, 52, 52-58, 52-61, 52-63, 52-64, 52-66, 52-76, and 52-77.
• Duchenne patients having the following mutations may be amenable to Exon 53 skipping therapeutics: exon(s) 3-52, 4-52, 5- 52, 6-52, 9-52, 10-52, 11-52, 13-52, 14-52, 15-52, 16-52, 17-52, 19-52, 21-52, 23-52, 24-52, 25-52, 26-52, 27-52, 28-52, 29-52, 30-52, 31-52, 32-52, 33-52, 34-52, 35-52, 36-52, 37-52, 38-52, 39-52, 40-52, 41-52, 42-52, 43-52, 45-52, 47-52, 48-52, 49-52, 50-52, 52, 54-58, 54- 61, 54-63, 54-64, 54-66, 54-76, and 54-77.
• dystrophin results in mechanical instability causing myofibers to weaken and eventually break during contraction.
• Patients with DMD first display skeletal muscle weakness in early chil dhood, which progresses rapidly to loss of muscle mass, spinal curvature known as kyphosis, paralysis and ultimately death from cardiorespiratory failure before 30 years of age.
• Skeletal muscles of DMD patients also develop muscle hypertrophy, particularly of the calf, evidence of focal necrotic myofibers, abnormal variation in myofiber diameter, increased fat deposition and fibrosis, as well as lack of dystrophin staining in immunohi s tol ogi cal sect! on s .
• the goal of gene therapy treatment provided herein is to slow or arrest the progression of DMD, or other muscular dystrophy disease, or to reduce the severity of one or more symptoms associated with DMD, or other muscular dystrophy disease.
• the goal of gene therapy provided herein is to reduce muscle degeneration, induce/improve muscle regeneration, and/or prevent/reduce downstream pathologies including inflammation and fibrosis that interfere with muscle regeneration and cause loss of movement, orthopedic complications, and, ultimately, respiratory and cardiac failure.
• 6-min distance walked ⁇ 300m, between 300 and 400m, or > 400m
• a timed function test to measure changes from baseline in time taken to stand from a supine position (1 to 8s (good), 8 to 20s (moderate), and 20 to 35s (poor)
• time to climb (4 steps)
• time to run/walk assessments (10 meters)
• my om etry to evaluate changes from baseline in strength of upper and lower extremities
• Efficacy may also be monitored by measuring changes (reduction) from baseline in serum creatine kinase (CK) levels (normal: 35-175 U/L, DMD: 500-20,000 U/L), an enzyme that is found in abnormally high levels when muscle is damaged, serum or urine creatinine levels (DMD: 10-25 pmol/L, mild BMD: 20-30 pmol/L, normal > 53 pmol/L, DMD) and exon skipping transgene (dystrophin) protein levels in muscle biopsies, including skipped and unskipped forms of dystrophin.
• Magnetic Resonance Imaging MRI may also be performed to assess fatty tissue infiltration in skeletal muscle (fat fraction) (Burakiewicz, J. et al. “Quantifying fat replacement of muscle by quantitative MRI in muscular dystrophy.” Journal of Neurology vol. 264,10 (2017): 2053-2067. doi: 10.1007/s00415-017-8547-3).
• Serum creatinine kinase levels may be further separated into its isoenzyme fractions, MM-CPK (skeletal muscle), BB-CPK (brain), and MB-CPK (heart).
• compositions comprising an amount of a nucleic acid composition, including, in particular, gene cassette containing vectors, viral vectors, and AAV vectors, comprising a nucleic acid sequence encoding a exon skipping transgene described herein that is effective to improve gross motor function or slow the loss of gross motor function, for example, as measured using the North Start Ambulatory Assessment to assess ambulatory function as compared to an untreated control or to the subject prior to treatment with the nucleic acid composition; or as assessed by a timed function test to measure time taken to stand from a supine position, or to demonstrate improvement by myometry, or reduction in serum creatinine kinase levels.
• DMD dilated cardiomyopathy
• LV left ventricle
• DMD may be associated with various ECG changes like sinus tachycardia, reduction of circadian index, decreased heart rate variability, short PR interval, right ventricular hypertrophy, S-T segment depression and prolonged QTc.
• Gene therapy treatment provided herein can slow or arrest the progression of DMD and other dystrophinopathies, particularly to reduce the progression of or attenuate cardiac dysfunction and/or maintain or improve cardiac function. Efficacy may be monitored by periodic evaluation of signs and symptoms of cardiac involvement or heart failure that are appropriate for the age and disease stage of the trial population, using serial electrocardiograms, and serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)).
• CMR cardiac magnetic resonance imaging
• ECG may be used to monitor changes from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis.
• FVC forced vital capacity
• FEV1 forced expiratory volume
• MIP maximum inspiratory pressure
• MEP maximum expiratory pressure
• PEF peak expiratory flow
• LVEF left ventricular ejection fraction
• LVFS left ventricular fractional shortening
• inflammation and fibrosis.
• ECG may be used to monitor conduction abnormalities and arrythmias.
• ECG may be used to assess normalization of the PR interval, R waves in VI, Q waves in V6, ventricular repolarization, QS waves in inferior and/or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS.
• nucleic acid compositions including compositions comprising gene expression cassettes and viral vectors, comprising a nucleic acid encoding a exon skipping transgene disclosed herein, and methods of administering those compositions that improve or maintain cardiac function or slow the loss of cardiac function, for example, by preventing reductions in decreasing LVEF below 45% and/or normalization of function (LVFS > 28%) as measured by serial electrocardiograms, and/or serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)). Measurements may be compared to an untreated control or to the subject prior to treatment with the nucleic acid composition.
• CMR cardiac magnetic resonance imaging
• nucleic acid compositions described here in and the methods of administering nucleic acid compositions results in an improvement in cardiac function or reduction in the loss of cardiac function as assessed by monitoring changes from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis.
• FVC forced vital capacity
• FEV1 forced expiratory volume
• MIP maximum inspiratory pressure
• MEP maximum expiratory pressure
• PEF peak expiratory flow
• peak cough flow peak cough flow
• LVEF left ventricular ejection fraction
• LVFS left ventricular fractional shortening
• inflammation and fibrosis.
• ECG may be used to monitor conduction abnormalities and arrythmias.
• ECG may be used to assess normalization of the PR interval, R waves in VI, Q waves in V6, ventricular repolarization, QS waves in inferior and/or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS.
• the goal of gene therapy treatments disclosed herein can be to improve cognitive function or alleviate symptoms of epilepsy and/or psychiatric disorders. Efficacy may be assessed by periodic evaluation of behavior and cognitive function that are appropriate for the age and disease stage of the trial population and or by quantifying and qualifying seizure events.
• nucleic acid compositions and methods of administering the exon skipping gene therapy compositions that improve cognitive function, reduce the occurrence or severity of seizures, alleviate symptoms of ADHD, obsessive- compulsive disorder, anxiety and/or sleep disorders.
• the present invention also relates to combination therapies appropriate for the treatment of certain types of muscular dystrophy such as DMD by administration of a dose of an exon skipping gene therapy vector, such as an AAV gene therapy vector, in combination with a second therapy for treating dystrophinopathies.
• an exon skipping gene therapy vector such as an AAV gene therapy vector
• kits for treatment of human subjects for any muscular dystrophy disease that is amenable to treatment with functional dystrophin via exon skipping gene therapy and a second therapy for treating or ameliorating one or more symptoms of a dystrophinopathy are provided herein.
• the second therapy is a mutation suppression therapy, an exon skipping therapy, a steroid therapy, an immunosuppressive/anti-inflammatory therapy, or a therapy that treats one or more symptoms of the dystrophinopathy.
• a mutation suppression therapy can be ataluren or gentamycin.
• an exon skipping therapy can be any one of the exon skipping therapies that results in skipping of one or more of exons, e.g. exon 2, 43, 44, 45, 50, 51, 52, 53, 55 of the human dystrophin (DMD) gene, to express a form of dystrophin protein depending on the patient’s DMD mutation.
• exon skipping therapy can skip exon 45, such as casimersen, SRP-5045, or DS-5141B.
• an exon skipping therapy can skip exon 50, such as SRP-5050.
• an exon skipping therapy can skip exon 51, such as eteplirsen or SRP-5051.
• an exon skipping therapy can skip exon 53, such as golodirsen, SRP-5053, viltolarsen. In some embodiments, an exon skipping therapy can skip exon 52, such as SRP-5052. In some embodiments, an exon skipping therapy can skip exon 44, such as SRP-5044 or NS- 089/NCNP-02. In some embodiments, an exon skipping therapy can skip exon 2, such as scAAV9.U7.ACCA.
• a steroid therapy can be prednisone, deflazacort, Vamorolone, or Spironolactone, or a combination thereof.
• the therapy that treats one or more symptoms of the dystrophinopathy can be a therapy that improves muscle mass and/or strength such as spironolactone, Follistatin, SERCA2a, EDG-5506, tamoxifen, Givinostat, ASP0367.
• the therapy that treats one or more symptoms of the dystrophinopathy can be a therapy that improves a cardiac condition such as ifetroban, bisoprolol fumarate, eplerenone, or a combination thereof.
• the therapy that treats one or more symptoms of the dystrophinopathy can be a therapy that treats a respiratory symptom such as idebenone.
• the therapy that treats one or more symptoms of the dystrophinopathy can be a therapy that provides orthopedic management, endocrinologic management, gastrointestinal management, urologic management, or a combination thereof.
• Patient primary endpoints may include monitoring the change from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), change from baseline in the NSAA, change from baseline in the Performance of Upper Limp (PUL) score, and change from baseline in the Brooke Upper Extremity Scale score (Brooke score), change from baseline in grip strength, pinch strength, change in cardiac fibrosis score by MRI, change in upper arm (bicep) muscle fat and fibrosis assessed by MRI, measurement of leg strength using a dynamometer, walk test 6-minutes, walk test 10-minutes, walk analysis - 3D recording of walking, change in utrophin membrane staining via quantifiable imaging
• Example 1 Construction of exon skipping expression cassettes in Cis plasmids.
• Target regions within exon 53, and at the junction of exon 52 and 53 were selected and antisense sequences were designed using an exonic splicing finder software tool, such as ESEfinder (exon.cshl.edu/ESE/), and other well-known methods. See FIG. 1A.
• Antisense sequences were flanked by mouse U7 promoter (SEQ ID NO: 6 or 15) and snRNA 3’ Flanking sequence (SEQ ID NO: 10 or 22) and cloned into cis plasmids (e.g. Construct Exon53-3). All expression cassettes cloned into cis plasmids were flanked by ITRs (one ITR is mutated so as to allow for self-complementary DNA).
• the AAV2-AS1 and -AS2 vectors were applied to RD cell cultures in serial doses as depicted in FIG. 2C.
• the AAV2-AS1, -AS2 and -AS3 vectors were also compared as applied to RD cell cultures in serial doses as seen in FIG. 2D. All vectors when transduced in the muscle cell line increased the expression of U7 snRNA as detected by ddPCR in a dose-
• snRNAs containing different antisense sequences express at approximately equivalent levels. (snRNA expression was normalized to TATA-binding protein RNA (TBP).)
• the level of percent skipped exon 53 was measured in the transduced RD cells by ddPCR.
• Treatment of RD cells with AAV2 encoding three snRNAs leads to 90% skipping of DMD exon 53 (FIG. 2E).
• ddPCR methodology was used and percent skipped calculated as described in FIG. 2F.
• Quantification of percent skipping of DMD exon 53 was compared in RD cells for single, dual, and triple snRNA vectors (FIG. 2G) showing increases in percent skipping as additional snRNAs are added to the expression cassette of the vector, suggesting a synergistic effect.
• Another method of measuring RNA expression miRNAscope, showed robust nuclear expression of U7 snRNAs in RD cells treated with high-dose MOI AAV2-3x53 AS vector (FIG. 2H).
• mice contain exon 52-deleted human DMD on chromosome 5 and do not express mouse dystrophin. Skipping of exon 53 restored the DMD reading frame, resulting in expression of human dystrophin protein.
• 3x53 AS construct Exon53-2
• 3xScramble low dose vectors were administered to 5-6-week-old male or female del52 mice by tail vein injection. 1 month or 3 months after vector administration, tissues were harvested for analysis.
• RNA/RNA extraction and ddPCR Total RNA, including snRNA, was extracted from tissues via Kingfisher Apex and the MagMAX mirVana Total RNA kit (Thermo). Levels of the three individual snRNAs were quantified by ddPCR using AS/snRNA specific primers and probe. Unskipped dystrophin was measured via ddPCR using a primer probe set specific to the exon 51/53 junction and skipped dystrophin was measured with primer/probes against the exon 51/54 junction. Percent skipped was calculated by dividing normalized skipped dystrophin copies by skipped plus unskipped dystrophin copies.
• Vectorized U7 snRNA anti-sense sequences (AAV.hu32-Construct Exon53-2) also induced significant restoration of dystrophin protein expression in quadriceps one and three months post dosing as determined by analysis by capillary electrophoresis (Jess, PROTEINSIMPLE). Briefly, total protein lysate was extracted from quadriceps and heart in SDS/Tris buffer. 3 pg of protein per sample were loaded onto plates for analysis by capillary electrophoresis (Jess, PROTEINSIMPLE, bio-techne). Dystrophin was detected with a primary antibody recognizing human dystrophin and an HRP-conjugated anti-mouse secondary antibody.
• %WT dystrophin was quantified by interpolation from a standard curve generated by mixing defined ratios of pooled WT lysate with pooled hDMDdel52/mdx lysates.
• FIG. 9A The expression of U7 snRNA (AS1-, or AS2- or AS3-U7snRNA measured by ddPCR) at 3 months post-administration is plotted in FIG. 9A.
• FIG. 9B depicts the expression of U7 snRNA 1 (ASl-U7snRNA measured by ddPCR) at 1 month and 3 months postadministration of high dose and low dose Construct Exon53-2 vectors.
• the biodistribution of AAV genomes per diploid cell in del52 mouse in skeletal muscle (gastrocnemius and tibialis anterior), heart, and liver was plotted for 3 month (FIG. 9C) and for 3 months LD and HD against 1 month (FIG. 9D) post-administration of LD and HD AAV.hu32-Construct Exon53- 2.
• Dystrophin protein expression (e.g. “skipped” dystrophin) was restored in del52 mouse muscle post administration of Hu32. Construct Exon53-2, and dystrophin accumulation increased slightly from 1 month to 3 month.
• FIG. 10A shows the quantification of dystrophin expression (determined by Jess, LD and HD).
• FIG. 10E The ratio of dystrophin normalized by actinin is calculated and plotted for 1 month and 3 month (FIG. 10F) post-administration.
• Dystrophin protein expression in quadriceps for 1 month and 3 month post-administration not normalized by actinin (determined by Jess) is shown in FIG. 10G, and normalized by actinin, (determined by Jess) compared to a standard curve of wild type (wt) mouse dystrophin is shown in FIG. 10H.
• FIG. 6A U7 snRNA expression in differentiated human DMDdel52 muscle cells was dose dependent (AS1-, or AS2- or AS3-U7snRNA measured by ddPCR).
• PacBio SMRTbell library was prepared from extracted vector DNA by following manufacturer’s instructions. Sequencing was conducted on the PacBio Sequel lie platform with v3.0 chemistry. HiFi reads were generated in fastq format. HiFi reads were aligned to the Hu32.3x.U7.Ex53 sc. AAV genome and non-vector genome through minimap2 version 2.24-rl 122. The coverage track for read alignments within the sc. AAV genome was displayed in the Integrative Genomics Viewer (IGV) software. Additionally, the distribution of lengths for reads mapping to the sc. AAV genome were plotted and visualized, as in the diagram depicting the self complementary genome of Construct Exon53-2 as FIG.
• IGV Integrative Genomics Viewer
• FIG. 7A top
• FIG. 7A bottom
• FIG. 7A bottom
• the distribution of lengths for HiFi reads that align to the Hu32.3x.U7.Ex53 genome show a concentrated peak of reads indicating that the majority of genomes are of appropriate length (FIG. 7C).
• mice express human dystrophin compensating for the lack of mouse dystrophin and represent a functional dystrophin (“normal”) control.
• the injection was through a single tail vein injection.
• the mice were tested on separate days for 2 limb hanging test (e.g., on Tuesdays), forelimb grip strength (e.g., on Wednesdays), and 4 limb hanging test (e.g., on Thursdays) from week 4 to week 12 post administration as indicated by the arrows in FIG. 11.
• 2 limb hanging test for the wire hanging test, the mouse was suspended above a metal wire located 40 cm above a cage with soft beddings. After the mouse grasped the wire with its forelimbs it was released, and the hanging time was measured.
• the test was completed after a hanging time of 600 s was achieved or after three sessions.
• the maximum hanging time was used for analysis.
• the results of the 2 limb hanging tests are plotted in FIGs. 12A and 12B, and a robust result was observed following administration of low or high dose exon skipping vector to hDMD Exon 52-deleted mice.
• the mouse was placed on a grid, which was then turned upside down, 15 cm above a cage filled with soft bedding. Also, this hanging test was completed after a hanging time of 600 s was achieved or after three sessions. The maximum hanging time was used for analysis.
• the results of the 4 limb hanging tests are plotted in FIGs. 12C and 12D.
• forelimb grip strength test grip strength of the fore limbs was assessed using a grid attached to an isometric force transducer (Columbus Instruments, Columbus, USA). The force transducer recorded the maximum force that is required to break the mouse’s grip from the mesh surface. In total five strength measurements, each containing of three pulls, was recorded. Three highest values were averaged and normalized to body weight. The results of the forelimb grip strength tests are plotted in FIGs. 12E and 12F.
• Antisense sequences were flanked by mouse U7 promoter (SEQ ID NO: 6 or 15) and snRNA 3’ Flanking sequence (SEQ ID NO: 22) and cloned into cis plasmids.
• trans plasmid contained rep 2/cap2 genes or rep 2/cap hu32 for the production of AAV2 pseduotype vector or AAV.hu32 pseudotype vector, respectively.
• nucleic acid sequences, or amino acid sequences, of recombinant components and composite elements utilized to make the expression cassettes or vectors, and other, are described in Table 2 and Table 3.
• Candidate antisense sequences for Exon51 skipping were tested analogously to the vectors described in Example 2 except that cis plasmids expressing AS-U7snRNA exon 51 skipping cassettes were transfected into human RD cells. mRNA was measured by RT-PCR and visualized on an agarose gel, as depicted in FIG. 14. Exon51 skipping of endogenous human dystrophin in RD cells treated with triple -AS-snRNA constructs is shown in FIG. 15A. RT-PCR amplifying both the skipped and unskipped dystrophin mRNA was semiquantified on an agarose gel (FIG. 15B). Several triple AS-snRNA constructs induced Exon51 skipping in RD cells. Exon 51 skipping efficacy in RD cells for exemplary AS- U7snRNAs (expressed as % skipped) is shown in FIG. 15C.
• FIG. 16A depicts both exon 51 and 53 skipping efficacies in Aexon52 RD cells.
• Results were confirmed by quantitative tapestation analysis which is a visualization of skipped and unskipped mRNA RT-PCR amplicon (transcript) (FIG. 16B), as performed on an automatic electrophoresis platform TapeStation 4200 (Agilent Technologies, Santa Clara, USA) with a D 1000 tape according to manufacturer instructions.
• Construct 51-12 when expressed from plasmids, induced robust exon 51 skipping in RD cells.
• Construct 51-12 mediated snRNA expression was also evaluated via AAV delivery by incubating RD-AAVR cells with increasing titers, D IO 4 , D 10 5 , and D IO 6 MOI AAV vectors.
• Construct 51-12 were quantified and plotted in FIGs. 17A, 17B, and 17C, respectively.
• the normalized ASO (AS-U7snRNA) expression depicted in FIG. 17G was calculated by normalizing dose-dependent expression of ASO1, ASO2, and ASO3 over TBP control as shown in FIGs. 17D, 17E, and 17F, respectively.
• FIGs. 18A and 18B depict robust exon 51 skipping induced by AAV2 delivered Construct 51-12. Results are confirmed by ddPCR analysis and plotted in FIG. 18C for exon skipped and FIG. 18D for unskipped. The normalized ratio of skipped/unskipped dystrophin mRNA over TBP control (FIGs. 18E and 18F) is plotted in FIG. 18G.
• FIGs. 19A and 19B depict exon 51 and exon 53 skipping induced by AAV.hu32 delivered constructs.
• RD-AAVR cells contain endogenous dystrophin while Aexon52 RD cells contain a mutated dystrophin gene, and visualization of skipped dystrophin appeared to be more robust the Aexon52 RD cells in these experiments.
• the normalized skipping efficacy of dystrophin mRNA was measured in RD-AAVR cells (FIG. 19C) and in 6D10 Aexon52 RD cells (FIG. 19D).
• Construct 51-12 performs well in both RD-AAVR cells and Aexon52 RD cells.
• FIG. 20A results were confirmed by ddPCR analysis and plotted in FIG. 20B for exon skipped dystrophin mRNA, FIG. 20C for skipped normalized by TBP control, FIG. 20D for exon unskipped, FIG. 20E for unskipped normalized by TBP control.
• FIG. 20F The normalized skipping efficacy in human DMDdel52 myoblast cell lines is shown in FIG. 20F.
• Exon skipping in human DMDdel52 myoblasts cells was further confirmed (using similar methods as in Example 4) with AAV8 and AAV.hu32 delivered Ex51-12 constructs at four different multiplicity of infection (MOI) levels. Robust exon 51 skipping is seen at three of the four MOIs (FIG. 20G). Results were confirmed by ddPCR analysis for snRNA expression normalized by TBP control (FIG. 20H) and for percent Ex51 -skipped dystrophin mRNA where the highest MOI resulted in percent skipped mRNA at over 70% skipped (FIG. 201).
• tissues were harvested for molecular, biochemical and histological analysis, including RNA analysis by digital droplet PCR, and protein analysis by IF staining, histology, Sirius Red staining and Simple Western JESS (FIG. 21C).
• RNA/RNA extraction DNA/RNA extraction, ddPCR and TapeStation. Dose-dependent vector biodistribution (AAV genomes per diploid cell) was also assessed in skeletal muscle (gastrocnemius, diaphragm, and tibialis anterior), heart, and liver (FIG. 22A). Briefly, total RNA and DNA were extracted from tissues using the Kingfisher Apex via the MagMAX mirVana Total RNA and MagMAX DNA Multi-Sample Ultra 2.0 kits (Thermo). Levels of the three individual snRNAs were quantified by digital droplet PCR (ddPCR) using AS/snRNA specific primers and probe.
• ddPCR digital droplet PCR
• Unskipped dystrophin was measured via ddPCR using a primer probe set specific to the exon 51/53 (del52 mice and immortalized myoblast cell) or 51/52 (RD cell) junction and skipped dystrophin was measured with primer/probes against the exon 50/53 (del52 mice and immortalized myoblast cell) or 50/52 (RD cell) junction.
• Unskipped dystrophin mRNA transcript measured by these primers is 458 bp in length and exon-skipped transcript is predicted to be 225 bp in length. Percent skipped was calculated by dividing skipped dystrophin copies by skipped plus unskipped dystrophin copies. AAV genomes were detected using transgene specific primers/probe and normalized by mouse glucagon copies.
• the exon 51 skipping results in del52 mice receiving high dose, middle dose and low dose were compared in heart and diaphragm (see FIG. 22E), gastrocnemius (GAS, see FIG. 22F), tibialis anterior (TA, see FIG. 22F) and liver as visualized and quantitated by Tapestation.
• GAS gastrocnemius
• TA tibialis anterior
• FIG. 22F liver as visualized and quantitated by Tapestation.
• AAVhu32-delivered Construct 51-12 led to robust exon51 skipping of dystrophin mRNA in various tissues in mice two months after vector administration. See, for example, FIG. 22F, where each lane represents an individual mouse (lanes with * indicate unsuccessful RNA extraction from the tissue).
• Percentage of DMD transcripts with skipped exon51 were quantified with ddPCR. (FIG. 22G) and percentage of skipped DMD transcripts correlates with AS-snRNA expression (FIG. 22H).
• Results were confirmed by ddPCR analysis in heart as shown in FIGs. 23A, 23B, 23C, 23D, 23E, 23F, and 23G, in diaphragm as showin in FIGs. 23H, 231, 23J, 23K, 23L, 23M, and 23N, in GAS as shown in FIGs. 230, 23P, and 23Q, in TA as shown in FIGs. 23R, 23S, and 23T, and in liver as shown FIGs. 23Uand 23V.
• FIGs. 24A, 24B, 24C, 24D, 24E, and 24F show the quantification of dystrophin expression (dystrophin/ a-actinin) in GAS, diaphragm, and heart (determined by Jess, high dose, middle dose, and low dose, each lane represents an individual mouse).
• Vectorized exon51 skipping restores dystrophin in muscle in vivo.
• Dystrophin is dose-dependently restored to the gastrocnemius following administration of AAVhu32.Ex51- 12 (FIG. 25A) which results in correction of dystrophic muscle pathology (Sirius Red, FIG. 25A).
• Quantification of percentages of muscle fibers expressing dystrophin (FIG. 25B), intensity of dystrophin immunostaining (FIG. 25C), and Sirius Red-positive area in gastrocnemius muscle (FIG. 25D).
• AAVhu32. Ex51-12 induces robust expression of dystrophin protein in heart (FIG. 25E).
• AAVhu32 delivery of Construct-Ex51-12 results in exon51 skipping in a humanized Del52 mouse model. Treatment restores dystrophin protein expression in Del52 mouse muscle and heart, at two months post dosing, while reducing fibrosis.
• Target regions within exon 53, and at the junction of exon 52 and 53 were selected and antisense sequences were designed using an exonic splicing finder software tool, such as ESEfinder (exon.cshl.edu/ESE/), and other well-known methods. See FIG. 1A.
• Antisense sequences were flanked by mouse U7 promoter (SEQ ID NO: 6 or 15) and snRNA 3’ Flanking sequence (SEQ ID NO: 10 or 22) and cloned into cis plasmids. All expression cassettes cloned into cis plasmids were flanked by ITRs (one ITR was mutated so as to allow for self-complementary DNA).
• trans plasmid contained rep 2/cap8 genes for the production of AAV8 pseduotype.
• Cells were harvested 5-days post-transduction and assayed for total AS snRNA expression and exon 53-skipping by ddPCR (BioRad) or RT-PCR visualized via automated electrophoresis (TapeStation, Agilent), and dystrophin protein expression by capillary electrophoresis (JessTM, Bio-Techne).
• dystrophin protein quantification total protein lysate was extracted from harvested cells in SDS/Tris buffer. 3 pg of protein per sample were loaded onto plates for analysis by capillary electrophoresis. Dystrophin was detected with a primary antibody recognizing human dystrophin and an HRP-conjugated anti-mouse secondary antibody. Area under curve (AUC) values for dystrophin peaks were quantified for each sample and normalized to an a-actinin loading control. WT dystrophin reference levels were determined from AUC values obtained from lysates of an identically differentiated control immortalized human myoblast cell line.
• mice of either sex were administered the test articles via tail vein IV at the 6-7 weeks of age. Animals were weighed on Study Day -1 and dosing volume was adjusted to ensure delivery of the appropriate body weight-normalized vector load according to the assigned dose/vector groups as depicted in FIG. 28. The animals were necropsied 3-month post-AAV delivery.
• Blood was collected for serum preparation and relevant tissues were harvested to evaluate vector biodistribution, snRNA (transgene) expression, DMD mRNA exon 53-skipping, dystrophin protein expression (as %WT dystrophin by capillary electrophoresis) and immunohistochemical and histopathological analyses to evaluate dystrophin at the sarcolemma and correction of fibrosis and muscle regeneration. Methods for relevant downstream analyses were described in Example 3 and Example 11.
• FIG. 29A and FIG. 29B The delivery of AAVHu32.Ex53 resulted in dose-dependent biodistribution and snRNA expression (FIG. 29A and FIG. 29B). DMD mRNA exon 53-skipping and dystrophin protein expression were observed in heart, diaphragm and skeletal muscles (FIG. 29C and FIG. 29D) Creatine kinase levels were reduced in the serum of mice treated at the high dose (FIG. 29E). Biomarker myosin light chain 3 (MYL3) was also measured in the serum (Ay oglu, et al. 2014, EMBO Mol Med 6: 918-936).
• MYL3 Biomarker myosin light chain 3
• mice treated with either high dose (HD) or low dose (LD) resulted in significantly reduced MYL3 levels in serum compared to vehicle control (FIG. 29J).
• Immunofluorescence analysis demonstrated the restoration of dystrophin at the sarcolemma in over 70% dystrophin positive fibers at the high dose (FIG. 29F and FIG. 29G).
• Immunofluorescent detection of embryonic Myosin Heavy Chain (eMyHC) showed a reduction in muscle regeneration of treated mice (FIG. 29F and FIG. 291). Sirius Red staining demonstrated a reduction in skeletal muscle fibrosis (GAS shown) (FIG. 29F and FIG. 29H).
• snRNA expression, DMD mRNA exon 53-skipping and dystrophin protein expression were all sustained or increased at 3 -month compared to 1- month time point (data not shown), demonstrating the durability of the treatment.
• FIG. 30A Biodistribution (FIG. 30A), snRNA expression (FIG. 30B), and exon 53-skipping (FIG. 30C) exhibited distinct dynamics in several muscles following AAVHu32.Ex53.3AS administration at mid dose. As demonstrated in FIG. 30D, Exon 53-skipping was a function of snRNA expression.
• mice were treated with either vehicle or AAVhu32.Ex53.3AS at two different doses (1E14 and 3E14 GC/kg) via tail vein IV at 6 weeks of age.
• Age-matched, untreated male hDMD/mdx mice were enrolled in the study as a control.
• 2-limb wire hanging, 4-limb grid hanging and forelimb grip strength were performed on separate days with a biweekly cadence starting 4 weeks post-dosing (FIG. 32A).
• mice of either sex were administered at 1E14 gc/kg ("mid dose") of AAVhu32-Ex53-2 vector.
• the animals were necropsied 12-month post-AAV delivery. Blood was collected for serum preparation and relevant tissues were harvested to evaluate vector biodistribution, snRNA (transgene) expression, DMD mRNA exon 53- skipping, dystrophin protein expression (as %WT dystrophin by capillary electrophoresis) and immunohistochemical and histopathological analyses to evaluate dystrophin restoration. Methods for relevant downstream analyses were described in Example 3 and Example 11.
• FIG. 33A left
• expression FIG. 33A, middle
• exon 53-skipping FIG. 33A, right
• FIG. 33B capillary Western
• FIG. 33C immunofluorescence
• FIG. 33D Immunofluorescence
• FIG. 33E increases in dystrophin positive fibers
• FIG. 33F decreases in tissue fibrosis
• FIG. 33F increases in acute muscle regeneration
• Non-human primate (NHP) study design All animals were screened for AAVhu32 total antibody (Tab) levels in serum and were found negative prior to enrolling in the study. NHP study was conducted as provided in FIG. 34. The animals received an immune suppression regimen of prednisolone sodium phosphate at a dose of 1 mg/kg administered orally, once daily, starting at Study Day -14 (SD-14) and ending at necropsy (SD84). The test articles were administered to animals by IV bolus under ketamine sedation through an intravenous catheter placed in right saphenous vein followed by a flush of at least 1 mL of sterile saline.
• AAVhu32.Ex53.3AS resulted in greater than 70% skipping of endogenous DMD mRNA exon 53 in the heart of NHPs, leading to corresponding reduction of WT dystrophin protein.
• Variable but detectable levels of skipping were observed in the skeletal muscle (GAS) and diaphragm of these animals.
• GAS skeletal muscle
• the 3-month intravenous administration of 1 x 10 14 GC/kg AAVhu32.Ex53.3AS in NHP resulted in efficient skipping in heart, diaphragm and skeletal muscles (FIG. 35A). Sequencing of the skipped RT-PCR band revealed a perfect junction between dystrophin exons 52 and 54 (FIG. 35B).
• Dystrophin levels ranged from about 20% to about 80% wild-type dystrophin and when plotted as averages for each dose at 2 months (8 weeks) and 6 months (26 weeks) (FIGS. 37H-J) showed durability in that time frame.
• the relationship between percent skipping and dystrophin expression levels was linear for heart expression however not linear for skeletal muscle as shown in FIGS. 37K-M.
• a Next Generation Sequencing data analysis was used to assess genome integrity of the scAAV particles.
• Animal studies were performed by injecting each test vector at a 1E14 vg/kg dose per mouse as depicted in FIG. 38.
• Nine hDMD/del52 mice were injected with mITRdelB - original AAVhu32-Ex53.3AS vector, for example, AAVhu32- Ex53-2; and nine hDMD/del52 mice were injected with full mITR, for example, AAVhu32- Ex53-4.
• tissues were harvested for DNA, RNA and protein analysis.
• PacBio sequencing revealed a single major population of AAV genome at the correct size in the scAAV vectors made with the mITR cis plasmid having the full mITR with a restored region (FIG. 39B). This signaled that restoring the region removed the deletion population of genomes, leaving a single uniform genome with the full mutant ITR.
• the results from the animal study showed on all levels that DNA, RNA, skipping, and protein reveals no significant differences between the two test vectors, indicating the restoration of the region deletion does not impact potency of the AAV vector (FIG. 40A-G). Therefore, restoration of the region deletion back to the full-length mutant ITR improves genome integrity of the scAAV vector without impacting efficacy of the vector.
• RNA-Seq analysis was performed by isolating RNA from the target tissue and creating an RNA library using e.g. an NEBNext Ultra II RNA library preparation kit (NEB #E7775). Gastrocnemius tissue from hDMDdel52/mt& mice (treated and untreated) were isolated and RNA libraries prepared for Illumina® sequencing according to the manufacturer's instructions (FIG. 41A). Following Next-generation sequencing (NGS), data was analyzed and plotted (e.g. as Sashimi plots) to evaluate RNA transcripts (read counts). Three months post administration of AAVHu32.Ex53.3AS treatment, a reduction in RNA transcripts having Exon 53 (shaded vertical bar) was seen compared to samples from untreated hDMDdel52/mt& mice (FIG. 41B).
• NGS Next-generation sequencing
• nucleic acid sequences of recombinant components and composite elements utilized to make the expression cassettes or vectors, and other, are described in Table 4.

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