Restore Dystrophin Expression Research Articles

Link between MHC Fiber Type and Restoration of Dystrophin Expression and Key Components of the DAPC by Tricyclo-DNA-Mediated Exon Skipping.

Exon skipping mediated by tricyclo-DNA (tc-DNA) antisense oligonucleotides has been shown to induce significant levels of dystrophin restoration in mdx, a mouse model of Duchenne muscular dystrophy. This translates into significant improvement in key disease indicators in muscle, cardio-respiratory function, heart, and the CNS. Here we examine the relationship between muscle fiber type, based on myosin heavy chain (MHC) profile, and the ability of tc-DNA to restore not only dystrophin but also other members of the dystrophin-associated glycoprotein complex (DAPC). We first profiled this relationship in untreated mdx muscle, and we found that all fiber types support reversion events to a dystrophin-positive state, in an unbiased manner. Importantly, we show that only a small fraction of revertant fibers expressed other members of the DAPC. Immunoblot analysis of protein levels, however, revealed robust expression of these components, which failed to correctly localize to the sarcolemma. We then show that tc-DNA treatment leads to nearly all fibers expressing not only dystrophin but also other key components of the DAPC. Of significance, our work shows that MHC fiber type does not bias the expression of any of these important proteins. This work also highlights that the improved muscle physiology following tc-DNA treatment reported previously results from the complete restoration of the dystrophin complex in all MHCII fibers with equal efficiencies.

From gRNA Identification to the Restoration of Dystrophin Expression: A Dystrophin Gene Correction Strategy for Duchenne Muscular Dystrophy Mutations Using the CRISPR-Induced Deletion Method.

The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51-53 of the DMD gene.

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients.

Abstract 415: Transgenic Expression of Dimethylarginine Dimethylaminohydrolase Attenuates Exercise-induced Fatigue in Duchenne Muscular Dystrophy Carrier Mice

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in dystrophin and characterized by muscle degeneration, cardiomyopathy, and impaired muscle nitric oxide (NO) production that disrupts muscle blood flow regulation and leads to excessive post-exercise fatigue. Interestingly, circulating levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in dystrophin-deficient subjects. Therefore, we hypothesized that excessive circulating ADMA impairs muscle NO production and thus negatively impacts exercise tolerance in DMD. The objectives of this study were to determine whether increased circulating ADMA is itself sufficient to affect exercise performance, and whether transgenic modulation of ADMA metabolism could improve exercise-induced fatigue in the mdx mouse model of DMD. Although infusion of exogenous ADMA did impair exercise performance in healthy, wild-type mice, transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), the enzyme that degrades ADMA, did not affect exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. This improvement in exercise tolerance was associated with reduced heart weight, improved cardiac contractile function, and improved chronotropic responsiveness to beta-adrenergic stimulation in DDAH-transgenic female mdx carriers. In contrast, DDAH transgene expression did not significantly affect skeletal muscle pathology in female mdx carriers. We conclude that DDAH transgene expression improves exercise tolerance in dystrophin-heterozygous females, possibly by limiting pathological cardiac remodeling and helping to maintain heart performance. These findings emphasize the importance of methylated arginines to the development of cardiomyopathy in female DMD carriers, and have interesting implications for current genetic therapies that may only partially restore dystrophin expression in the hearts of male DMD patients.

CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Lentiviral vectors can be used for full-length dystrophin gene therapy

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a ‘template-switching’ lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

Muscle stem cells (MuSCs) hold great therapeutic potential for muscle genetic disorders, such as Duchenne muscular dystrophy (DMD). The CRISP/Cas9-based genome editing is a promising technology for correcting genetic alterations in mutant genes. In this study, we used fibrin-gel culture system to selectively expand MuSCs from crude skeletal muscle cells of mdx mice, a mouse model of DMD. By CRISP/Cas9-based genome editing, we corrected the dystrophin mutation in expanded MuSCs and restored the skeletal muscle dystrophin expression upon transplantation in mdx mice. Our studies established a reliable and feasible platform for gene correction in MuSCs by genome editing, thus greatly advancing tissue stem cell-based therapies for DMD and other muscle disorders.

Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs.

Duchenne muscular dystrophy is the most common genetic muscular dystrophy. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and to progressive degeneration of skeletal muscle. We have demonstrated that the exon skipping method safely and efficiently brings to the expression of a functional dystrophin in dystrophic CD133+ cells injected scid/mdx mice. Golden Retriever muscular dystrophic (GRMD) dogs represent the best preclinical model of Duchenne muscular dystrophy, mimicking the human pathology in genotypic and phenotypic aspects. Here, we assess the capacity of intra-arterial delivered autologous engineered canine CD133+ cells of restoring dystrophin expression in Golden Retriever muscular dystrophy. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected Golden Retriever muscular dystrophy dogs. The occurrence of T-cell response in three Golden Retriever muscular dystrophy dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophin.

In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

G.O.18 - Restoration of dystrophin expression and motor function in using exosomes-based non-immunogenic genetic therapy

G.O.18 - Restoration of dystrophin expression and motor function in using exosomes-based non-immunogenic genetic therapy

Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is one of the most common inherited genetic diseases and is caused by mutations to the DMD gene that encodes the dystrophin protein. Recent advances in genome editing and gene therapy offer hope for the development of potential therapeutics. Truncated versions of the DMD gene can be delivered to the affected tissues with viral vectors and show promising results in a variety of animal models. Genome editing with the CRISPR/Cas9 system has recently been used to restore dystrophin expression by deleting one or more exons of the DMD gene in patient cells and in a mouse model that led to functional improvement of muscle strength. Exon skipping with oligonucleotides has been successful in several animal models and evaluated in multiple clinical trials. Next-generation oligonucleotide formulations offer significant promise to build on these results. All these approaches to restoring dystrophin expression are encouraging, but many hurdles remain. This review summarizes the current state of these technologies and summarizes considerations for their future development.

Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors.

Duchenne muscular dystrophy (DMD), an incurable and a progressive muscle wasting disease, is caused by the absence of dystrophin protein, leading to recurrent muscle fiber damage during contraction. The inflammatory response to fiber damage is a compelling candidate mechanism for disease exacerbation. The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects. Recent reports show the therapeutic potential of inhibiting or enhancing pro- or anti-inflammatory factors released from DMD skeletal muscles, resulting in significant recovery from muscle atrophy and dysfunction. We discuss and review the recent findings of DMD inflammation and opportunities for drug development targeting specific releasing factors from skeletal muscles. It has been speculated that nonsteroidal anti-inflammatory drugs targeting specific inflammatory factors are more effective and have less side effects for DMD compared with steroidal drugs. For example, calcium channels, reactive oxygen species, and nuclear factor-κB signaling factors are the most promising targets as master regulators of inflammatory response in DMD skeletal muscles. If they are combined with an oligonucleotide-based exon skipping therapy to restore dystrophin expression, the anti-inflammatory drug therapies may address the present therapeutic limitation of low efficiency for DMD.

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Activation of Wnt3a signaling promotes myogenic differentiation of mesenchymal stem cells in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling. MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting. Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation. Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD.

504. Restoration of Dystrophin Expression by Gene Editing with S. aureus Cas9 in Models of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common fatal genetic disease characterized by progressive muscle wasting, loss of ambulation, and typically death in the third decade of life due to respiratory and cardiac complications. DMD results from deleterious mutations in the dystrophin gene that disrupts the translational reading frame and causes a complete lack of dystrophin protein. Becker muscular dystrophy (BMD) is similar to DMD in that it is the result of deletions in the dystrophin gene. However these deletions maintain the translational reading frame and result in the production of an internally truncated but partially functional dystrophin protein. The BMD phenotype is typically less severe than DMD, and thus converting DMD to a BMD-like phenotype by restoring the dystrophin reading frame is a widely explored therapeutic strategy. CRISPR/Cas9 can target precise loci to make specific DNA sequence changes in the genome. We have previously used S. pyogenes Cas9 (SpCas9) to restore dystrophin expression in immortalized myoblasts from DMD patients by deleting dystrophin exon 51 to repair the disrupted reading frame. A promising therapeutic application of this strategy involves in vivo viral delivery of the CRISPR/Cas9 system by AAV, however AAV cannot efficiently package the large SpCas9 gene along with full size promoters. Therefore we have made use of AAV delivery of the smaller S. aureus Cas9 (SaCas9) to delete exon 23 in the mouse dystrophin gene in vivo in the mdx mouse model of DMD and showed restored dystrophin expression and functional recovery. Here we are continuing this work by preparing a SaCas9 system targeted to the human dystrophin gene. gRNAs were designed to target the intronic regions flanking exon 51 and tested in vitro in HEK293T cells as well as immortalized DMD patient myoblasts that are correctable by removal of exon 51. The expected deletion was confirmed by PCR and sequencing of the genomic DNA and dystrophin cDNA. Western blot of lysates from differentiated cells confirmed restoration of dystrophin protein expression. We have also demonstrated exon 51 deletion in vivo following AAV delivery of this CRISPR/Cas9 system to the muscles of a mouse model containing the full length human dystrophin gene. Ongoing work involves testing this approach in a novel dystrophic mouse model carrying a mutated version of the human dystrophin gene that is correctable by exon 51 deletion. This work is important to the continued development of a translational strategy for gene editing as a potentially curative treatment for DMD.

482. Local and Systemic Gene Editing in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a highly prevalent genetic disorder leading to muscle wasting, loss of ambulation, and premature death by the third decade of life. DMD is caused by gene deletions, duplications, or nonsense mutations leading to the loss of dystrophin, an essential musculoskeletal protein. Gene therapy has held tremendous promise for the treatment of monogenic disorders, yet an effective gene replacement therapy has been elusive. Genome editing has been established as a potential approach to correct the dystrophin gene in cultured human cells by excising non-essential exons from the dystrophin gene producing a shortened yet in-frame dystrophin protein (1). In contrast to gene replacement therapy, genome editing repairs the causative mutation in the native genomic context with the potential for permanent gene repair. Recently, we and others have demonstrated that CRISPR/Cas9 genome editing in neonatal and adult mouse models of DMD restores dystrophin expression, improves muscle biochemistry, and strengthens muscle force generation (2-4). However, further optimization of the approach for systemic gene correction is still needed.To target the dystrophin gene in the mdx mouse, the 3.2kb S. aureus Cas9 and two guide RNAs (gRNA) targeting intronic regions surrounding exon 23 were packaged into an adeno-associated virus (AAV). Double stranded breaks created by Cas9 were repaired with the relatively efficient non-homologous end joining pathway leading to excision of the nonsense mutation in exon 23. AAV vectors were injected intramuscularly into the tibialis anterior muscle in adult mice and intravenously into neonatal mice and adult mice and characterized for gene deletions, dystrophin restoration, and improvements in muscle physiology.Local correction restored overall dystrophin levels to 8% by western blot with 67% of muscle fibers positive for dystrophin by immunofluorescence. Repeated cycles of eccentric contraction showed 60% resistance to damage compared to sham-treated mice. Systemic correction was achieved through IP injection into P2 neonates with dystrophin restoration primarily in the cardiac muscle and skeletal muscle surrounding the peritoneal cavity. IV administration in adult mice restored dystrophin expression in the cardiac muscle. Improved systemic distribution and correction was achieved with intravenous administration into P2 neonates with AAV8 or AAV9 (Fig. 1Fig. 1).Figure 1Intravenously administered AAV restores dystrophin in cardiac and skeletal muscle in P2 neonates after 8 weeks of treatement. a) Cardiac muscle. b) Tibialis anterior muscle. Scale bar = 200µm, green - dystrophin, blue - DAPI.View Large Image | Download PowerPoint SlideThis study establishes CRISPR/Cas9-based gene editing as a promising approach for the treatment of DMD. Ongoing work to improve the efficiency and safety of in vivo gene editing includes the incorporation of muscle specific promoters, minimization of vector packaging, and optimization of AAV serotype.1. Ousterout et al. Nat Comm 2015. | 2. Nelson et al. Science 2015 | 3. Tabebordbar et al. Science 2015 | 4. Long et al. Science 2015

317. Screening S. Aureus CRISPR-Cas9 Paired-Guide RNAs for Efficient Targeted Deletion in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a recessive X-linked neuromuscular disorder that results in progressive muscle degeneration and premature death. Most patients have exonic deletions in the dystrophin gene that result in a frameshift and nonfunctional protein. In contrast, Becker muscular dystrophy (BMD) patients carry a range of exonic deletions in dystrophin that do not disrupt the reading frame, leading to a much milder disease phenotype. Thus, multiplex CRISPR/Cas9 targeted deletions that restore the reading frame could convert DMD genotypes into BMD-like genotypes and potentially treat this disease. Previously, this strategy has been demonstrated in vitro with zinc finger nucleases, TALENs, and S. pyogenes Cas9 leading to restoration of dystrophin expression in DMD patient myoblasts. However, these genome-editing enzymes are limited by the difficulty of delivering large transgenes with viral vectors in vivo. Alternatively, the smaller Cas9 ortholog from S. aureus (SaCas9) can be packaged with paired sgRNAs in an all-in-one AAV vector for in vivo gene therapy. Recently, three groups have demonstrated that AAV-SaCas9 can mediate targeted deletions in mdx mice and restore dystrophin expression. As a first step towards developing a genome editing therapeutic for DMD, we conducted an efficiency screen to identify highly active paired sgRNAs for targeted deletion of exon 51 of the dystrophin gene. First, a sensitive, digital droplet PCR assay to quantify exon 51 deletion was validated with DMD patient samples. An initial pilot study of 15 sgRNAs identified a pair (01+09) that mediated 18% Exon 51 deletion in HEK293T cells three days post-transfection. Next, from the set of 10,553 21-nt SaCas9 guides targeting human DMD introns 50 and 51, we selected 53 sgRNAs (675 pairs) that met the following filtering requirements: an endogenous 5’ G, a 3’ T in the NNGRR(T) PAM, cross-reactivity with the non-human primate (NHP) genome, and no off-by-1 or off-by-2 mismatch sites in the human genome. These filters were selected to improve U6 promoter expression, improve SaCas9 cleavage efficiency, enable pre-clinical animal model studies, and minimize off-target editing concerns, respectively. Notably, the NHP cross-reactivity requirement skewed the targeted deletion lengths towards larger sizes, all greater than 12.4kb. In order to test smaller deletions, an additional 174 pairs of human-only sgRNAs were designed for deletion lengths of 0.8-14kb. From the 850 guide pairs screened, 78 pairs with a deletion efficiency Z-score >1.5 were selected for follow-up validation. Interestingly, a few individual sgRNAs consistently appeared among the top performing pairs, regardless of whether they were paired with a generally less effective partner sgRNA. The top hit (guides 68+84), a human-only guide pair with a deletion size of 2.3kb, demonstrated a reproducible deletion efficiency of 32%. The 6 best human-only guide pairs and the 6 best NHP cross-reactive guide pairs have been cloned together with SaCas9 in all-in-one AAV vectors for testing in ex vivo and in vivo models of DMD.