Products and methods for treating muscular dystrophy

Abstract

Products and methods are provided for treating or preventing muscular dystrophy in patients with mutations in any of exons 6, 7, 8, or 9 in the DMD gene. Gene therapy vectors, such as adeno-associated virus (AAV) vectors, and methods of using these vectors to deliver nucleic acids containing DMD antisense sequences in modulating or restoring expression of the transcript of the DMD gene and a functional form of the dystrophin protein, are provided. The products and methods are used to treat, ameliorate, and / or prevent muscular dystrophy, such as Duchenne muscular dystrophy or Becker muscular dystrophy.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Patent Application No. 63 / 178,648, filed April 23, 2021, which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to the field of gene therapy for the treatment and / or prevention of muscular dystrophy. More specifically, the present disclosure provides products and methods for treating or preventing muscular dystrophy in patients resulting from mutations in DMD exons 6, 7, 8, and / or 9. The present disclosure provides nucleic acids comprising nucleotide sequences for antisense-mediated exon skipping, skipping frame-disrupting exons and restoring the reading frame, thereby allowing expression of functional dystrophin protein. Gene therapy vectors, such as adeno-associated virus (AAV) vectors, comprising the nucleic acids, and methods of using these vectors to express dystrophin genes and proteins are provided. The products and methods are used for the treatment and / or prevention of muscular dystrophy, such as Duchenne muscular dystrophy or Becker muscular dystrophy.

[0003] Incorporation by reference of sequence listing This application contains, as a separate part of the disclosure, a sequence listing in computer readable form (Filename: 56493_SeqListing.txt, 20,439 bytes, ASCII text file created on April 22, 2022), which is incorporated by reference in its entirety herein. [Background technology]

[0004] Muscular dystrophies (MD) are a group of inherited degenerative disorders that primarily affect voluntary muscles. The group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD have an onset in infancy or childhood, whereas others may not appear until middle age or later. The disorders differ in the distribution and degree of muscle weakness (some forms of MD also affect the heart muscle), age at onset, rate of progression, and mode of inheritance.

[0005] MD is a group of diseases with no identifiable cure that severely impacts individuals, families, and communities. The costs are immense. Individuals suffer from emotional strain and reduced quality of life associated with loss of self-esteem. Extreme physical difficulties due to loss of limb function result in hardship in activities of daily living. Family relationships suffer from economic losses and interpersonal challenges. Affected siblings feel trapped and spousal conflicts often lead to divorce, especially when the blame for muscular dystrophy can be placed at the feet of one of the parental partners. The burden of the quest to find a cure is often a lifelong, highly intensive endeavor that impairs and tests every aspect of life. Beyond the family, the community bears the economic burden from the need for additional facilities to accommodate the handicaps of the muscular dystrophy population in special education, specialized transportation, and the costs of re-hospitalization to treat recurrent respiratory tract infections and cardiac complications. The economic responsibility is shared by state and federal agencies, with the responsibility extended to the taxpayer community.

[0006] One form of MD is Duchenne muscular dystrophy (DMD). DMD, an X-linked degenerative muscle disorder, is the most common severe childhood muscular dystrophy affecting approximately 1:5200 male births (Non-Patent Document 1). Symptoms of generalized muscle weakness first appear at 3-5 years of age, progress to inability to walk by 13 years of age, and death from cardiomyopathy or respiratory failure typically occurs by the third decade of life (Non-Patent Documents 2, 3). Dystrophin is a large (427 kDa) multifunctional protein localized in the lateral subfascial region of muscle fibers that plays an important role in protecting sarcomas from mechanical damage caused by muscle contraction (Non-Patent Document 4) and is encoded by the DMD gene (Non-Patent Document 5), but DMD is caused by mutations that disrupt its open reading frame.

[0007] Another form of MD is Becker muscular dystrophy (BMD). BMD is a mild allelic disorder resulting from the presence of partially functional dystrophin protein resulting from mutations that maintain the open reading frame (ORF) (Non-Patent Documents 6, 7). BMD, like DMD, is a genetic disease that gradually weakens and reduces the muscles of the body. BMD affects the muscles of the hips, pelvis, thighs, and shoulders, as well as the heart, but is known to cause less severe problems than DMD. Due to the variety of in-frame mutations that result in various partially functional proteins, BMD has a wide phenotypic spectrum, for example, with the inability to walk from late teens to late adulthood.

[0008] Promising therapeutic approaches to DMD are based on replacing a functional version of DMD or its repair at the DNA or pre-mRNA level. Both approaches aim at restoring the open reading frame, leading to the expression of a partially functional BMD-like dystrophin. Gene replacement studies using modified adeno-associated viruses (AAV) have been reported (Non-Patent Documents 8-10), but the packaging capacity of AAV transgenes is limited to about 5 kb. Since the DMD cDNA is 11.4 kb, current viral vectors utilize one of the microdystrophin cDNAs, which has several internal deletions but is in-frame (Non-Patent Document 11). An alternative approach is to restore the mRNA reading frame by delivering antisense sequences that bind to key exon-defining elements in the pre-mRNA, inhibiting the recognition of specific exons by the spliceosome and resulting in the exclusion of the targeted exon from the mature RNA. Such antisense sequences can consist of antisense oligonucleotides (AONs) or phosphorodiamidate morpholino oligomers (PMOs) such as eteplisene, the first such therapeutic agent approved by the FDA for the treatment of DMD caused by mutations that can result in exon 51 skipping (Non-Patent Documents 12-14).

[0009] Despite the many lines of research following the identification of the DMD gene, treatment options are limited. Thus, there remains a need in the art for treatments for MD, such as DMD and BMD, including treatment of one or more mutations in the DMD gene. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mendell et al.,Ann Neurol 71,304-313(2012) [Non-Patent Document 2] Passamano et al., Acta Myol 31,121-125(2012) [Non-licensed document 3] Duchenne,The Pathology of Paralysis with Muscular Degeneration(Paralysie Myosclerotique),or Paralysis with Apparent Hypertrophy.Br Med J 2,541-542(1867)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0011] The present disclosure provides products and methods for preventing, delaying disease progression or severity, and / or treating disease in patients with mutations in one or more of exons 6, 7, 8, and / or 9 of the DMD gene. More specifically, the present disclosure provides products and methods that use a U7 snRNA approach to induce skipping of exons 6, 7, 8, and / or 9 of the DMD gene.

[0012] More specifically, the disclosure provides products and methods for treating or preventing muscular dystrophy in patients resulting from mutations in DMD exons 6, 7, 8, and / or 9. The disclosure provides nucleic acids comprising nucleotide sequences for antisense-mediated exon skipping, skipping frame-disrupting exons and restoring the reading frame, thereby allowing expression of functional dystrophin protein. Gene therapy vectors, such as adeno-associated virus (AAV) vectors, comprising the nucleic acids, and methods of expressing dystrophin genes and proteins using these vectors are provided. The products and methods are used for treating and / or preventing muscular dystrophies, such as Duchenne muscular dystrophy or Becker muscular dystrophy.

[0013] The present disclosure provides nucleic acids comprising a nucleotide sequence or combination of nucleotide sequences designed to target one or more of exons 6, 7, and 8 of the human DMD gene to effect antisense-mediated exon skipping and treat, ameliorate, or prevent muscular dystrophy in patients resulting from mutations in DMD exons 6, 7, 8, and / or 9.

[0014] The present disclosure provides a nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20; (c) a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 20; (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 1 to 20; and (e) A nucleotide sequence that binds to any one of the sequences shown in SEQ ID NOs: 21 to 25.

[0015] In some embodiments, the nucleic acid comprises a combination of at least two nucleotide sequences, the combination being (i) a nucleotide sequence targeting the human DMD gene at exon 6, and a nucleotide sequence targeting the human DMD gene at exon 7, (ii) a nucleotide sequence targeting the human DMD gene at exon 6, and a nucleotide sequence targeting the human DMD gene at exon 8, and (iii) a nucleotide sequence targeting the human DMD gene at exon 7, and a nucleotide sequence targeting the human DMD gene at exon 8.

[0016] In some embodiments, the nucleic acid comprises a combination of at least three nucleotide sequences, the combination comprising a nucleotide sequence targeting the human DMD gene at exon 6, a nucleotide sequence targeting the human DMD gene at exon 7, and a nucleotide sequence targeting the human DMD gene at exon 8.

[0017] In some embodiments, the nucleic acid is (a) a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 26 to 29; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 26 to 29; (c) a nucleotide sequence comprising any one of SEQ ID NOs: 26 to 29; and (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 26 to 29.

[0018] The present disclosure provides an associated recombinant adenovirus (rAAV) comprising any of the nucleic acids described herein. In some aspects, the rAAV is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAV-anc80, rAAVrh.74, rAAVrh.8, rAAVrh.10, or rAAV-B1. In some aspects, the rAAV is rAAV9. In some aspects, the rAAV is self-complementary.

[0019] The disclosure provides compositions comprising any one or more of the nucleic acids described herein and a carrier, diluent, excipient, and / or adjuvant. The present disclosure provides a composition comprising any of the vectors described herein and a carrier, diluent, excipient, and / or adjuvant. In some aspects, the vector is AAV or rAAV.

[0020] The present disclosure provides a method of treating, preventing, or ameliorating muscular dystrophy in a subject in need thereof, the method comprising administering to the subject an effective amount of (a) any one or more of the nucleic acids described herein; (b) any vector described herein; or (c) administering any composition comprising a nucleic acid or vector described herein.

[0021] In some embodiments, the vector is AAV or rAAV. In some embodiments, the rAAV is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAV-anc80, rAAVrh.74, rAAVrh.8, rAAVrh.10, or rAAV-B1. In some embodiments, the rAAV is rAAV9. In some embodiments, the rAAV is self-complementary. In some embodiments, the administering is via a systemic route. In some embodiments, the systemic route is by injection, infusion, or implantation. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

[0022] In some aspects of the methods, the level of functional dystrophin gene expression or protein expression in the subject's cells is increased after administering the nucleic acid, rAAV, or composition compared to the level of functional dystrophin gene expression or protein expression before administering the nucleic acid, rAAV, or composition.

[0023] In some aspects of the methods, expression of functional dystrophin in cells is detected by measuring dystrophin protein levels by Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of the nucleic acid, rAAV, or composition.

[0024] In some aspects of the method, the level of serum creatine kinase is decreased after administration of the nucleic acid, rAAV, or composition compared to the level of serum creatine kinase before administration of the nucleic acid, rAAV, or composition.

[0025] In some aspects of the methods, the treatment results in improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject. In some embodiments of the method, the progression of muscular dystrophy in the subject is delayed or muscle function in the subject is improved after administration of the nucleic acid, rAAV, or composition, as measured by a 6-minute walk test, a time to stand test, an ascend 4 steps test, an ascend and descend 4 steps test, a North Star Ambulatory Assessment (NSAA), a forced vital capacity (FVC) test, a timed 10 meter test, a timed 100 meter test, a hand held dynamometry (HHD) test, a timed Up and Go test, a Gross Motor Subtest Scaled (Bayley-III) score, a maximal isometric voluntary contraction test (MVICT), or a combination of two or more thereof.

[0026] In some embodiments, the therapeutic methods of the present disclosure further comprise administering a second therapy or a combination therapy. In some embodiments, the methods further comprise administering a glucocorticoid.

[0027] The present disclosure also provides (a) any one or more of the nucleic acids described herein; (b) any vector described herein; or (c) the use of any composition comprising a nucleic acid or vector described herein, For the preparation of a medicament for the treatment of muscular dystrophy or for treating muscular dystrophy in a human subject in need thereof.

[0028] In some embodiments, the vector is AAV or rAAV. In some embodiments, the rAAV is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAV-anc80, rAAVrh.74, rAAVrh.8, rAAVrh.10, or rAAV-B1. In some embodiments, the rAAV is rAAV9. In some embodiments, the rAAV is self-complementary. In some embodiments, the pharmaceutical is designed for use via a systemic route. In some embodiments, the systemic route is by injection, infusion, or implantation. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

[0029] In some aspects of the use, the level of functional dystrophin gene expression or protein expression in the subject's cells is increased after administration of the nucleic acid, rAAV, or composition compared to the level of functional dystrophin gene expression or protein expression before administration of the nucleic acid, rAAV, or composition.

[0030] In some aspects of the use, the level of expression of functional dystrophin in cells is detected by measuring dystrophin protein levels by Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of the nucleic acid, rAAV, or composition.

[0031] In some aspects of the use, the level of serum creatine kinase is decreased after administration of the nucleic acid, rAAV, or composition compared to the level of serum creatine kinase before administration of the nucleic acid, rAAV, or composition.

[0032] In some aspects of use, the treatment results in improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in a subject. In some aspects of the use, the progression of muscular dystrophy in the subject is delayed or muscle function in the subject is improved after administration of the nucleic acid, rAAV, or composition as measured by the 6-minute walk test, the time to stand test, the 4 steps up test, the 4 steps up and down test, the North Star Gait Assessment (NSAA), the forced vital capacity (FVC) test, the timed 10 meter test, the timed 100 meter test, the handheld dynamometry (HHD) test, the timed up and go test, the gross movement subtest equivalent (Bayley-III) score, the maximal isometric voluntary contraction test (MVICT), or a combination of two or more thereof.

[0033] In some embodiments, the disclosed uses further comprise a second or combination therapy. In some embodiments, the uses further comprise administering a glucocorticoid. Other features and advantages of the present disclosure will become apparent from the following drawings and detailed description, but it should be understood that the drawings, detailed description and examples, while illustrating aspects of the disclosed subject matter, are given by way of example only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the drawings, detailed description and examples. [Brief description of the drawings]

[0034] [Figure 1A] Figure 1 shows a comparison of the nucleotide sequences of DMD exons 6, 7, and 8 in human, mouse, and dog. Figure 1 also shows the location of antisense and targeting sequences targeting these exons. Targeting sequences are underlined. Highlighted and bold bases indicate differences in mouse and dog sequences compared to the human sequence (lowercase = intron sequence, uppercase = exon sequence). Figure 1A shows human, mouse, and dog exon 6 sequences. Figure 1B shows the target and antisense sequences for exon 6. Figure 1C shows human, mouse, and dog exon 7 sequences. Figure 1D shows the target and antisense sequences for exon 7. Figure 1E shows human mouse, and dog exon 8 sequences. Figure 1F shows the target and antisense sequences for exon 8. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 2A]We show how a combination of antisense sequences targeting exons 6, 7, and / or 8 can mediate exon 6-8 skipping in WT FibroMyoD. Figure 2A shows RT-PCR results from transduction of WT FibroMyoD using a combination of antisense sequences (indicated in the right panel) in AAV1 (2.5E11vg / 6 cm well). Primers amplifying the WT and skipped transcripts were located in exons 5 and 10. All the different constructs led to multi-exon skipping. Exon 9 is often spontaneously skipped as shown in the untreated control cell line. Several bands were obtained after treatment, representing two transcripts: (1) a transcript containing exons 5, 9, and 10 (top box containing the two transcripts boxed in red, ESE8_2, ESE7, and an antisense to the published exon 6 (i.e., ESE6, Vulin et al., Mol. Ther. 2012 Nov;20(11):2120-33.doi:10.1038 / mt.2012.181.Epub 2012 Sep 11.PMID:22968479, Bish et al., Mol. Ther. 2012 Mar;20(3):580-9.doi:10.1038 / mt.2011.264.Epub 2011 Dec 6. PMID:22146342), or treatment with ESE8_2, ESE7, and SD6), and (2) transcripts containing exons 5 and 10 (lower box containing 8 transcripts boxed in green, treatment with various combinations of antisenses shown on the right in the panel). Both transcripts containing exons 5, 9, and 10, as well as exons 5 and 10, are therapeutic because they restore the reading frame in DMD. Figure 2B shows Sanger sequencing of transcripts from the green lower box corresponding to exon 5, 10 transcripts. Figure 2C shows Sanger sequencing of transcripts from the red upper box corresponding to exon 5, 9, 10 transcripts. [Figure 2B] Same as above. [Figure 2C-1] Same as above. [Figure 2C-2] Same as above. [Figure 3A] The U7snRNA vector approach to exon skipping is shown. U7snRNA is used as a carrier to target pre-messenger RNA. It consists of a loop used for nucleocytoplasmic export, a recognition sequence that binds to Sm protein used for efficient assembly between U7snRNA and target pre-mRNA, and an antisense sequence that targets pre-mRNA. It has its own promoter and 3' downstream sequence. The U7 cassette is then cloned into an AAV plasmid to produce the vector. Two orientations are shown as examples: a forward construct containing U7-antisense sequences targeting exons 6, 7, and 8 (Figures 3A and 3C, U7 ESE8_2_ESE7_SD6_forward), and a reverse orientation of a similar construct (Figures 3B and 3D, U7 ESE8_2_ESE7_SD6_reverse). Nucleic acids containing SEQ ID NOs: 26 and 28 are shown in Figures 3A-D. The lengths of the exons and introns are indicated by blue arrows, plus the numbers in red correspond to amino acids. The PCR amplicons from exons 5–10 and 5–9 differ by 129 bp. [Figure 3B] Same as above. [Figure 3C-1] Same as above. [Figure 3C-2] Same as above. [Figure 3C-3] Same as above. [Figure 3C-4] Same as above. [Figure 3D-1] Same as above. [Figure 3D-2] Same as above. [Figure 3D-3] Same as above. [Figure 3D-4] Same as above. [Figure 3D-5] Same as above. [Figure 3D-6] Same as above. [Figure 4A]Generation and characterization of a novel mouse model, the hDMDm7 model, carrying a nonsense mutation in exon 7 of the hDMD gene. Figure 4A shows a schematic diagram of human DMD exons 6, 7, and 8. The lightning bolt symbol represents the position where the CRISPR / Cas9 complex cleaves human DMD exon 7. Figure 4B shows Sanger sequence confirmation of the introduced nonsense mutation after genome editing. Figure 4C shows RT-PCR results from the hDMDm7 mouse model, confirming the presence of hDMD transcripts despite the nonsense mutation at both 4 weeks and 3 months. As expected, transcripts without exon 9 were also detected (lower band). Figure 4D shows Western blot results from control (Bl6 mice) and hDMDm7 mice. Dystrophin is present in WT mice but not in hDMDm7 mice (C-terminal antibody: PA1-21011, Thermo Fisher). Full-length dystrophin (upper arrow), loading control (lower arrow). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 5A]Figure 5 shows the histopathology and force production of the tibialis anterior (TA) muscle in transgenic mouse models of DMD (hDMDm7 or delCH2) compared to control mice (hDMD). Figure 5A shows H&E staining performed on the tibialis anterior (TA), gastrocnemius (Gas), triceps (Tri), and diaphragm (Dia) muscles from 3- and 6-month-old WT hDMD and hDMDm7(delCH2) mice (n=5). In both 3- and 6-month-old animals, central nucleation was observed in hDMDm7 mice, but not in WT hDMD mice. In addition, fibrosis was also observed in the diaphragm in 6-month-old hDMDm7 mice (asterisk). Figure 5B shows TA muscle strength assessment measured by normalized specific force after tetanic contraction. Figure 5B shows that muscle strength from 3- and 6-month-old hDMDm7(delCH2) mice decreases over time compared to 3- and 6-month-old wild-type mice (hDMD). Figure 5C shows loss of force in the TA muscle after repeated eccentric contractions. hDMDm7(delCH2) mice lost approximately 55% of their TA strength at 3 months of age and approximately 65% ​​of their TA strength at 6 months of age as measured by this assay. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A]U7_U7 ESE8_2 SD7 SD6 Forward or Reverse SC rAAV (4E11vg) was delivered by intramuscular injection in hDMDm7 mice. 4E11vg was injected into the TA of hDMDm7 mice. One month after injection, RNA was isolated and RT-PCR was performed. Figure 6A shows that both untreated wild-type mice (hDMD, lanes 1 and 2) and mice in a mouse model of DMD (hDMDm7, lanes 3 and 4) expressed transcripts for exons 5, 6, 7, 8, 9, and 10, as well as exons 5, 6, 7, 8, and 10, as indicated by the top two arrows. Mice injected with AAV1.U7 ESE8_2 SD7 SD6 forward (lanes 5–10) showed varying amounts of exon skipping, as demonstrated by transcripts of exons 5, 6, and 10, as well as exons 7–9 (upper box in green, with less efficient skipping of exon 6) and exons 6–9 (lower box in red, with more efficient skipping of exon 6). These results differ slightly from the in vitro data shown in Figure 2A, where exon 6 was excluded but exon 9 was present in one of the transcripts after exon skipping. Transcripts that include exon 6 but lack exons 7, 8, and / or 9 do not restore the reading frame, whereas transcripts that include (1) exons 5, 9, and 10, or (2) exons 5 and 10 restore the reading frame. 6B-C show Sanger sequencing of the transcripts confirming expression of exons 5, 6, and 10 (FIG. 6B) and exons 5 and 10 (FIG. 6C) transcripts following treatment with antisense constructs. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 7-1] 1 shows the antisense, U7, and DMD target sequences of the present disclosure, with the promoter (yellow), smOPT (green), loop (blue), and 3'UTR regions (pink) highlighted in different colors. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 8-1] Some examples of U7 antisense construct sequences that contain multiple antisense sequences of the present disclosure are shown. The promoter (yellow), smOPT (green), loop (blue), and 3'UTR regions (pink) are highlighted in different colors. In each construct, there are three antisense sequences, each with their own promoter, smOPT, loop, and 3'UTR sequences. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9] Figure 1 shows the efficacy of exon skipping after intramuscular injection of three hDMDm7 mice into the left tibialis anterior (LTA) and right tibialis anterior (RTA) muscles. Mice were injected with scAAV1.nESE8_SD7_SD6(TT745-3), designed to skip exons 6, 7, and 8. Results are from RT-PCR performed on RNA extracted from treated muscles. Band sequences were confirmed by Sanger sequencing. Transcripts with exons 5, 6, 10 and exons 5, 10 are in-frame and therefore therapeutic. In conclusion, scAAV1.nESE8_SD7_SD6(TT745-3) is able to skip exons 7, 8, and 9 and partially skip exon 6. Transcripts omitting or lacking exons 6, 7, 8, and 9 are therapeutic, as they restore the reading frame of dmd. [Figure 10A]Results are shown from hDMDm7 mice receiving intramuscular injections of AAV.U7snRNA (construct TT744-3) into both TA muscles at 7 weeks of age. Physiological experiments were performed 3 months later to measure TA muscle strength. Figure 10A shows results from measurements of specific muscle strength in healthy control mice (hDMD-saline), delCH2 / hDMDm7 treated mice (delCH2-TT744-3), and delCH2 / hDMDm7 untreated mice (delCH2-saline), respectively (from left to right). Figure 10B shows results from measurements of force from successive eccentric contractions from the same group of mice. TT745-3 can improve the specific muscle strength of the tibialis anterior muscle and improve the resistance of the tibialis anterior muscle to eccentric forces, supporting the therapeutic benefit of this vector. [Figure 10B] Same as above. [Figure 11] Representative images of 3-month-old hDMDm7 (referred to as DelCH2 in the images) mice treated (see the far right panel labeled AAV.U7snRNA (construct TT744-3) "DelCH2+TT744-3-2001-LTA-20x") and untreated (saline, see the middle panel labeled "DelCH2+Sal-2306-LTA-20x") are shown. A healthy control mouse (hDMD1044) is shown in the far left panel for comparison. Dystrophin is stained red by immunofluorescence staining. Images are 20x magnification of LTA. These results show that treatment with the TT744-3 construct induced dystrophin expression in the tibialis anterior muscle 3 months after injection. [Figure 12A]Quantification of immunofluorescence staining for dystrophin from 20x magnification images of saline for therapeutic injection of mouse LTA and RTA. Figure 12A shows automated fiber quantification for fibers with 50% more dystrophin in treated mice compared to untreated mice (DelCH2). Compared to WT, treated mice have about 15 dystrophin-positive fibers with 50% higher dystrophin intensity. Figure 12B shows automated quantification of dystrophin intensity. Treated mice have an overall intensity of about 550 compared to about 480 for untreated mice, supporting the fact that this construct allows more dystrophin expression after treatment. Taken together, these results show that treatment with the TT744-3 construct introduced dystrophin expression in the tibialis anterior muscle 3 months after injection. [Figure 12B] Same as above. [Figure 13] Quantification of the percentage of transcripts from hDMDm7 / DelCH2 mice injected intramuscularly with saline (left bar) or construct TT744-3 (right bar). Treatments show 25% skipping (blue corresponding to Δ6-9, therapeutic transcripts) and 75% non-skipped transcripts (orange corresponding to pause, non-skipped transcripts). Taken together, this data supports the efficacy of TT744-3 to mediate the skipping of exons 6, 7, 8, and 9, restoring the reading frame of dmd allowing dystrophin expression. [Figure 14] Total LTA images (10x) from three mice stained by immunofluorescence for dystrophin are shown. hDMD LTA dystrophin (far left panel) shows dystrophin staining in a 5-month-old mouse and serves as a positive control. LTA staining for dystrophin 5 months after injection (DelCH2+saline, center panel) shows almost no dystrophin expression. LTA staining for dystrophin 5 months after injection (DelCH2+TT744-3, right panel) shows approximately 40% dystrophin-positive fibers. These results show that treatment with TT744-3 restored dystrophin expression in 40% of fibers supporting its therapeutic potential. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The products and methods described herein are used to prevent disease, delay disease progression, and / or treat muscular dystrophy in patients with one or more mutations in the DMD gene. More specifically, the present disclosure provides products and methods that use the U7snRNA approach to induce skipping of exons 6, 7, 8, and / or 9 of the DMD gene.

[0036] In some aspects, the products and methods described herein induce expression of dystrophin lacking the second portion of actin binding domain 1 (ABD1) to treat patients with mutations in exons 6, 7, 8, and / or 9 of the DMD gene that encodes dystrophin. Such products and methods force expression of a shorter dystrophin protein isoform.

[0037] Garcia and coworkers have investigated the use of vectors that allow for the simultaneous skipping of both exons 6 and 8 (US2012 / 0077860 (Garcia); Bish et al., Mol. Ther. 20(3):580-9, 2012; Vulin et al., Mol. Ther. 20(11):2120-33, 2012; Barbash et al., Gene Therapy 20:274-82, 2013; and Le Guiner et al., Mol. Ther. 22(11):1923-35; 2014). Such vectors allow expression of a shorter dystrophin (presumably the delta CH2 protein) in a GRMD canine model, resulting in improvements in skeletal muscle and heart.

[0038] The present disclosure provides novel antisense sequences designed to target each of exons 6, 7, and 8 in the human DMD gene to treat patients with mutations in exons 6, 7, 8, and / or 9 of the DMD gene encoding dystrophin. In some embodiments, the present disclosure includes a nucleic acid comprising at least two antisense sequences, each antisense sequence preceded by a U7 promoter. In some embodiments, the present disclosure includes a nucleic acid comprising at least three antisense sequences, each antisense sequence preceded by a U7 promoter. In some embodiments, the present disclosure includes at least three antisense sequences, each antisense sequence preceded by a U7 promoter, each antisense sequence targeting a different exon, for example, one targeted exon 6, one targeted exon 7, and one targeted exon 8. In some embodiments, the present disclosure includes multiple copies of a construct comprising at least two or at least three or more antisense sequences. In some aspects, the disclosure includes vectors containing multiple copies of such constructs, each construct containing at least two or at least three antisense sequences. In some aspects, the disclosure includes methods and uses of these nucleic acids (and vectors and compositions containing such nucleic acids) in the manufacture of a medicament for the treatment, amelioration, or prevention of MD, or for use in the treatment of MD.

[0039] The present disclosure provides antisense targeting sequences (shown in Table 1 below) and / or combinations of antisense targeting sequences embedded in modified U7 small nuclear RNA (U7snRNA) (Gorman et al., Proc Natl Acad Sci USA 95, 4929-4934 (1998)). In some embodiments, each antisense sequence is preceded by a U7 promoter sequence, which becomes part of the small nuclear ribosomal protein complex (snRNP) that protects the antisense sequence from degradation and allows it to accumulate in the nucleus where splicing occurs (Suter et al., Hum Mol Genet 8, 2415-2423 (1999)). In some embodiments, the U7snRNA, which contains an internal promoter that allows continuous transcription of the downstream antisense sequence, is packaged into an AAV for wide range tissue delivery. This approach has been shown to be useful in vitro and in mouse and dog models of DMD (Wein et al., Nature Medicine 20, 992-1000 (2014); Goyenvalle et al., Hum Mol Genet 21, 2559-2571 (2012); Barbash et al., Gene Ther 20, 274-282 (2013); Bish et al., Mol Ther 20, 580-589 (2012); Vulin et al., Mol Ther 20, 2120-2133 (2012)). Thus, in some aspects, the present disclosure provides such U7snRNA for the prevention, treatment, or amelioration of a disease or disorder caused by a mutation in the DMD gene. In some aspects, the DMD gene is a human DMD gene.

[0040] [Table 1]

[0041] U7 can be cloned in two orientations: forward and reverse. When U7 is in the forward orientation, the forward antisense sequence is used. When U7 is in the reverse orientation, the reverse antisense sequence is used. Lowercase letters in the sequence represent bases in intron regions. Uppercase letters in the sequence represent bases in exon regions.

[0042] The present disclosure provides antisense, U7, and target sequences for the DMD gene (shown in Table 2 below).

[0043] [Table 2-1]

[0044] [Table 2-2]

[0045] [Table 2-3]

[0046] [Table 2-4]

[0047] The present disclosure provides nucleic acids comprising combinations of antisense targeting sequences (shown in Table 3 below). In some embodiments, the sequences are embedded in modified U7 small nuclear RNA (U7snRNA). Such constructs shown in Table 3 below are not limiting and the nucleic acids include any of the antisense sequences shown in any of SEQ ID NOs: 1-20, or variants thereof, or other possible combinations of antisense sequences targeting any of the nucleotide sequences shown in any of SEQ ID NOs: 21-25.

[0048] [Table 3-1]

[0049] [Table 3-2]

[0050] Thus, the present disclosure provides a nucleic acid comprising any of the sequences shown in Tables 1 to 3. More specifically, the present disclosure provides a nucleic acid comprising a nucleotide sequence comprising at least 70% or 80% identity to a sequence shown in any one of SEQ ID NOs: 1 to 20 and 26 to 29, a nucleotide sequence complementary to a nucleotide sequence comprising at least 70% or 80% identity to a sequence shown in any one of SEQ ID NOs: 1 to 20 and 26 to 29, a nucleotide sequence comprising a sequence shown in any one of SEQ ID NOs: 1 to 20 and 26 to 29, a nucleotide sequence complementary to a nucleotide sequence comprising a sequence shown in any one of SEQ ID NOs: 1 to 20 and 26 to 29, or a nucleotide sequence binding to a sequence shown in any one of SEQ ID NOs: 21 to 25.

[0051] In some embodiments, the present disclosure provides a nucleic acid comprising a combination of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more of the following nucleotide sequences: (a) a nucleotide sequence comprising at least 70% or 80% identity to a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29; (b) a nucleotide sequence complementary to a nucleotide sequence comprising at least 70% or 80% identity to a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29; (c) a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29; (d) a nucleotide sequence complementary to a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29; or (e) a nucleotide sequence binding to a sequence set forth in any one of SEQ ID NOs: 21-25.

[0052] In some aspects, the disclosure includes a nucleic acid comprising a nucleotide sequence having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs:1-20 and 26-29.

[0053] In some aspects, the disclosure includes a nucleic acid comprising a nucleotide sequence complementary to a nucleotide sequence having at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs:1-20 and 26-29.

[0054] In some embodiments, the disclosure includes a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29. In some embodiments, the disclosure includes a nucleotide sequence complementary to a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 1-20 and 26-29. In some embodiments, the disclosure includes a nucleic acid comprising a nucleotide sequence that binds to a sequence set forth in any one of SEQ ID NOs: 21-25.

[0055] Thus, the present disclosure provides a nucleic acid comprising a nucleotide sequence comprising at least 80% identity to a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence complementary to a nucleotide sequence comprising at least 80% identity to a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence comprising a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence complementary to a nucleotide sequence comprising a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, and a nucleotide sequence binding to a sequence shown in any one of SEQ ID NOs: 21-25.

[0056] In some embodiments, the nucleic acid comprises a combination of at least two nucleotide sequences, the combination comprising a nucleotide sequence targeting the human DMD gene at exon 6 and a nucleotide sequence targeting the human DMD gene at exon 7, a nucleotide sequence targeting the human DMD gene at exon 6 and a nucleotide sequence targeting the human DMD gene at exon 8, and a nucleotide sequence targeting the human DMD gene at exon 7 and a nucleotide sequence targeting the human DMD gene at exon 8. In some embodiments, the nucleic acid comprises a combination of at least three nucleotide sequences, the combination comprising a nucleotide sequence targeting the human DMD gene at exon 6, a nucleotide sequence targeting the human DMD gene at exon 7, and a nucleotide sequence targeting the human DMD gene at exon 8. In some further embodiments, the nucleic acid comprises a combination of at least four nucleotide sequences, at least five nucleotide sequences, at least six nucleotide sequences, at least seven nucleotide sequences, at least eight nucleotide sequences, at least nine nucleotide sequences, or at least ten or more nucleotide sequences targeting the human DMD gene at exon 6, 7, and / or 8.

[0057] In some embodiments, such a nucleotide sequence includes any combination of one or more of a sequence having at least 80% identity to a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence complementary to a nucleotide sequence having at least 70% or 80% identity to a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence including a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, a nucleotide sequence complementary to a nucleotide sequence including a sequence shown in any one of SEQ ID NOs: 1-20 and 26-29, and a nucleotide sequence binding to a sequence shown in any one of SEQ ID NOs: 21-25.

[0058] In some aspects, such combinations of nucleotide sequences include a nucleotide sequence comprising at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 26-29, a nucleotide sequence complementary to a nucleotide sequence comprising at least 70% or 80% identity to a sequence set forth in any one of SEQ ID NOs: 26-29, a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 26-29, a nucleotide sequence complementary to a nucleotide sequence comprising a sequence set forth in any one of SEQ ID NOs: 26-29.

[0059] In some embodiments, the disclosure includes such nucleic acids under the control of a U7 promoter. In some embodiments, the disclosure includes delivery of such nucleic acids in a U7 small nuclear RNA (snRNA). In some embodiments, the U7 promoter includes a nucleotide sequence that includes at least 70%, 80%, or 90% identity to the sequence set forth in SEQ ID NO: 30 or 34.

[0060] In some aspects, the present disclosure provides small nuclear ribonucleic acids (snRNAs), also commonly referred to as U-RNAs, that affect DMD expression. snRNAs are a class of small RNA molecules found in the splicing specks and Cajal bodies of the cell nucleus in eukaryotic cells. Small nuclear RNAs are associated with a set of specific proteins, and the complexes are called small nuclear ribonucleoproteins (snRNPs, often pronounced "snurps"). Each snRNP particle is composed of an snRNA component and several snRNP-specific proteins, including Sm proteins, a family of nuclear proteins. snRNAs, along with their associated proteins, form ribonucleoprotein complexes (snRNPs) that bind to specific sequences on pre-mRNA substrates. They are transcribed by either RNA polymerase II or RNA polymerase III. snRNAs are often divided into two classes based on both common sequence features and associated protein factors, such as RNA-binding LSm proteins. The first class, known as the Sm class of snRNAs, is composed of U1, U2, U4, U4atac, U5, U7, U11, and U12. The Sm class of snRNAs are transcribed by RNA polymerase II. The second class, known as the Lsm class of snRNAs, is composed of U6 and U6atac. The Lsm class of snRNAs are transcribed by RNA polymerase III and, in contrast to the Sm class of snRNAs, never leave the nucleus.

[0061] In some embodiments, the present disclosure provides U7snRNA molecules that interfere with DMD gene expression. U7snRNA is normally involved in histone pre-mRNA 3' end processing, but in some embodiments, it is converted into a versatile tool for splicing regulation or as an antisense RNA that is continuously expressed in cells [Goyenvalle et al., Science 306[5702):1796-9(2004)]. Furthermore, when embedded in gene therapy vectors, these small RNAs can be permanently expressed in target cells after a single injection [Levy et al., Eur. J. Hum. Genet. 18(9):969-70(2010); Wein et al., Hum. Mutat. 31(2):136-42,(2010); Wein et al., Nat. Med. 20(9):992-1000(2014)]. The potential of the U7 snRNA system in neuromuscular disorders using AAV approaches has been investigated in vivo (AAV.U7) [Levy et al., Eur. J. Hum. Genet. 18(9): 969-70 (2010); Wein et al., Hum. Mutat. 31(2): 136-42 (2010); Wein et al., Nat. Med. 20(9): 992-1000 (2014)]. A single injection of this AAV9.U7 targeting the defective RNA in a mouse model of Duchenne muscular dystrophy results in long-term correction of the disease in all muscles, including the heart and diaphragm. The ability to target the heart is important because DM1 patients exhibit cardiac abnormalities.

[0062] In some embodiments, the DNA encoding the U7snRNA gene comprising the DMD inhibitory nucleic acid is delivered in a vector. However, in some other embodiments, such DNA is not delivered in an AAV or other vector. Thus, the present disclosure includes other means of delivering the DNA encoding the antisense constructs described herein. In some embodiments, such delivery includes, but is not limited to, delivery via liposomes, nanoparticles, or chemical transfection. Chemical transfection introduces DNA via calcium phosphate, lipid, or protein complexes.

[0063] Thus, the antisense sequences of the present disclosure are carried by snRNA in some embodiments and are delivered using viral vectors such as adeno-associated virus (AAV) or recombinant AAV (rAAV). The advantage of this approach is that the antisense sequences are embedded within the small nuclear ribonucleoprotein (snRNP) complex, thereby protecting it from degradation and causing accumulation in the nucleus where splicing occurs. Furthermore, when embedded in a gene therapy vector, these small RNAs can be permanently expressed in target cells after a single injection. AAV is a small virus that naturally infects humans without causing any known disease. The virus induces a very mild immune response and can infect both dividing and quiescent cells. The modified forms used in gene therapy persist in an episomal state without being integrated into the genome of the host cell. Thus, AAV vectors have an excellent safety profile, making them very attractive for gene therapy.

[0064] The present disclosure, in some embodiments, utilizes AAV to deliver inhibitory U7snRNA to deliver DMD antisense sequences that bind to key exon-defining elements in pre-mRNA and inhibit recognition of specific exons by the spliceosome, resulting in the exclusion of the targeted exon from the mature RNA and expression of dystrophin. U7snRNA is normally involved in histone pre-mRNA 3' end processing, but in some embodiments is converted into a general tool for splicing regulation or as an antisense RNA that is continuously expressed in cells [Goyenvalle et al., Science 306[5702):1796-9(2004)]. Furthermore, when embedded in gene therapy vectors, these small RNAs can be permanently expressed in target cells after a single injection [Levy et al., Eur. J. Hum. Genet. 18(9):969-70(2010); Wein et al., Hum. Mutat. 31(2):136-42,(2010); Wein et al., Nat. Med. 20(9):992-1000(2014)]. The potential of the U7 snRNA system in neuromuscular disorders using AAV approaches has been investigated in vivo (AAV.U7) [Levy et al., Eur. J. Hum. Genet. 18(9): 969-70 (2010); Wein et al., Hum. Mutat. 31(2): 136-42 (2010); Wein et al., Nat. Med. 20(9): 992-1000 (2014)]. A single injection of this AAV9.U7 targeting the defective RNA in a mouse model of Duchenne muscular dystrophy results in long-term correction of the disease in all muscles, including the heart and diaphragm. The ability to target the heart is of great importance since DM1 patients exhibit cardiac abnormalities.

[0065] Thus, the present disclosure includes the production and administration of an AAV vector comprising U7 snRNA for delivery of a DMD antisense sequence, such as a nucleotide sequence or a combination of at least two nucleotide sequences selected from the group consisting of: (a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (c) a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 20 and 26 to 29; (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 1 to 20 and 26 to 29; and (e) A nucleotide sequence that binds to any one of the sequences shown in SEQ ID NOs: 21 to 25.

[0066] The disclosure also includes the production and administration of AAV vectors comprising U7snRNA for delivery of DMD antisense sequences, such as a nucleotide sequence or a combination of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more nucleotide sequences selected from the group consisting of: (a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (c) a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 20 and 26 to 29; (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 1 to 20 and 26 to 29; and (e) A nucleotide sequence that binds to any one of the sequences shown in SEQ ID NOs: 21 to 25.

[0067] In some aspects, the present disclosure provides one or more copies of the various antisense sequences described herein combined into a single vector. In some aspects, the present disclosure provides one or more copies of the various antisense sequences described herein, or combinations of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, and ten or more of the various antisense sequences combined into a single vector. Thus, the present disclosure includes vectors comprising the nucleic acids of the present disclosure or combinations of the nucleic acids of the present disclosure. Such vectors include, for example, viral vectors such as adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, equine associated virus, alphavirus, poxvirus, herpes virus, herpes simplex virus, poliovirus, sindbis virus, vaccinia virus, or synthetic viruses, such as chimeric viruses, mosaic viruses, or pseudotyped viruses, and / or viruses that include foreign proteins, synthetic polymers, nanoparticles, or small molecules to deliver one or more nucleic acids disclosed herein. In some aspects, the vector is AAV. Thus, in some embodiments of the present disclosure, the U7snRNA is delivered via a viral vector, such as AAV.

[0068] In some aspects, the AAV lacks the rep and cap genes. In some aspects, the AAV is a recombinant AAV (rAAV). In some aspects, the rAAV is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

[0069] In some embodiments, the viral vector is an AAV1 (i.e., an AAV containing AAV1 inverted terminal repeats (ITRs) and AAV1 capsid protein), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid protein), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid protein), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid protein), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid protein), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid protein), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid protein), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid protein), AAV9 (i.e., an AAV containing AAV9 ITRs and AAV9 capsid protein), AAV10 (i.e., an AAV containing AAV10 ITRs and AAV10 capsid protein), AAV11 (i.e., an AAV containing AAV11 ITRs and AAV11 capsid protein), AAV12 (i.e., an AAV containing AAV12 ITRs and AAV12 capsid protein), AAV13 (i.e., an AAV containing AAV13 ITRs and AAV13 capsid protein), AAV14 (i.e., an AAV containing AAV14 ITRs and AAV14 capsid protein), AAV15 (i.e., an AAV containing AAV15 ITRs and AAV15 capsid protein), AAV16 (i.e., an AAV containing AAV16 ITRs and AAV16 capsid protein), AAV17 (i.e., an AAV containing AAV17 ITRs and AAV1 and AAV9 capsid protein), AAVrh74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein), AAV11 (i.e., an AAV containing AAV11 ITRs and AAV11 capsid protein), AAV12 (i.e., an AAV containing AAV12 ITRs and AAV12 capsid protein), AAV13 (i.e., an AAV containing AAV13 ITRs and AAV13 capsid protein), AAV-anc80, AAVrh.74, AAVrh.8, AAVrh.10, or AAV-B1.

[0070] AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length, including an inverted terminal repeat (ITR) of 145 nucleotides. For example, as noted herein above, there are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, and the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al. al., J. Virol., 45:555-564 {1983), the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829, the AAV-5 genome is provided under GenBank Accession No. AF085716, the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is provided by Gao et al. al, J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is reviewed in Virology, 330(2):375-383 (2004). Cis acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), direct the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene.The rep protein has multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0071] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. In some embodiments, the rep and cap proteins are provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°-65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0072] In some embodiments, the DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell that is permissive for infection with a helper virus (e.g., adenovirus, E1 deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into an infectious viral particle. Techniques for producing rAAV particles are standard in the art, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell. Production of rAAV requires that the following components are present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAVrep gene may be derived from any AAV serotype from which a recombinant virus may be derived, or may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-anc80, AAVrh.74, AAVrh.8, and AAVrh.10. In some embodiments, the AAV DNA in the rAAV genome is derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-anc80, AAVrh.74, AAVrh.8, AAVrh.10, and AAV-B1. Other types of rAAV variants, such as rAAVs with capsid mutations, are also included in the present disclosure. For example, as reviewed in Marsic et al., Molecular Therapy 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of the various AAV serotypes shown above are known in the art. The use of cognate components is specifically contemplated. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0073] In some embodiments, the recombinant AAV genome of the present disclosure comprises one or more AAV ITRs flanking a polynucleotide sequence, e.g., one or more antisense sequences that bind to key exon-defining elements of pre-mRNA and inhibit recognition of a specific exon by the spliceosome, resulting in the exclusion of the target exon of DMD from the mature RNA. Thus, in some embodiments, the rAAV genome of the present disclosure comprises one or more AAV ITRs flanking a polynucleotide encoding, for example, one or more DMD antisense sequences. Commercial vendors such as Ambion Inc. (Austin, TX), Darmacon Inc. (Lafayette, CO), InvivoGen (San Diego, CA), and Molecular Research Laboratories, LLC (Herndon, VA) custom manufacture inhibitory RNA molecules. In addition, custom siRNA molecules can be manufactured using commercially available kits, such as the SILENCER™ siRNA Construction Kit (Ambion Inc., Austin, TX) or the psiRNA System (InvivoGen, San Diego, CA).

[0074] Thus, in some embodiments, the U7 snRNA gene comprising the DMD inhibitory nucleic acid is cloned into a rAAV vector. Thus, embodiments of the present disclosure include an rAAV genome comprising a nucleic acid comprising a nucleotide sequence or a combination of at least two nucleotide sequences selected from the group consisting of: (a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (c) a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 20 and 26 to 29; (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 1 to 20 and 26 to 29; and (e) A nucleotide sequence that binds to any one of the sequences shown in SEQ ID NOs: 21 to 25.

[0075] In some embodiments, the rAAV genome comprises a nucleic acid that comprises a combination of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more nucleotide sequences selected from the group consisting of: (a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (b) a nucleotide sequence complementary to a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 20 and 26 to 29; (c) a nucleotide sequence comprising any one of the sequences set forth in SEQ ID NOs: 1 to 20 and 26 to 29; (d) a nucleotide sequence complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 1 to 20 and 26 to 29; or (e) A nucleotide sequence that binds to any one of the sequences shown in SEQ ID NOs: 21 to 25.

[0076] In some embodiments, the viral vector is pseudotyped AAV and comprises ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV comprising AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 8 (i.e., AAV comprising AAV2 ITRs and AAV8 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 1 (i.e., AAV comprising AAV2 ITRs and AAV1 capsid proteins).

[0077] In some aspects, the AAV comprises a recombinant capsid protein, such as a capsid protein comprising a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-anc80, AAVrh74, AAVrh.8, or AAVrh.10, AAV10, AAV11, AAV12, AAV13, or AAV-B1. Other types of rAAV variants, such as rAAVs with capsid mutations, are also included in the present disclosure. For example, as reviewed in Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of these various AAV serotypes shown herein above are known in the art.

[0078] In some embodiments, a DNA plasmid is provided that contains the rAAV genome described herein. The DNA plasmid is transferred to a cell that is permissive for infection with a helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into an infectious viral particle. Techniques for producing rAAV particles are standard in the art, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell. Production of rAAV requires that the following components are present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and the helper virus functions. The AAVrep gene may be derived from any AAV serotype from which a recombinant virus may be derived, or may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-B1, and AAVrh74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety.

[0079] In some embodiments, packaging cells are provided. The method of generating packaging cells is to generate cell lines that stably express all components necessary for the production of AAV particles. Retroviral vectors are generated by the removal of retroviral gag, pol, and env genes. These are replaced with therapeutic genes. In order to generate vector particles, packaging cells are essential. Packaging cell lines provide all viral proteins necessary for capsid production and virion maturation of the vector. Thus, packaging cell lines are generated such that they contain gag, pol, and env genes. After inserting the desired genes into the retroviral DNA vector and maintaining the appropriate packaging cell line, it is now a simple matter to prepare the retroviral vector.

[0080] For example, a plasmid (or a plurality of plasmids) containing a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0081] Thus, in some embodiments, packaging cells that produce infectious rAAV are further provided. The packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 cells). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblast cells), WI-38 cells (human fetal fibroblast cells), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0082] In some embodiments, the method for generating packaging cells is to create a cell line that stably expresses all components necessary for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy et al., 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0083] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol. 158:97-129). Various approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988). Samulski et al. al., J. Virol., 63: 3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, and corresponding U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine, 13: 1244-1250 (1995), Paul et al. al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy, 3:1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; U.S. Patent No. 6,258,595; and McCarty, Mol. Ther., 16(10):1648-1656 (2008). The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions of the documents relating to rAAV production. The manufacture and use of various types of rAAV are specifically contemplated and exemplified. Thus, recombinant AAV (i.e., infectious encapsulated rAAV particles) are provided herein.In some embodiments, the genome of the rAAV lacks the AAVrep and cap genes, i.e., there is no AAVrep or cap DNA between the ITRs of the genome of the rAAV. In some embodiments, the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV (ssAAV), or a recombinant self-complementary AAV (scAAV).

[0084] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 cells). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblast cells), WI-38 cells (human fetal fibroblast cells), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0085] rAAV can in some embodiments be purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and are disclosed, for example, in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0086] In some aspects, the present disclosure provides compositions comprising a vector, such as a nucleic acid or viral vector, as described herein. Thus, compositions are provided that comprise a delivery vehicle (such as a rAAV) as described herein. In various aspects, such compositions also comprise a pharma- ceutically acceptable carrier. In various aspects, such compositions also comprise other components, such as diluents, excipients, and / or adjuvants. Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acid salts; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic, or polyethylene glycol (PEG).

[0087] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, together with various other ingredients as listed above as necessary, and then filtering and sterilizing. Generally, dispersions are prepared by mixing sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients as listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0088] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the targeted individual, and the cell type, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per mL.6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 , about 1×10 15 , about 1×10 16 , or even more DNase resistant particles (DRP) [or viral genomes (vg)].

[0089] In some embodiments, the dose of rAAV administered is about 1.0×10 10 vg / kg ~ approx. 1.0×10 16 In some embodiments, the concentration is 1.0×10 10 vg / kg is also designated as 1.0E10vg / kg, which is simply an alternative way of indicating scientific notation. 11 is equivalent to E11. In some embodiments, the dose of rAAV administered is about 1.0 x 10 11 vg / kg ~ approx. 1.0×10 15 In some embodiments, the dose of rAAV is about 1.0×10 10 vg / kg, approx. 2.0×10 10 vg / kg, approx. 3.0×10 10 vg / kg, approximately 4.0×10 10 vg / kg, approximately 5.0×10 10 vg / kg, approximately 6.0×10 10 vg / kg, approximately 7.0×10 10 vg / kg, approximately 8.0×10 10 vg / kg, approximately 9.0×10 10 Approximately 1.0×10 11 vg / kg, approx. 2.0×10 11 vg / kg, approx. 3.0×10 11 vg / kg, approximately 4.0×10 11 vg / kg, approximately 5.0×10 11 vg / kg, approximately 6.0×10 11 vg / kg, approximately 7.0×10 11 vg / kg, approximately 8.0×10 11vg / kg, approximately 9.0×10 11 vg / kg, approximately 1.0×10 12 vg / kg, approx. 2.0×10 12 vg / kg, approx. 3.0×10 12 vg / kg, approximately 4.0×10 12 vg / kg, approximately 5.0×10 12 vg / kg, approximately 6.0×10 12 vg / kg, approximately 7.0×10 12 vg / kg, approximately 8.0×10 12 vg / kg, approximately 9.0×10 12 vg / kg, approximately 1.0×10 13 vg / kg, approx. 2.0×10 13 vg / kg, approx. 3.0×10 13 vg / kg, approximately 4.0×10 13 vg / kg, approximately 5.0×10 13 vg / kg, approximately 6.0×10 13 vg / kg, approximately 7.0×10 13 vg / kg, approximately 8.0×10 13 vg / kg, approximately 9.0×10 13 vg / kg, approximately 1.0×10 14 vg / kg, approx. 2.0×10 14 vg / kg, approx. 3.0×10 14 vg / kg, approximately 4.0×10 14 vg / kg, approximately 5.0×10 14 vg / kg, approximately 6.0×10 14 vg / kg, approximately 7.0×10 14 vg / kg, approximately 8.0×10 14 vg / kg, approximately 9.0×10 14 vg / kg, approximately 1.0×10 15 vg / kg, approx. 2.0×10 15 vg / kg, approx. 3.0×10 15 vg / kg, approximately 4.0×10 15 vg / kg, approximately 5.0×10 15 vg / kg, approximately 6.0×10 15 vg / kg, approximately 7.0×10 15 vg / kg, approximately 8.0×10 15 vg / kg, approximately 9.0×10 15 vg / kg, or approximately 1.0 × 10 16 vg / kg.

[0090] In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 1.0×10 15 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 5.0×10 14 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 1.0×10 14 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 5.0×10 13 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 1.0×10 13 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 5.0×10 12 In some embodiments, the dose is about 1.0×10 11 vg / kg ~ approx. 1.0×10 12 vg / kg. In some embodiments, the initial dose is followed by a second, larger dose. In some embodiments, the initial dose is followed by a second, same dose. In some embodiments, the initial dose is followed by one or more smaller doses. In some embodiments, the initial dose is followed by multiple doses of the same or greater doses.

[0091] Methods of transducing target cells in vivo or in vitro with a delivery vehicle (such as rAAV) are contemplated. Transduction of cells with the rAAV of the present disclosure results in sustained expression of antisense sequences that bind to key exon-defining elements in the pre-mRNA of the DMD gene, inhibiting recognition of the specific exon by the spliceosome and removing the target exon from the mature DMD RNA. Thus, the present disclosure provides rAAVs expressing antisense sequences that bind to key exon-defining elements in the pre-mRNA, inhibiting recognition of the specific exon by the spliceosome and excluding the target exon from the mature RNA in the subject, and methods of administering / delivering the rAAVs. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. These methods include transducing cells and tissues (including but not limited to tissues such as muscle) with one or more rAAVs described herein. Transduction can be performed with gene cassettes that contain cell-specific control elements. The term "transduction" is used to refer to the administration / delivery of u7snRNA comprising an antisense sequence to a target cell, either in vivo or in vitro, e.g., via a replication-deficient rAAV as described herein, which results in the expression of a functional form of dystrophin protein by the target cell.

[0092] The in vivo method includes administering an effective dose or effective doses of a composition comprising a delivery vehicle (such as rAAV) to a subject (including a human subject) in need thereof. Thus, methods are provided for administering an effective dose (or doses administered essentially simultaneously or at intervals) of the rAAV described herein to a subject in need thereof. If the dose(s) are administered prior to the onset of the disorder / condition, the administration is prophylactic. If the dose(s) are administered after the onset of the disorder / condition, the administration is therapeutic. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / condition being treated, delays or prevents the progression of the condition of the disorder / condition, delays or prevents the progression of the condition of the disorder / condition, reduces the extent of the disease, results in remission (partial or complete) of the disorder / condition, and / or prolongs survival.

[0093] In some embodiments, the compositions and methods of the present disclosure are used in treating, ameliorating, or preventing diseases such as muscular dystrophy (MD). In various embodiments, such MD is Duchenne muscular dystrophy (DMD). DMD, an X-linked degenerative muscle disorder, is the most common severe childhood muscular dystrophy, affecting approximately 1:5200 male births (Mendell et al., Ann Neurol 71,304-313(2012)). Symptoms of generalized muscle weakness first appear between 3 and 5 years of age, progressing to inability to walk by age 13 years, and death from cardiomyopathy or respiratory failure typically occurs by the third decade of life (Passamano et al., Acta Myol 31, 121-125 (2012); Duchenne, The Pathology of Paralysis with Muscular Degeneration (Paralysie Myosclerotique), or Paralysis with Apparent Hypertrophy. Br Med J 2, 541-542 (1867)). Dystrophin is a large (427 kDa) multifunctional protein that is localized in the lateral subfascial region of muscle fibers and plays an important role in protecting sarcolemma from mechanical damage caused by muscle contraction (Petrof et al., Proc Natl Acad Sci USA 90, 3710-3714 (1993)), and is encoded by the DMD gene (Juan-Mateu et al., PLoS One 10, e0135189 (2015)), but DMD is caused by a mutation that destroys its open reading frame. In various other embodiments, such MD is Becker muscular dystrophy (BMD). The presence of a partially functional dystrophin protein occurs together with mutations that maintain the open reading frame (ORF), resulting in the milder allelic disorder BMD (Wein et al., Nature Medicine 20, 992-1000 (2014); Monaco, Trends Biochem Sci 14, 412-415 (1989)). BMD, like DMD, is a genetic disorder that causes the muscles of the body to become gradually weaker and smaller.BMD affects the muscles of the hips, pelvis, thighs, and shoulders, as well as the heart, but is known to cause less severe problems than DMD. Due to a variety of in-frame mutations resulting in a variety of partially functional proteins, BMD has a broad phenotypic spectrum, including, for example, the inability to walk from late teens to late adulthood.

[0094] In families known to have pathological DMD or BMD mutations, the disclosed methods, in various embodiments, are methods of preventing disease, which are performed prior to the onset of disease, while in various other embodiments, the disclosed methods are performed after diagnosis, and thus are methods of treating or ameliorating the disease.

[0095] Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the method. Outcome measures are described, for example, in Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4 th The method is reviewed in Chapters 32, 35, and 43 of The American Academy of Neurology, Volume 1 (2005), and Burgess et al., Methods Mol. Biol., 602:347-393 (2010). Outcome measures include, but are not limited to, one or more of the following: elimination of the targeted exon from mature RNA, reduction or elimination of mutant DMD mRNA or protein in affected tissues, and expression of a functional form of dystrophin. Expression of functional dystrophin in cells is detected by measuring dystrophin protein levels by methods known in the art, including but not limited to Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of rAAV to determine improvement.

[0096] In some embodiments, the level of functional dystrophin gene expression or protein expression in the cells of the subject is increased after administration of the rAAV compared to the level of functional dystrophin gene expression or protein expression before administration of the rAAV, hi some embodiments, expression of a functional form of dystrophin is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or more than at least about 100%. In various embodiments, improved muscle strength, improved muscle function, and / or improved mobility and stamina represents an improvement of at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or greater than at least about 100%.

[0097] Other outcome measures include measuring the subject's serum creatine kinase (CK) levels before and after treatment. Elevated CK levels are a hallmark of muscle damage. In Duchenne patients, CK levels are significantly increased above the normal range (10-100 times normal levels since birth). Elevated CK levels in blood samples usually mean that muscles are being destroyed by abnormal processes such as muscular dystrophy or inflammation. Thus, a positive therapeutic outcome for treatment with the disclosed method is a decrease in serum creatine kinase levels after administration of rAAV compared to serum creatine kinase levels before administration of rAAV.

[0098] Other outcome measures include determining whether the subject has improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof after treatment. Such outcome measures are important in determining the progression of muscular dystrophy in subjects, and are measured by various tests known in the art. Some of these tests include, but are not limited to, the 6-minute walk test, the stand-up test, the 4-step up test, the 4-step up and down test, the North Star Gait Assessment (NSAA) test, the 10-meter timed test, the 100-meter timed test, the hand-held dynamometry (HHD) test, the timed up and go test, the gross movement subtest equivalent (Bayley-III) score, the maximum isometric voluntary contraction test (MVICT), or a combination of two or more thereof.

[0099] Combination therapy is also contemplated by the present disclosure. Combination, as used herein, includes simultaneous or sequential therapy. Combination of the methods described herein with standard medical treatments and supportive care, as well as combinations with treatments such as glucocorticoids, are specifically contemplated. All types of glucocorticoids are included for use in the combination therapy disclosed herein. Such glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.

[0100] Administration of an effective dose of a nucleic acid, viral vector, or composition of the present disclosure may be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraventricular, intrathecal, intraosseous, rectal, or vaginal. In some embodiments, an effective dose is delivered by a systemic route of administration, i.e., systemic administration. Systemic administration is a route of administration to the circulatory system such that the entire body is affected. Such systemic administration is, in various embodiments, via enteral administration (absorption of the drug via the gastrointestinal tract) or via parenteral administration (generally injection, infusion, or implantation). In various embodiments, an effective dose is delivered by a combination of routes. For example, in various embodiments, an effective dose is delivered intravenously and / or intramuscularly, or intravenously and intraventricularly, etc. In some embodiments, an effective dose is delivered sequentially or sequentially. In some embodiments, an effective dose is delivered simultaneously. In various aspects, the route of administration and serotype of the AAV components of the rAAV of the present disclosure (particularly the AAV ITRs and capsid proteins) are selected and / or matched by one of skill in the art taking into consideration the condition or state of the disease or disorder being treated, the condition, state, or age of the subject, and the target cells / tissues expressing the nucleic acid or protein.

[0101] In particular, the actual administration of the delivery vehicle (such as rAAV) of the present disclosure can be accomplished by using any physical method that will transport the delivery vehicle (such as rAAV) to the target tissue of the animal. Administration includes, but is not limited to, direct injection into muscle, bloodstream, and / or nervous system or liver. Simply resuspending rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV can be modified to target rAAV to a specific target tissue of interest, such as nerve. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a local formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been developed in advance and can be used in carrying out the present disclosure. The delivery vehicle (such as rAAV) can be used with any pharma- ceutically acceptable carrier to facilitate administration and handling.

[0102] Dispersions of the delivery vehicle (such as rAAV) can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed are all readily available by standard techniques in the art.

[0103] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, sorbitol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0104] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, together with various other ingredients as listed above as necessary, and then filtering and sterilizing. Generally, dispersions are prepared by mixing sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other required ingredients as listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0105] The present disclosure also provides kits that include the nucleic acids, vectors, or compositions of the present disclosure or are manufactured according to the processes of the present disclosure. In the context of the present disclosure, the term "kit" refers to two or more components, one of which corresponds to the nucleic acids, vectors, or compositions of the present disclosure, and the other of which corresponds to a container, recipe, instructions, etc. Thus, in various aspects, a kit is a set of products sufficient to achieve a particular goal and can be sold as a single unit.

[0106] The kit may include one or more recipients (vials, ampoules, containers, syringes, bottles, bags, etc.) of any suitable shape, size, and material containing the nucleic acid, vector, or composition of the disclosure, in suitable dosage amounts for administration (see above). The kit may further include directions or instructions for use (e.g., in the form of a leaflet or instruction manual), a means for administering the nucleic acid, vector, or composition (e.g., a syringe, pump, injector, etc.), a means for reconstituting the nucleic acid, vector, or composition, and / or a means for diluting the nucleic acid, vector, or composition.

[0107] In one embodiment, such a kit includes the nucleic acid or vector in a diluent packaged in a container, such as a sealed bottle, or a container, with a label affixed to the container or included in the package that describes the use of the nucleic acid or vector. In one embodiment, the diluent is in the container such that the amount of head space in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of head space is negligible (i.e., almost nonexistent).

[0108] In some embodiments, the formulation comprises a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the formulation from harmful conditions, such as those that occur during heating or freezing, and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose, sugar alcohols such as mannitol, amino acids such as glycine or glutamic acid, and proteins such as human serum albumin or gelatin.

[0109] In some embodiments, the formulation includes an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to a composition to inhibit the growth of microorganisms that may be introduced upon repeated puncturing of the vial or container in which it is used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0110] In some embodiments, the kits include labels and / or instructions that describe the use of the reagents provided in the kit. The kits also optionally include catheters, syringes, or other delivery devices for delivering one or more of the compositions used in the methods described herein.

[0111] The present disclosure also provides unit dose or multi-dose kits. In some aspects, the present disclosure provides kits including single-chamber and multi-chamber pre-filled syringes.

[0112] This entire document is intended to be related as a unified disclosure, and it is understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes all embodiments of the disclosure that are somewhat narrower in scope than, for example, the variations specifically mentioned above. With respect to aspects of the disclosure described as genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with "a" or "an", these terms are understood to mean "one or more" unless the context clearly requires a more limited meaning. When aspects of the disclosure are described as "comprising" a feature, it is also contemplated that the embodiment "consists of" or "consists essentially of" the feature.

[0113] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.

[0114] As used herein, the term "and / or" is inclusive of the meaning of "and", "or" and "all or any other combination of the elements connected by that term". The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, the present invention also includes specific numbers, for example, about 10 includes 10. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0115] Throughout this specification and the claims which follow, unless the context otherwise requires, the words "comprise" and variations such as "comprises" and "comprising" should be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. As used herein, the term "comprising" can be substituted with the terms "containing" or "including," or is used in conjunction with the term "having" herein.

[0116] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0117] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. It is to be understood that this disclosure is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the presently disclosed subject matter, which is defined solely by the claims.

[0118] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illuminate the invention and do not state limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0119] All publications and patents cited throughout the body of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., are hereby incorporated by reference in their entirety. To the extent that material incorporated by reference is inconsistent or contradictory with the present specification, the present specification takes precedence over such material.

[0120] A better understanding of the present disclosure and its advantages will be gained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the disclosure. EXAMPLES

[0121] Aspects and aspects of the present disclosure are illustrated by the following examples. Example 1 Materials and Methods Constructs DNA encoding the U7 snRNA gene constructs, including the promoter, antisense, smOPT, loop, and 3'UTR sequences, as shown in Tables 2 and 3, were cloned into AAV vectors. All plasmid constructs were sequence verified.

[0122] Design and production of U7snRNA AAV vectors Vectors were generated at the Viral Vector Core at The Research Institute at Nationwide Children's Hospital. Serotype 1 and 9 recombinant adeno-associated virus (rAAV1, rAAV9) vectors were generated in human embryonic kidney 293 cells by a modified cross-packaging approach using an adenovirus-free triple plasmid DNA transfection (CaPO4 precipitation) method (Rabinowitz et al., J. Virol. 2002;76:791-801).

[0123] An immortalized and conditionally inducible fibroMyoD cell line Expression of the MyoD gene in mammalian fibroblasts results in the transdifferentiation of the cells into the myogenic lineage. Such cells can further differentiate into myotubes, where they express muscle genes, including the DMD gene. An immortalized cell line was generated that conditionally expresses MyoD under the control of a tetracycline-inducible promoter. This was achieved by stable transfection of a primary fibroblast cell line with lentiviral tet-inducible MyoD, which contains the human telomerase gene (TER). The resulting stable line is capable of initiating MyoD expression by treatment with doxycycline. Such a cell line was generated from a DMD patient carrying a mutation within exons 6, 7, and 8.

[0124] WT immortalized human fibroblasts that could transdifferentiate into muscle lineage cells under the control of doxycycline were generated by transduction with both a telomerase-expressing vector and a tet-inducible MyoD-expressing vector [Chaouch, S., et al. Human gene therapy 20, 784-790 (2009)]. The transformed human fibromyoblasts (FM) were then transduced with scAAV1 vectors carrying different U7 constructs incorporating antisense sequences against exons 6, 7, and 8.

[0125] hdmd7(delCH2) mouse model Mice carrying a nonsense mutation in hDMD exon 7 within the hdmd locus were developed using CRISPR / Cas genome editing tools. A schematic showing the mutation is shown in Figure 4A, where the numbers indicate the length of each exon and intron (the numbers of each exon (shown in red) correspond to amino acids). PCR amplicons from exons 5-10 and 5-9 differ by 129 bp (i.e., the length of exon 9).

[0126] This hdmd7(delCH2) mouse model shows the absence of dystrophin at the protein level, and displays central nucleation and muscle weakness (Figure 5A-C). Multi-exon skipping of exons 6, 7, and 8 was designed to restore the reading frame in this hDMDm7 mouse. As demonstrated in the Golden Retriever Canine Model (GRDM) (Leguiner et al., 2014 Nov;22(11):1923-35; Vulin et al., Mol Ther. 2012 Nov;20(11):2120-33), skipping of exons 6-8 resulted in the expression of a truncated but highly functional dystrophin. Dogs in these studies were able to express transcripts lacking exons 6-8 and were able to walk or run (Leguiner et al., 2014 (supra); Vulin et al., 2012 (supra)).

[0127] Male C57BL / 6 ES cells were transfected with the CRISPR / Cas9 tool and template to introduce a nonsense mutation in exon 7 (see Figure 4A-B). The insertion was confirmed by PCR. One superior clone was found, expanded, and injected into several dozen albino BL / 6 blastocysts. The injected blastocysts were implanted into recipient mice. The dystrophin gene from the chimeric males was confirmed by PCR and then RT-PCR. Colonies were expanded and maintained as heterozygotes. Figure 4C shows the absence of dystrophin expression in muscles from both 4-week-old and 3-month-old hemizygous hDMDm7 mice.

[0128] Use of Laboratory Animals and Animal Testing All experimental procedures involving animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Nationwide Children's Hospital.

[0129] Intramuscular and tail vein injections In some embodiments, intramuscular injection was used. For intramuscular injection studies, mice were injected with a vector DNA dose in the 4.11E11 vector genome (vg) into the tibialis anterior (TA) muscle and sacrificed one month later. At the time of sacrifice, the TA, gastrocnemius, triceps, diaphragm, and heart were removed and frozen in liquid nitrogen-cooled isopentane.

[0130] In some embodiments, injections are performed using a tail vein device. The tail is warmed via a light bulb to enlarge the vein. Once visible, AAV vectors or controls (saline or phosphate buffered saline (PBS)) are injected using a 33G gas-tight Hamilton syringe in a total of 300 μl of saline or PBS, or saline or PBS alone. After injection, a sterile cotton pad is placed at the injection site and pressure is applied and held until bleeding stops.

[0131] Muscle preparation Standard techniques were used for dissection of mice. Muscles were harvested and either snap frozen or mounted for cryosection preparation and cut at 10 μM for immunofluorescence and H&E staining. Right tibialis anterior (ta), gastroc, quad and triceps muscles were analyzed in all treatment groups. Tissues / organs for histopathology examination were harvested and fixed in 10% neutral buffered formalin (10% NBF).

[0132] Exon skipping analysis Total RNA was isolated from tissue samples and analyzed by reverse transcription (RT) followed by PCR. Total RNA was isolated from frozen muscle sections (15 x 40 μM sections) using Trizol according to the manufacturer's protocol (Life Technologies, 15596018). For each sample, 1 μg of total RNA was used to generate cDNA by RT-PCR using random hexamer primers according to the manufacturer's protocol (Thermo Scientific, Ferk1672), and then used for 35 cycles of single PCR using 1 μg of RNA and a mixture of random hexamers and oligo(dT) for each RT reaction. The mRNA was then amplified via primers specific for the analyzed region. PCR amplification was performed using 2× master mix (Thermo Scientific, K0172) and 150 ng of RT product as template. After electrophoresis, the PCR products were separated by electrophoresis on a 2% agarose gel and imaged using a Gel Logic 200 imaging system (Kodak).

[0133] Sanger sequencing To confirm the presence of point mutations and exon skipping products, amplification PCR was generated and run on a 2% agarose gel. Bands were extracted using a Zymogen gel DNA recovery kit (D4001 / D4002, Zymogen) according to standard protocols. DNA was then sent for Sanger sequencing to the Ohio State University Comprehensive Cancer Center Shared Resources Genomics Shared Resources, where sequencing was performed.

[0134] Immunoblot analysis Protein extraction was started with 25 sections (40 μM) and 100 μL of lysis buffer containing base buffer, phosphatase inhibitor (PhosStop, Roche, 4906845001) and protease inhibitor (Halt Protease Inhibitor Cocktail, Fisher, 78430). Steel beads were added to the tissue, which was homogenized for 2 min at a speed of 30 / s using a Tissuelyser II (Qiagen). The lysate was then incubated on ice, spun down, the supernatant removed and stored at -80 °C until immunoblotting, and cell debris was discarded. For immunoblotting, 50-150 μg of muscle tissue protein was loaded with loading dye (1 × laemmli) on a 3-8% Tris-acetate gel (Life Technologies, EA0378BOX). The gel was run at 80 V for 30 min and then at 120 V for 4 h. Proteins were transferred to nitrocellulose membranes (Fisher, 09-301-108) overnight at 4°C in transfer buffer (Invitrogen, NP00061) at 50V. The membranes were then exposed with rat monoclonal anti-dystrophin primary antibody (1:200, Abcam, ab15277) and mouse monoclonal β-actinin primary antibody (1:5000, Fisher, MA122863), followed by IRDye α-rabbit 680 and α-mouse 800 (Licor, 926-68071 and 926-32210) secondary antibodies. The membranes were scanned on an Odyssey CLx and imaged using Image Studio 14.

[0135] Force generation and protection from eccentric contractions Physiological testing was performed on the TA muscles of 3- and 6-month-old mice. The force testing procedure was performed using a modified version of Hakim's procedure (Hakim et al., J Appl. Physiol. (1985) 2011; 110: 1656-1663, Wein et al., Nat. Med. 2014; 20: 992-1000). Initial anesthesia of mice was performed by intraperitoneal injection of a cocktail containing 5 times their weight (i.e., mouse weight) of 25 mg / mL ketamine and 2 times their weight of 2.5 mg / mL xylazine. Skin fascia and connective tissue were removed from around the tibialis anterior (TA) muscle. During surgery, the TA muscle was constantly moistened with 0.9% saline. A knot was tied to the distal TA tendon with a 4-0 suture, and the tendon was cut. The excess suture was then re-tied, leaving a loop for attaching the tendon to a force transducer. The mouse was then placed on a platform maintained at 37 °C for the duration of the experiment. The limbs were secured to the platform by passing pins through knee caps and taping the feet. The loop of suture attached to the TA tendon was then attached to a 205B dual mode servo motor transducer (Aurora Scientific, Aurora, ON, Canada). Finally, two electrical probes were placed on the biceps femoris muscle near the sciatic nerve for stimulation.

[0136] Resting force was set between 3-4 g over a 10 min equilibration period. The TA muscle was then stimulated at its optimal length (Lo, mm) and active tetanic force was recorded to perform absolute force measurements using a LabView-based DMC program (Aurora Scientific). The muscle was then run through a 10-step passive stretch protocol during which stimulation was applied to determine the force drop after repeated eccentric contractions. At each step, the TA muscle was passively taut at 10% of Lo. Upon completion of the protocol, mice were administered a lethal dose of ketamine / xylazine and the TA muscle was removed and weighed. Cross-sectional area was measured as mass (g) / (muscle density × fiber length ratio × Lo), yielding a muscle density of 1.06 mg / mm3 and a fiber length ratio of 0.6 in the TA (Burkholder et al., J Morphol 1994;221:177-190). Muscle weight and cross-sectional area using Lo were applied to the absolute force measurements to obtain specific muscle force measurements. Statistical significance was assessed using the Kruskal-Wallace test assuming non-parametric data in GraphPad Prism (version 6.03 for Windows, GraphPad Software, San Diego California USA). For all data, the mean and standard deviation of each measurement were determined, and measurements beyond ±1 standard deviation were subsequently excluded as outliers. The tenth eccentric contraction recording was used to determine outliers.

[0137] statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad). Data normality was tested using D'agostino-Pearson if sample size was sufficient or using the Shapiro-Wilk test. If data had a Gaussian distribution, one-way ANOVA test was used. Otherwise, Kruskal-Wallis assay was performed. This method was applied to all experiments except eccentric contraction-induced injury (ECC), where two-way ANOVA analysis followed by Bonferroni post-hoc test was performed.

[0138] Example 2 Antisense sequences and vectors We have developed products and methods for performing virus-mediated skipping of exons 6, 7, and 8. The products and methods were modified compared to the U7snRNA system reported in Goyenvalle et al., Science, 306(5702):1796-1799(2004) or Goyenvalle et al., Mol. Ther., 20(6):179601799(2004). The U7snRNA was modified to contain several targeted antisense sequences to interfere with splicing at exons 6, 7, and 8. Specifically, antisense sequences targeting exons 6, 7, and 8 were designed as shown in Figures 1A-F and shown in Table 4 below.

[0139] [Table 4]

[0140] U7snRNA constructs were generated that contained antisense sequences targeting exons 6, 7, and 8. Each U7snRNA construct contained one of the target sequences. Self-complementary (sc) AAV vectors were then generated whose genomes contained one or more of the U7snRNA constructs.

[0141] Recombinant scAAV was produced in HEK293 cells by a modified cross-packaging approach using a plasmid containing the desired vector genome by an adenovirus-free triple plasmid DNA transfection (CaPO4 precipitation) method [Rabinowitz et al., J. Virol., 76:791-801 (2002)]. The vector was generated by cotransfecting AAV helper plasmid and adenovirus helper plasmid in a manner similar to that previously reported [Wang et al., Gene. Ther., 10:1528-1534 (2003)]. The adenovirus helper plasmid (pAdhelper) expresses the adenovirus type 5 E2A, E4ORF6, and VA I / II RNA genes required for high-titer rAAV production.

[0142] Vectors were purified from clarified 293 cell lysates by sequential iodixanol gradient purification and anion exchange column chromatography using a linear NaCl salt gradient as previously described [Clark et al., Hum. Gene Ther, 10:1031-1039 (1999)]. Vector genome (vg) titers were measured using QPCR-based detection with specific primer / probe sets utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems) as previously described (Clark et al., supra). Vector stock titers ranged from 1 to 10 × 10 12 The range was vg / mL.

[0143] Example 3 Efficacy of U7 snRNA-mediated skipping in WT cell lines and hDMD mouse models The first exon skipping analysis was performed by RT-PCR using a self-complementary recombinant AAV (scrAAV) vector to transduce WT immortalized human fibromyoblasts. WT immortalized human fibroblasts capable of transdifferentiating into muscle lineage cells under the control of doxycycline were generated by transduction with both a telomerase expression vector and a tet-inducible MyoD expression vector [Chaouch, S., et al. Human gene therapy 20, 784-790 (2009)]. The transformed human fibromyoblasts (FM) were then transduced with scrAAV vectors carrying different U7 constructs incorporating antisense sequences against exons 6, 7, and 8.

[0144] RT-PCR results are shown in FIG. 2A for sc rAAV-U7 constructs with three different antisense sequences (e.g., ESE8_2, ESE7, SD6 (combination shown as "136") or ESE8, SD7, and SD6 (combination shown as "256"). The U7 ese8_2 ese7 sd6 construct was included in the vector genome in either the forward or reverse orientation (FIG. 3A-D). Each U7 construct had an antisense directed against exon 6, 7, or 8 (as shown in the plasmid map in FIG. 3A-D), including three exon 6, 7, and 8-targeting U7 snRNA polynucleotide constructs in the sequence: U7 ESE8_2 / ESE8 construct, U7 ESE7 / SD7 construct, and U7 SD6 construct. ESE8_2 / ESE8, SD7 / ESE7, SD6 forward or reverse SC rAAV (abbreviated elsewhere herein as U7_U7 ESE8_2 SD7 SD6 forward or reverse SC rAAV) achieved a higher percentage of exon 6, 7, and 8 skipping.

[0145] In subsequent experiments, exon skipping efficiency was analyzed in vivo in the hdmd7 mouse model described herein above. The most efficient AAV-U7 vector, rAAV1.U7_U7 ESE8_2 SD7 SD6 forward or reverse, containing antisense sequences targeting each of exons 6, 7, and 8, was selected for initial intramuscular injection in hDMDm7 mice.

[0146] The results are shown in Figures 6A-C. Figure 6A shows the RT-PCR results of exon skipping of exons 7, 8, and 9 (green box (upper box of Figure 6A)) or exons 6, 7, 8, and 9 (red box (lower box of Figure 6A)). Lanes 1-2 show spontaneous amplification in WThDMD mice with transcripts having either exons 5, 6, 7, 8, 9, and 10, or exons 5, 6, 7, 8, and 10. Lanes 3-4 show spontaneous amplification in hDMDm7 mice with the same transcripts. Lanes 5-10 show the results of skipping after treatment with U7 ESE8_2SD7 SD6 forward. Exons 5, 6, and 10 (green box (upper box of Figure 6A)), exons 5 and 10 (red box (lower box of Figure 6A)) are present, confirming the efficiency of skipping. FIG. 6B shows Sanger sequencing confirming the skipping of exons 7, 8, and 9 (green boxes (upper boxes in FIG. 6A)) and exons 6, 7, 8, and 9 (red boxes (lower boxes in FIG. 6A)) in both transcripts. The transcript sequenced in FIG. 6B lacks exons 7, 8, and 9, and the transcript sequenced in FIG. 6C lacks exons 6, 7, 8, and 9. These results confirmed that the vector containing the antisense sequence was able to mediate exon skipping of exons 6, 7, 8, and 9. These results differ slightly from the in vitro data obtained from FIG. 2A, where exon 6 was excluded but exon 9 was present in one of the transcripts after exon skipping. Transcripts containing exon 6 but lacking exons 7, 8, and / or 9 do not restore the reading frame, whereas transcripts containing exons 5, 9, and 10, as well as transcripts containing exons 5 and 10, both restore the DMD reading frame.

[0147] Figure 9 shows the efficacy of exon skipping after intramuscular injection of three hDMDm7 mice into the left tibialis anterior (LTA) and right tibialis anterior (RTA) muscles. Mice were injected with scAAV1.nESE8_SD7_SD6(TT745-3), designed to skip exons 6, 7, and 8. Results are from RT-PCR performed on RNA extracted from treated muscles. Band sequences were confirmed by Sanger sequencing. Transcripts with exons 5, 6, 10 and exons 5, 10 are in-frame and therefore therapeutic. In conclusion, scAAV1.nESE8_SD7_SD6(TT745-3) can skip exons 7, 8, and 9 and partially skip exon 6. Transcripts omitting or lacking exons 6, 7, 8, and 9 are therapeutic because they restore the reading frame of dmd.

[0148] Physiological experiments were performed to measure TA muscle strength 3 months after injection into mice. Figure 10A-B show results from hDMDm7 mice receiving intramuscular injections of AAV.U7snRNA (construct TT744-3) into both TA muscles at 7 weeks of age. Figure 10A shows results from measurements of specific muscle strength in, respectively (from left to right), healthy control mice (hDMD-saline), delCH2 / hDMDm7 treated mice (delCH2-TT744-3), and delCH2 / hDMDm7 untreated mice (delCH2-saline). Figure 10B shows results from measurements of force from consecutive eccentric contractions from the same group of mice. TT745-3 was able to improve the specific muscle strength of the tibialis anterior muscle and improve the resistance of the tibialis anterior muscle to eccentric forces, supporting the therapeutic benefit of this vector.

[0149] Dystrophin expression in mouse muscles was examined after treatment. Figure 11 shows representative images of treated (AAV.U7snRNA (construct TT744-3), see the rightmost panel labeled "DelCH2+TT744-3-2001-LTA-20x") and untreated (saline, see the center panel labeled "DelCH2+Sal-2306-LTA-20x") 3-month-old hDMDm7 (referred to as DelCH2 in the images) mice. A healthy control mouse (hDMD1044) is shown in the leftmost panel for comparison. Dystrophin is stained red by immunofluorescence staining. Images are 20x magnification of LTA. These results show that treatment with the TT744-3 construct introduced dystrophin expression in the tibialis anterior muscle 3 months after injection.

[0150] Quantification of dystrophin expression in mice after treatment was also performed. 12A-B show quantification of immunofluorescence staining of dystrophin from 20x magnification images of saline in therapeutic injection of mice LTA and RTA. FIG. 12A shows automated fiber quantification for fibers with 50% more dystrophin in treated mice compared to untreated mice (DelCH2). Compared to WT, treated mice have about 15 dystrophin-positive fibers with 50% higher dystrophin intensity. FIG. 12B shows automated quantification of dystrophin intensity. The intensity of treated mice is about 550 overall compared to about 480 overall for untreated mice, supporting the fact that this construct allows more dystrophin expression after treatment. Taken together, these results show that treatment with the TT744-3 construct introduced dystrophin expression in the tibialis anterior muscle 3 months after injection.

[0151] Quantification of the percentage of transcripts from hDMDm7 / DelCH2 mice injected intramuscularly with saline (left bar) or construct TT744-3 (right bar) was also performed (Figure 13). Treatment showed 25% skipping (blue corresponding to 6-9, therapeutic transcripts) and 75% non-skipped transcripts (orange corresponding to pause, non-skipped transcripts). Taken together, this data shows the efficacy of TT744-3 to mediate the skipping of exons 6, 7, 8, and 9, restoring the reading frame of dmd allowing dystrophin expression.

[0152] Dystrophin expression was measured 5 months after injection. Figure 14 shows total LTA images (10x) from three mice stained by immunofluorescence for dystrophin. hDMD LTA dystrophin (far left panel) shows dystrophin staining in 5-month-old mice and serves as a positive control. LTA staining for dystrophin 5 months after injection (DelCH2+saline, center panel) shows almost no dystrophin expression. LTA staining for dystrophin 5 months after injection (DelCH2+TT744-3, right panel) shows approximately 40% dystrophin-positive fibers. These results show that treatment with TT744-3 restores dystrophin expression in 40% of fibers, supporting its therapeutic potential.

[0153] Example 4 Dystrophin expression after treatment in human patients As described above, a therapeutic exon skipping viral vector was created that contains three U7 snRNAs with antisense sequences targeting exons 6, 7, and 8 of the DMD gene. Following dose-finding studies in mice, the first human clinical trials were initiated after demonstrating non-toxicity in non-human primates. As discussed above, when the targeted exon is skipped, the functional form of dystrophin is expressed by this therapy.

[0154] While the present disclosure has been described in terms of specific embodiments, it will be understood that variations and modifications thereof may occur to those skilled in the art. Accordingly, limitations on the present disclosure should be construed as being set forth only in the appended claims.

[0155] All documents referenced in this application are incorporated herein by reference in their entirety.

Claims

1. A nucleic acid comprising: (a) a nucleotide sequence that contains at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 1 to 20; (b) a nucleotide sequence complementary to said nucleotide sequence comprising at least 80% identity to said sequence set forth in any one of SEQ ID NOs: 1 to 20; (c) a nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 1 to 20; (d) a nucleotide sequence complementary to the nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 1 to 20; and (e) a nucleotide sequence that binds to a sequence set forth in any one of SEQ ID NOs: 21 to 25.

2. A combination of at least two nucleotide sequences, said combination comprising: (i) a nucleotide sequence targeting the human DMD gene at exon 6, and a nucleotide sequence targeting the human DMD gene at exon 7; (ii) a nucleotide sequence targeting the human DMD gene at exon 6, and a nucleotide sequence targeting the human DMD gene at exon 8, and (iii) the nucleic acid of claim 1, comprising a nucleotide sequence targeting the human DMD gene at exon 7 and a nucleotide sequence targeting the human DMD gene at exon 8.

3. The nucleic acid of claim 1, comprising a combination of at least three nucleotide sequences, the combination comprising a nucleotide sequence targeting the human DMD gene at exon 6, a nucleotide sequence targeting the human DMD gene at exon 7, and a nucleotide sequence targeting the human DMD gene at exon 8.

4. (a) a nucleotide sequence that contains at least 70% identity to a sequence set forth in any one of SEQ ID NOs: 26 to 29; (b) a nucleotide sequence complementary to said nucleotide sequence comprising at least 70% identity to said sequence set forth in any one of SEQ ID NOs: 26-29; (c) a nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 26 to 29; and (d) a nucleotide sequence complementary to the nucleotide sequence comprising the sequence set forth in any one of SEQ ID NOs: 26-29. The nucleic acid of claim 1, comprising a nucleotide sequence selected from the group consisting of:

5. 2. An associated recombinant adenovirus (rAAV) comprising the nucleic acid of claim 1.

6. The rAAV of claim 5, wherein the rAAV is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, rAAV-anc80, rAAVrh.74, rAAVrh.8, rAAVrh.10, or rAAV-B1.

7. The rAAV of claim 5, wherein the rAAV is rAAV9.

8. The rAAV of claim 5, wherein the rAAV is self-complementary.

9. A composition comprising the nucleic acid of claim 1 and a carrier, diluent, excipient, and / or adjuvant.

10. A composition comprising the rAAV of claim 5 and a carrier, diluent, excipient, and / or adjuvant.

11. A composition described in claim 9 or 10 for treating, preventing, or ameliorating muscular dystrophy in a subject.

12. The composition of claim 11, configured to be administered via a systemic route.

13. The composition of claim 12, wherein the systemic route is by injection, infusion, or implantation.

14. The composition of claim 11, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

15. The composition of claim 11, wherein the level of functional dystrophin gene expression or protein expression in the subject's cells is increased after administration of the composition compared to the level of functional dystrophin gene expression or protein expression before administration of the composition.

16. The composition of claim 15, wherein expression of functional dystrophin in the cells is detected by measuring dystrophin protein levels by Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of the composition.

17. 12. The composition of claim 11, wherein the level of serum creatine kinase is decreased after administration of the composition compared to the level of serum creatine kinase before administration of the composition.

18. 12. The composition of claim 11, which results in improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject.

19. 6-minute walk test, stand-up test, ascend 4 steps test, ascend and descend 4 steps test, North Star Ambulatory Assessment (NSAA), forced vital capacity (FVC) test, timed 10 meter test, timed 100 meter test, hand held dynamometry (HHD) test, timed Up and Go test, Gross Motor Subtest 12. The composition of claim 11, wherein the progression of muscular dystrophy in the subject is delayed or muscle function in the subject is improved after administration of the composition as measured by a Bayley-III Scaled score, a Maximal Isometric Voluntary Contraction Test (MVICT), or a combination of two or more thereof.

20. The composition of claim 11 used in combination with a second or combination therapy.

21. 21. The composition of claim 20 for use in combination with a glucocorticoid.

22. For the preparation of a medicament for the treatment of muscular dystrophy, (a) a nucleic acid according to any one of claims 1 to 4, or (b) the rAAV according to any one of claims 5 to 8 Use of.

23. The use of claim 22, wherein the pharmaceutical agent is administered via a systemic route.

24. 24. The use according to claim 23, wherein the systemic route is by injection, infusion or implantation.

25. 23. The use according to claim 22, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

26. The use described in claim 22, wherein the level of functional dystrophin gene expression or protein expression in a subject's cells is increased after use of the pharmaceutical agent compared to the level of functional dystrophin gene expression or protein expression before use of the pharmaceutical agent.

27. 27. The use described in claim 26, wherein expression of functional dystrophin in the cells is detected by measuring the dystrophin protein levels by Western blot, immunofluorescence, or immunohistochemistry in biopsied muscles before and after administration of the pharmaceutical agent.

28. 23. The use of claim 22, wherein the level of serum creatine kinase is reduced after administration of the pharmaceutical agent compared to the level of serum creatine kinase before administration of the pharmaceutical agent.

29. The use of claim 22, wherein the pharmaceutical agent results in improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in a subject.

30. 23. The use of claim 22, wherein the progression of muscular dystrophy in the subject is slowed or muscle function in the subject is improved after administration of the pharmaceutical agent as measured by the 6 minute walk test, the time to stand test, the 4 step up test, the 4 step up and down test, the North Star Gait Assessment (NSAA), the forced vital capacity (FVC) test, the timed 10 meter test, the timed 100 meter test, the handheld dynamometry (HHD) test, the timed up and go test, the gross movement subtest equivalent (Bayley-III) score, the maximal isometric voluntary contraction test (MVICT), or a combination of two or more thereof.

31. 23. The use of claim 22, further comprising the use of a second therapy or a combination therapy.

32. 32. The use according to claim 31 , comprising the use of a glucocorticoid.

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