Annual Meeting of American Society of Gene Therapy - May 2014

Ex Vivo Gene Therapy of Duchenne Muscular Dystrophy Using iPSCs from Duchenne Muscular Dystrophy Patients

Chantale Maltais, William-Édouard Gravel, Carl Lebel, Jacques P. Tremblay. Molecular Medecine, Laval University, Québec, Canada

Duchenne Muscular Dystrophy is a hereditary myopathy characterized by a progressive muscular degeneration, due to the absence of dystrophin. Among the possible therapies there is the autologous transplantation of myoblasts derived from induced pluripotent stem cells (hiPSCs) derived from the dystrophic patient himself. Indeed, hiPSCs represents a renewable source of myogenic autologous cells and would thus not induce an immune response of the patient against the transplanted cells. However, there is currently no effective and reproducible protocol of differentiation of hiPSCs into myoblasts that results in considerable transplantation success. We aim to induce myogenesis of hiPSCs with recombinant transcription factor proteins fused with a cell penetrating peptide, i.e., Tat. These recombinant proteins were produced in bacteria and purified. Our results showed that these transcription factors were able to enter into mesenchymal-like cells. The capacity of these recombinant proteins to induce a myogenic differentiation is currently under study. When these therapeutic approaches will be established, they could be clinically applied to treat dystrophic patients.

 A Novel Utrophin-Based Genome Engineering Strategy for Duchenne Muscular Dystrophy Utilizing Recombinases and CRISPR/Cas9

Jonathan M. Geisinger, Christopher B. Bjornson, Tawny L. Neal, Michele P. Calos. Genetics, Stanford University School of Medicine, Stanford, CA

Duchenne muscular dystrophy (DMD) is a degenerative disease caused by mutations in the dystrophin gene that ultimately results in the absence of the functional protein. The number of different mutations, coupled with the sheer size of the dystrophin gene, makes DMD a particularly difficult disease for which to develop treatments. While there has been some promise with exon-skipping-based treatments, greatly shortened micro-dystrophin cDNAs, and myoblast transplantation, there still remains the concern that expression of wild-type dystrophin in a DMD patient will be immunogenic and cause corrected fibers to be destroyed. However, another gene, utrophin, has significant homology to dystrophin, and is not mutated in DMD. Utrophin is widely expressed at the sarcolemma during muscle development, where it fulfills the same role as dystrophin. However, utrophin is restricted to the neuromuscular junction in adult muscle. Additionally, the utrophin mRNA has been shown to be translationally repressed by several miRNAs acting on the 3' untranslated region (3'UTR). Thus, we sought to develop a genome engineering-based strategy for a cellular therapy in which the patient's own cells would be modified to produce enough utrophin to repair the dystrophin deficiency. As a proof-of-principle, we chose to use induced pluripotent stem cells (iPSCs) from the mdx mouse, which is dystrophin-deficient. We have previously shown that we can reprogram fibroblasts to iPSCs bearing a single copy of our reprogramming cassette, subsequently introduce a transgene, and then excise the reprogramming cassette using a combination of the phiC31 and Bxb1 integrases and Cre resolvase. In this work, we chose peroxisome proliferator-activated receptor gamma coactivator-1 alpha as our transgene because it is a known upregulator of utrophin and has other beneficial effects. Subsequently, we utilize the CRISPR/Cas9 system to replace the 3'UTR of utrophin with a GFP-luciferase reporter construct with a different 3'UTR in frame with the utrophin gene, both to relieve translational repression and facilitate tracking of utrophin expression. This strategy is also applicable to human cells. These data demonstrate the feasibility of using genome engineering to exploit and modify transcriptional and translational regulation to generate a cell-based therapy for Duchenne muscular dystrophy.

Engineered Nuclease Mediated Genetic Correction in iPSCs Derived From Duchenne Muscular Dystrophy Patient

Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Takashi Yamamoto, Knut Woltjen, Akira Watanabe, Hidetoshi Sakurai, Shinya Yamanaka, Akitsu Hotta. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; JSPS Research Fellow, Tokyo, Japan; Hiroshima University, Hiroshima, Japan; Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan; Yamanaka iPS Cell Special Project, JST, Kyoto, Japan; Gladstone Institute of Cardiovascular Disease, San Francisco; PRESTO, JST, Kawaguch, Japan

Duchenne Muscular Dystrophy (DMD) is the most common and severe inherited neuromuscular disease caused by the loss-of-function mutations in Dystrophin gene. Currently, no effective treatment available for DMD patients, and the large size of the Dystrophin gene hampers the delivery for gene augmentation therapy. Exon skipping to modulate mRNA splicing patterns using antisense oligonucleotide is a promising approach, however, the effect of antisense oligos is transient. Recently, targeted genome editing using engineered nucleases, such as TAL effector nucleases (TALENs) and CRISPR/Cas9, have been demonstrated to be effective and efficient to modify the target region of the genome. These promising technologies suggest the potential for the development of gene correction therapy for DMD.
In this study, we took advantages of patient-derived induced pluripotent stem cells (iPSCs) as a platform for testing various gene correction approaches to demonstrate the proof-of-concept for DMD gene thrapy. Firstly, we derived integration-free iPS cell lines from a DMD patient who lacks the exon 44 in theDystrophin gene. To correct the truncated dystrophin, we devised two approaches. One is to restore the reading frame by re-insertion of small deletion or insertion by engineered nucleases without a donor template. Another is to knock-in the deleted exon 44 with a repair template.
We constructed several TALENs and CRISPR/gRNAs to target the same site in exon 45, and demonstated high recombination efficiencies by the both systems. Isolated clones were then analized by Sanger sequencing to confirm the frame shift, and by PCR and Southern blotting to identify the knock-in clones. Since recent reports suggest the high frequencies of off-target mutagenesis by CRISPR system, we looked at the candidate off-target sites by T7 Endonuclease I assay and also examined the genome-wide protein-coding sequences by exome sequencing to assess the risk of off-target mutagenesis. So far, we obaserved no severe off-target mutagenesis introduced by both TALEN and CRISPR treatments. Finally, we differentiated the corrected DMD-iPSC clones into skeltal muscle cells in vitro by forced expression of MyoD or in vivo by injecting cells into NOD/SCID mice to form teratoma. We successfully detected the expression of dystrophin protein from two correction approaches. Our "genomic surgery" approaches using TALEN or CRISPR is effective in patient-derived iPSCs, and gene-corrected iPSCs hold a promise to serve as a cell source for cell transplantation therapies for DMD.

Parathyroid Hormone Administration and Parathyroid Hormone Type 1 Receptor Activation Accelerate Muscle Differentiation and Muscle Regeneration in Muscular Dystrophy

Shigemi Kimura, Kowasi Yoshioka. Department of Child Development, Kumamoto University Graduate School, Kumamoto, Japan

We have established genetically engineered embryonic stem cells (ZHTc6-MyoD) that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD. After removal of tetracycline, almost all of the cells differentiated into myotubes, but a few cells later re-formed colonies. DNA microarray analysis showed that the expression of parathyroid hormone type 1 receptor (PTH1R) in the colonies that differentiated into muscle lineage cells is higher in the colonies of undifferentiated ZHTc6-MyoD cells. In addition to the PTH1R required for the muscle differentiation of ZHTc6-MyoD cells, parathyroid hormone (1-34) [PTH (1-34)] further accelerated muscle differentiation. In human and mouse skeletal muscle tissue, most PTH1R-positive cells also expressed Pax7 and CD34, indicating that these cells originated from satellite cells.
The administration of 60 μg/kg per day of PTH (1-34) to mdx mice (dystrophin-deficient mice) was started at 4 weeks of age. The four limbs hanging wire test was performed on day 10. The mean hang time of PTH-treated mdx mice (n=6) was 132±61.6 sec. In contrast, the mean hang time of saline-injected mdx mice (n=7) was significantly shorter, at 49.5±44.9 sec (P=0.03). The treadmill test was performed on day 43. Mice were forced to run on a treadmill with the belt parallel to the floor at a speed of 25 cm/sec for 100 min. The distance that they ran was recorded and compared. All C57BL/10 mice (n=6) ran for 100 min, and the mean distance was 1486±7.4 m. PHT-treated mdx (n=6) mice ran a mean distance of 843.6±352 m, although one of the PHT-treated mice did run for 100 min. None of the saline-injected mdx mice (n=7) ran for 100 min, and the mean distance covered was 425±190 m. The C57BL/10 mice (normal mice) ran significantly longer than the saline-injected mdx mice (P<0.01), and the saline-injected mdx mice ran significantly shorter than the PTH-treated mdx mice (P=0.03). The mice were sacrificed on day 60 for histological studies. The diameters of myofibres of C57BL/10 (n=5), saline-injected mdx (n=7), and PTH-treated mdx mice (n=6) at the age of 13 weeks were 33.62±10.6, 25.41±10.4, and 31.16±12.9 μm, respectively. The saline-injected mdx mice had smaller round fibres (regenerative fibres) as compared to the PTH-treated mdx mice. There were significant differences (P<0.01) between the fibre widths of the groups. The percentage of myofibres with central nuclei in C57BL/10 (n=5), saline-injected mdx (n=7) and PTH-treated mdx mice (n=6) were 0.34±0.2, 93.2±2.0 and 83.01±4.0 % respectively. There were also significant differences (P<0.01) between the groups. These results showed that there was improvement in the histologic findings in PTH-treated mdx mice. We, therefore, concluded that PTH administration improved muscle weakness by enhancing muscle fibre regeneration. It appears that PTH administration may be effective in the treatment of muscle diseases such as Duchenne muscular dystrophy. This is the first report that PTH1R and PTH accelerated the differentiation of muscle lineage cells.

Microdystrophin and Follistatin Combinatorial Gene Delivery To Treat a Severe Mouse Model of Duchenne Muscular Dystrophy

Timothy D. Colgan, Kate T. Murphy, Hongwei Qian, Gordon S. Lynch, Jeff S. Chamberlain, Paul Gregorevic. Laboratory for Muscle Research & Therapeutics Development, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; Basic and Clinical Myology Laboratory, The University of Melbourne, Melbourne, Victoria, Australia; Department of Neurology, The University of Washington School of Medicine, Seattle, WA

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder that results in ambulatory reduction of affected children and premature death from cardiac and/or respiratory failure. DMD is caused by a variety of mutations that result in the loss of dystrophin protein. As DMD is a single gene disorder, gene therapies have been pursued with the intention of restoring dystrophin expression in order to ameliorate the dystrophic pathology. Using recombinant serotype 6 adeno-associated vectors (rAAV6), an expression cassette encoding for a miniaturized version of dystrophin (microdystrophin) was systemically delivered to the musculature of the dystrophin-/-:utrophin-/- (double knockout: dko) mouse model of DMD. Though microdystrophin expression increased muscle strength and longevity of treated dystrophic mice when compared to their untreated littermates, wild-type levels of strength and lifespan were not obtained. Alternate strategies that increase muscle mass and strength have subsequently been explored. Follistatin binds and inhibits TGF-β ligands myostatin and activin, negative regulators of muscle mass. Follistatin expression has been reported to increase muscle mass and strength. We tested the hypothesis that the co-delivery of follistatin and microdystrophin expression cassettes would ameliorate the dystrophic pathology to a greater extent than either gene alone.
We administered rAAV6:FST317 (follistatin) to the hindlimb muscles of C57Bl/6 mice, and the mdx (dystrophin-/-) and dko models of dystrophinopathy. We observed treatment increased follistatin expression and mass in all strains, but that the expression of follistatin and the hypertrophic response was diminished in severely dystrophic mice. We hypothesize that this differential expression and hypertrophic effect could be accounted for by fibre turnover in severely dystrophic mice. We identified that dystrophic muscles receiving a combinatorial delivery had more follistatin than those treated with follistatin alone. We sought to investigate whether our observations from treating at the single-limb level would translate to treating the entire musculature. To test our hypothesis cohorts of dko mice received an intravenous injection of either rAAV6:microdystrophin, rAAV6:FST317 or both vectors. Five weeks post-injection we observed improved muscle mass and body condition scores in mice receiving the combinatorial treatment. The physiological and therapeutic benefits attributed to initially stabilizing the muscle fibre before inducing muscle growth are also currently being interrogated and will be reported on. These results highlight factors to consider when developing therapeutics, including that the efficacy of potential interventions is influenced by the severity of the disease model. Our findings also emphasize the potential for a combinatorial gene therapy to ameliorate dystrophic pathology.

Immune Tolerance Induction in Canine X-Linked Muscular Dystrophy With rAAV9-Microdystrophin Transduction

Hiromi Hayashita-Kinoh, Yuko Nitahara-Kasahara, Hironori Okada, Tomoko Chiyo, Naoko Yugeta, Takashi Okada, Shin'ichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan

Background: Duchenne muscular dystrophy (DMD) is a congenital disease causing progressive deterioration of skeletal and cardiac muscles because of mutations in the dystrophin gene. We have previously reported that local injection of rAAV2 or rAAV8 to canine skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Here we used pregnant dog and fetuses of the CXMDJ to investigate two strategy of inducing immune tolerance to the rAAV as well as the muscle transduction profiles of rAAV9-microdystrophin.
Methods: For fetal transduction, we tried two methods to induce immune tolerance against rAAV. First, direct injection of 1x1012 of AAV9-microdystrophin and 1x1011 of rAAV9-Luc into amniotic fluid at embryonic day 35 fetuses (oral ingestion of rAAV). Second, pregnant CXMDJ heterozygote with embryonic day 30 fetuses was injected with 1x1012 of rAAV-CMV-microdystrophin along with 1x1011 of rAAV-CAG-Luciferase by intravenous injection (trans-placental rAAV transduction). The yolk sacs and umbilical cords were sampled at delivery, and then transduced rAAV copy numbers were estimated by qPCR. To examine the immune tolerance to the rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. To expect systemic microdystrophin expression, we additionally injected rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks old dystrophic dog. Skeletal muscles (tibialis anterior) of the rAAV-injected animals were collected by biopsy for expression analysis. In addition, rAAV-injected CXMDJ and non-injected CXMDJ were compared each other to assess gait function and lameness in the hind limb.
Results: To rAAV injection to amniotic fluid, higher amount of AAV genome was detected in the umbilical cord, whereas in yolk sack in trans-placental rAAV transduction. Expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in both of the rAAV oral ingestion and trans-placental transduced dogs, suggesting the successful induction of immune tolerance against rAAV. rAAV-derived microdystrophin expression were confirmed by immunohistochemistry in the transduced affected dogs from additional rAAV injection. Monthly analysis of the transduced dystrophic dogs demonstrated superior gait function to non-injected age-matched CXMDJ.
Conclusion: Our results demonstrate that induction of immune tolerance against rAAV with long-term transgene expression can be achieved by both of the direct injection of rAAV to amniotic fluid and intravenous injection of rAAV into pregnant dog. These strategies would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated fetal gene delivery strategies. Furthermore, we plan immune tolerance induction after the delivery to meet clinical settings.

Genetic Correction of Duchenne Muscular Dystrophy by Multiplex CRISPR/Cas9-Based Gene Editing

David G. Ousterout, Ami M. Kabadi, Pratiksha I. Thakore, Charles A. Gersbach. Department of Biomedical Engineering, Duke University, Durham, NC; Institute for Genome Sciences and Policy, Duke University, Durham, NC; Department of Orthopaedic Surgery, Duke University, Durham, NC

The recently described CRISPR/Cas9 gene editing platform presents a powerful tool to correct the genetic basis of hereditary diseases. CRISPR/Cas9 systems create targeted genomic modifications through a protein-RNA complex consisting of the Cas9 nuclease and a co-expressed single guide RNA (sgRNA) molecule. This complex can be readily programmed to target a 20 bp complementary sequence at any chromosomal locus. We exploited the multiplex gene editing properties of the CRISPR/Cas9 system to restore the expression of the dystrophin gene that is mutated in Duchenne muscular dystrophy (DMD). Multiplexed sgRNAs were targeted to sequences flanking the exons within the mutational hotspot at exons 45-55 to generate large deletions of one or more exons from the genomic DNA (Figure 1a). These deletions were chosen to correct the dystrophin reading frame and restore functional dystrophin protein expression. To demonstrate the utility of this method, skeletal myoblasts from DMD patients were treated with sgRNAs and Cas9 to correct patient-specific mutations. We show that gene editing by CRISPR/Cas9 resulted in restored expression of dystrophin mRNA transcripts and protein by deletion of either exon 51 (Figure 1b) or exons 45-55 (Figure 1c). Furthermore, CRISPR/Cas9 gene editing did not have significant toxic or off-target effects in human cells as observed by stable gene editing frequencies, minimal cytotoxicity of several sgRNAs, and limited mutagenesis of predicted off-target sites for a selected sgRNA. Human dystrophin was detectedin vivo following transplantation of genetically corrected patient cells into immunodeficient mice (Figure 1d). By genetically deleting exons, this strategy introduces a uniform genetic change resulting in a predictable protein product. This is in contrast to our previous genome editing approach that corrected the reading frame by introducing random insertions and deletions within exons through DNA repair by non-homologous end joining1. Significantly, the unique multiplex gene editing capabilities of the CRISPR/Cas9 system enabled the efficient generation of large deletions of this mutational hotspot region that can correct up to 62% of patient mutations through a universal gene correction strategy.

Optimised AAV-Microdystrophin Gene Therapy in the GRMD Dog Model of Duchenne Muscular Dystrophy: Intravenous Delivery, Long-Term Expression, Functional Improvement and Absence of Adverse Cellular Immune Reactions

George Dickson, Caroline Le Guiner, Marie Montus, Laurent Servais, Yan Cherel, Jean-Yves Hogrel, Carlier Pierre, Carole Masurier, Oumeya Adjali, Fréderic Barnay-Toutain, Takis Athanasopoulos, Taeyoung Koo, Alberto Malerba, Fulvio Mavilio, Thomas Voit, Philippe Moullier. Royal Holloway - University of London, London, United Kingdom; Atlantic Gene Therapy Institute, Nantes, France; Genethon Laboratories, Evry, France; AFM Institut de Myologie, Paris, France

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there are no strongly effective treatments but new gene-based therapies are currently being developed with particular advances in using exon skipping and other RNA-based approaches, conventional gene replacement strategies, and cell-based gene therapy. In the case of DMD, putting aside exon skipping therapy, a number of groups are testing gene therapy with adeno-associated virus vectors expressing engineered microdystrophins (AAV-MDs). In our hands, highly sequence optimised AAV-MDs are available for use in mouse, dog and ultimately humans, expressed using a strong synthetic promoter specific for skeletal and cardiac muscle cells have been tested in detail in mdx mice, and in the GRMD dog. Here we report the outcome of a detail study of AAV2/8-canine MD delivery (5x1012 vg/kg) by isolated limb perfusion in dogs from ages 3 months to 6 months. No immunosuppression was applied. In skeletal muscles of the treated limb at 3-months after vector administration we report widespread vector biodistribution, and sustained high level MD expression (up to 95% of fibres). In addition the treated limb exhibited very significantly improved parameters of muscle turnover (regenerative fibres), fibrosis (Collagen type I), 1H-MRI and 31P-NMR spectroscopy, and muscle strength. In the context of immune parameters, AAV administration elicited anti-AAV2/8 antibodies and a transient elevation of serum cytokines, but no evidence of cellular immunity (IFNγ elispot) to microdystrophin or to the AAV vector was observed. This study strongly supports the hypothesis that the current optimised microdystrophin is highly functional, not only in mice, but also in a large animal model, and that AAV2/8 vector delivery results in sustained expression of microdystrophin without adverse immune responses. The current optimised AAV-microdystrophin configuration thus lays a sound basis for a translation programme towards clinical trials in DMD patients. Current studies are focussed on systemic intravenous delivery of AAV-canine MD vectors body-wide in the GRMD model.

Successful Use of Out-of-Frame Exon 2 Skipping Induces IRES-Driven Expression of the N-Truncated Dystrophin Isoform: Promising Approach for Treating Other 5' Dystrophin Mutations

Nicolas Wein, Adeline Vulin, Maria S. Falzanaro, Cristina Al-Khalili Szigyarto, Baijayanta Maiti, Andrew Findlay, Kristin N. Heller, Mathias Uhlen, Baskar Bakthavachalu, Sonia Messina, Giuseppe L. Vita, Francesca Gualandi, Steve D. Wilton, Lin Yang, Diane M. Dunn, Daniel Schoenberg, Robert B. Weiss, Michael T. Howard, Alessandra Ferlini, Kevin M. Flanigan. The Center for Gene Therapy and the Departments of Pediatrics and Neurology, Nationwide Children's Hospital and The Ohio State University, Columbus, OH; Section of Microbiology and Medical Genetics, Department of Medical Sciences, University of Ferrara, Ferrara, Italy; Department of Proteomics and Nanobiotechnology, KTH-Royal Institute of Technology, Stockholm, Sweden; Department of Neurology, Washington University School of Medicine, St Louis, MO; Center for RNA Biology and Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH; Department of Neuroscience, University of Messina, Messina, Italy; Centre for Comparative Genomics, Murdoch University, Perth, Australia; Department of Computer Science, University of Kentucky, Lexington, KY; Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, UT

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to the most common childhood muscle disease, the severe and progressive Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of the DMD gene typically do not result in Duchenne muscular dystrophy, but instead result in milder dystrophinopathy syndromes as originally observed in patients with very mild clinical features despite nonsense mutations in exon 1. We have previously shown that amelioration of disease severity result from the expression of a highly functional N-truncated dystrophin beginning in exon 6 of the DMD. Here we demonstrate that this protein represents a novel dystrophin isoform resulting from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. In vitro studies with bicistronic reporter assays demonstrate translation at levels approximately 60% of the well-known viral IRES (eMCV), suggesting a relatively strong activity. Activity in humans was confirmed in patient muscle tissues using ribosome profiling and mass-spectrometric peptide sequencing. The resultant N-truncated dystrophin protein produced from this IRES, lacks the first calponin homology domain of the canonical actin binding domain 1. Nevertheless, it is highly functional, raising the possibility of the therapeutic use of this isoform. We use a novel out-of-frame exon-skipping approach to generate a truncated reading frame upstream of the IRES in both patient-derived cell lines and in a new DMD mouse model, leading to synthesis of a functional N-truncated isoform. In the mouse, this expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. Together these results support a novel therapeutic approach for patients with mutations within the 5' exons of DMD.

Novel Formulation Enhances Nucleic Acid Cargoes Delivery To Muscle

Gang Han, Xianjun Gao, Qingsong Wang, Limin Cao, Ben Gu, Haifang Yin. Research Center of Basic Medical Science, Tianjin Medical University, Tianjin, China

Duchenne muscular dystrophy (DMD) is one of the most common and severe forms of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Currently there is no treatment available in clinic. Although antisense oligonucleotides (AOs) – mediated exon skipping approach has shown great promise for DMD patients from encouraging phase IIb clinical trials, the lack of clear success of recent GSK phase III trials further underline the importance of developing ways to further enhance the systemic delivery efficiency of current AOs.
To address this issue, in our current study, we screened a number of formulations, which have been extensively applied in clinic with high safety profiles, in mdx mice to identify effective and safe delivery systemis for AOs currently in clinical trials. From local intramuscular screening in mdx mice, we identified four top candidates which can increase the exon skipping efficiency of phosphorodiamidate morpholino oligomer (PMO), with up to 6 fold increase in dystrophin expression in tibialis anterior (TA) treated with formulation F9 compared with that of saline. Subsequent systemic administration studies revealed that F9 can significantly enhance the exon skipping efficacy of PMOs across different dosing regimens. Notably, approximately 6.5 fold higher level of dystrophin was restored with PMOs in the presence of formulation F9 compared to saline at different tested dosing regimens. Further investigation indicated that the increased exon skipping efficiency of PMOs is attributed to the enhanced delivery of PMOs to muscle conferred by F9. More importantly, F9 not only enhanced the delivery of PMOs but also applied to other nucleic acid cargoes e.g. 2'-O-methylphosphorothioate RNA (2'OMe RNA) and siRNA. Longer-term study further established the safety and efficacy of F9 in enhancing different cargoes to muscle. Our findings demonstrate the potential of formulation F9 as an effective and safe formulation for cargoes in DMD and other muscle-related disorders.

Production in Bacteria and Purification of ZFN Proteins Targeting Exon 50 of the Human Dystrophin Gene

Jean-Paul Iyombe, Joël Rousseau, Jacques P. Tremblay. Laboratoire de Biologie Moléculaire, Faculté des Sciences Pharmaceutiques, Université de Kinshasa, Kinshasa, Congo; Médecine Moléculaire, Faculté de Médecine, Université Laval, Québec, Canada

Gene therapy without gene transfer using specific restriction endonucleases is a therapeutic approaches aimed at the development of a cure for Duchenne muscular dystrophy (DMD). We aim to correct the human dystrophin gene with two Zinc Finger Nuclease (ZFN) proteins targeting exon 50. The two plasmids coding for the ZFNs were first demonstrated to be active by transfecting them in HEK 293T cells containing a surrogate plasmid coding for mCherry and a conditional eGFP gene that required the nuclease activity of the pair of ZFNs to be expressed. The presence of green fluorescent cells confirmed the nuclease activity. The presence of mutations in exon 50 following the double stand breaks and their repair by Non Homologous End Joining (NHEJ) was also detected with the Surveyor enzyme test in HEK 293T cells. Following the confirmation of the activity of this pair of ZFNs, we have produced these ZFN proteins in E. cloni 10Gbacteria using a rhamnose inducible promoter. The proteins (6His-SUMO-NLS-FLAG-ZFNs), which where produced,also contained a 6XHis tag to permit its purification, a SUMO sequence to increase its solubility and a FLAG to permit its detection by immunochemiluminescence. The proteins were purified on a nickel affinity column. The 6XHis tag and the SUMO peptide were then removed from the purified proteins with the SUMO Express protease. Anin vitrogel shift assay showed that the resulting ZFN proteins were able to bind specificallywith aplasmid containing the target sequence in exon 50. These proteins were also able to cut in vitro a oligonucleotide containing the targeted sequences. These ZFN proteins were also transduced in HEK 293T cells and in myoblasts of Duchenne Muscular Dystrophy patients with and without a transfecting agentPro-Deliver-IN (Biosciences).The transfection efficacy was however lower without the transfection agent. The capacity of these recombinant proteins to cut the dystrophin gene in these cells and restore by NHEJ the normal reading frame in myoblasts of patients with a deletion in the preceding exons is currently under investigation.

 Intravenous Delivery of a rAAV9 U7snRNA Vector Targeting Exon 2 Results in Widespread Dystrophin Expression in the Dup2 DMD mouse model

Tabatha R. Simmons, Adeline Vulin, Nicolas Wein, Andrea M. Rutherford, Paul M. Janssen, Kevin M. Flanigan. The Center for Gene Therapy, Nationwide Childrens Hospital, Columbus, OH; Department of Physiology, The Ohio State University, Columbus, OH; Department of Pediatrics, The Ohio State University, Columbus, OH; Department of Neurology, The Ohio State University, Columbus, OH

Exon skipping therapies for Duchenne muscular dystrophy (DMD) that are currently being tested in patients with frameshifting exon deletions aim to modulate splicing and restore an open mRNA reading frame. This leads to translation of an internally truncated Becker muscular dystrophy (BMD)-like protein. Skipping of a single copy of an exon duplication, in contrast, would be expected to restore a full-length dystrophin protein. We sought to test the efficacy of virally-mediated duplication skipping in the novel Dup2 mouse, developed to model the most common single-exon duplication (exon 2) seen in DMD patients. A targeting construct was created containing four copies of a modified U7snRNA, each of which targets either the splice donor or acceptor sites of exon 2 (U7sn RNA-ACCA). 2.2x1012 vg of rAAV9.U7snRNA.ACCA were introduced into 8 week old mice (n=20) by a single tail vein injection. Animals were sacrificed at 4 weeks post-injection for analysis of RNA splicing and protein expression. RT-PCR reveals widespread exon 2 skipping, with the simultaneous presence of all three predicted transcripts – duplicated exon 2 (Dup2), wild-type, and deleted exon 2 (Del2) – in variable proportions in all muscles tested. Dystrophin expression was confirmed in all muscles (tibilias anterior, gastrocnemius, triceps, heart and diaphragm) by immunoblot as well as by immunofluorescence, which showed proper localization of the dystrophin signal to the sarcolemmal membrane. Treatment normalized hindlimb and forelimb grip strength and partially corrected extensor digitorum longus and cardiac papillary muscle force deficits seen in untreated Dup2 mice. These results demonstrate the utility of the Dup2 mouse model as a tool for testing potential duplication exon-skipping strategies. They confirm that IV delivery of rAAV9.U7snRNA is able induce exon 2 skipping and to drive the production of a functional dystrophin protein, suggesting a promising strategy for future clinical development.

Exons 45-55 Skipping of Human Dystrophin Transcripts Using Cocktail Antisense Oligonucleotides

Yusuke Echigoya, William Duddy, Joshua Lee, Vincent Mouly, Toshifumi Yokota. Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada; Therapies for Striated Muscle Disease, Institute of Myology, Paris 6, Paris, France; Muscular Dystrophy Canada Research Chair, Edmonton, AB, Canada

Duchenne muscular dystrophy (DMD), one of the most common fatal genetic disorders, is caused by mutations in the dystrophin (DMD) gene. Currently, antisense oligonucleotide (AO)-mediated exon skipping, which restores the reading frame by eliminating specific exons, is a promising therapeutic approach for DMD. Remaining challenges of the current single exon skipping are the restricted applicability to patients and unclear stability/function of resulting in-frame short dystrophin proteins. A potential solution is multiple exon skipping targeting exons 45-55 at the mutation hot spot region. It is particularly noted that skipping of exons 45-55 could treat approximately 63% of DMD patients with deletion mutations in theory. In addition, the entire exons 45-55 deletion is reported to be mostly associated with milder symptoms or an asymptomatic course, indicating that the shortened dystrophin lacking this specific region is more stable/functional. Recently, we have demonstrated a proof-of-concept of exons 45-55 skipping with cocktail AOs in a dystrophic mouse model. However, sequence-specific AOs have to be optimized for the human DMD gene. In the present study, we designed AOs for the human dystrophin transcript with a new predictive tool. Immortalized human DMD skeletal muscle cells harboring deletion mutations within exons 45-55 (deletion of exons 45-52, exons 48-50, and exon 52) were transfected with cocktails containing 3, 6 or 10 phosphorodiamidate morpholino oligomers (PMOs) using Endo-Porter transfection reagent. Skipped dystrophin transcripts with the junction of exons 44 and 56 were observed in all the DMD cell lines we tested, accompanied by dystrophin protein expression. We demonstrate for the first time that skipping of the entire exons 45-55 region is feasible by using the optimal AO cocktails in the human DMD gene. Our observation will facilitate clinical development of exons 45-55 skipping therapy for the treatment of DMD.

Human α7 Integrin Gene (ITGΑ7) Delivered By Adeno-Associated Virus Reverses the Phenotype of the Double Knock Out (DKO) Mouse Devoid of Dystrophin and Utrophin

Kristin N. Heller, Chrystal L. Montgomery, Kimberly M. Shontz, Paul M. L. Janssen, K. Reed Clark, Jerry R. Mendell, Louise R. Rodino-Klapac. Center for Gene Therapy, Nationwide Childrens Hospital, Columbus, OH; Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing the human α7 integrin gene (ITGΑ7) using adeno-associated virus (AAV) delivery. α7 integrin is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. The presence of α7 in DMD muscle offers an advantage over direct dystrophin replacement because it will evade an immune response to a new protein. We have previously shown that delivery of rAAVrh.74.MCK.ITGΑ7 to the lower limb of mdx mice through isolated limb perfusion of the femoral artery resulted in overexpression of α7 at the sarcolemma and protected skeletal muscle from contraction induced injury. In this study, we tested the ability of rAAVrh.74.MCK.ITGA7 to improve a more severe model of DMD by treating mdx/utrn-/- dko mice systemically in 2-4 day old pups and performing functional studies in the diaphragm and EDL. In the mdx/utrn-/- dko mouse study, systemic delivery resulted in α7 expression in multiple muscle groups. The increase in ITGA7 significantly protected against loss of force following eccentric contraction-induced injury in the EDL muscle at eight weeks post injection and improved normalized specific force in the EDL and diaphragm muscles. The additional α7 integrin in dko mice showed a reduction in kyphosis, an increase in myofiber diameter, improvement in body weight and importantly extension of lifespan by an additional 8 wks. These data also indicate that early treatment with ITGA7 preserves force in a severe DMD model and predicts greater therapeutic potential for treating patients in early stages of disease progression. Taken together, we have shown that AAVrh.74.ITGΑ7 gene transfer demonstrates promise as a viable treatment for DMD.

Assessment of the Dystrophin Gene Exon 53 Skipping Using DMD Patient-Derived Fibroblasts for Exploratory Clinical Trial of Antisense Drug NS-065/NCNP-01

Takashi Saito, Tetsuya Nagata, Satoshi Masuda, Jun Tanihata, Maki Ohata, Akemi Tamaura, Miyuki Kanazawa, Narihiro Minami, Kanako Goto, Yukiko Hayashi, Kazu Iwasawa, Kaoru Tatezawa, Koichi Fukuda, Tsutomu Mizutani, Reiko Shimizu, Maiko Suzuki, Kazuo Yamaguchi, Hisateru Tachimori, Ichizo Nishino, Yu-ichi Goto, Hirofumi Komaki, Shin'ichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience (NIN), National Center of Neurology and Psychiatry (NCNP), Kodaira, Japan; Clinical Research Unit, NCNP, Kodaira, Japan; Department of Mental Retardation and Birth Defect Research, NIN, NCNP, Kodaira, Japan; Department of Neuromuscular Research, NIN, NCNP, Kodaira, Japan; Translational Medical Center, NCNP, Kodaira, Japan; Department of Mental Health Administration, National Institute of Mental Health, NCNP, Kodaira, Japan; Department of Child Neurology, NCNP, Kodaira, Japan; (From Dec 2013), Pharmaceuticals and Medical Devices Agency, Chiyoda-ku, Japan; Department of Pediatrics, School of Medicine, Tokyo Women's Medical University, Shinjuku, Japan

The NS-065/NCNP-01 is an antisense oligonucleotide drug that has been developed to skip the dystrophin gene exon 53 and to treat Duchenne Muscular Dystrophy (DMD) patients with mutations, which respond to this drug. This antisense drug has a morpholino backbone, and has been confirmed potent efficacy and high safety in pre-clinical studies. We, National Center of Neurology and Psychiatry, and Nippon Shinyaku Co., Ltd., had jointly developed this drug since 2009, and then started an investigator-initiated clinical trial from June 2013 (UMIN Clinical Trial ID: UMIN000010964). This trial is an exploratory phase-1, first-in-human study designed to assess the safety, tolerability and pharmacokinetics in eligible DMD patients. It is also intended to assess the efficacy and dose responses by evaluating the expression of dystrophin protein. To verify eligibility for the exon 53 skipping, confirmation of exon 53-skipped mRNA and truncated dystrophin protein by an in vitro NS-065/NCNP-01 treatment using the patient-derived cells, was established as one of the key inclusion criteria. DMD patients, who had correctable mutation by exon 53 skipping and had fulfilled the other inclusion criteria, underwent a muscle and skin biopsy simultaneously to obtain pretreatment muscle specimens and fibroblasts culture. We transformed the fibroblasts into myogenic cells by MYOD-transduction, and treated by NS-065/NCNP-01 in vitro, then evaluated by RT-PCR, mRNA sequence and western blot analysis. We have confirmed so far that seven recruited patients satisfy the established criteria: the exon 53-skipped mRNA and the truncated dystrophin protein were produced after an in vitro NS-065/NCNP-01 treatment. We would report that the in vitro assay of antisense drug using DMD patient-derived cells have provided valuable eligibility information regarding patient enrollment in this first-in-human, exon 53-skipping clinical trial

The Use of Human iPSC Derived Cardiomyocytes for Systematic Comparison of Cardiac Transduction Efficiency of AAV Serotypes

Zejing Wang, Xuan Guan, Véronique Blouin, Virginie Francois, Philippe Moullier, Martin K. Childers. Clinical Research, Fred Hutchinson Cancer Research Center, Seattle; Medicine, University of Washington, Seattle; Rehabilitation Medicine, University of Washington, Seattle; UMR1089 and Atlantic Gene Therapy Institute, Université de Nantes, Nantes Cedex 1, France; Institute for Stem Cell and Regenerative Medicine, Seattle

Adeno-associated viral (AAV) vectors-mediated gene replacement represents a promising therapeutic treatment in modern medicine. It has demonstrated efficacy both in preclinical studies and clinical trials for a number of acquired and inherited diseases. Several AAV serotypes, including AAV2, 5, 6, 8 and 9, have been tested for their ability in transducing cardiac muscle for their potential of treating heart diseases. Inconsistent findings on their efficiency have been reported due to the use of different model systems, vector administration routes and doses. A direct comparison of these serotypes under the same condition is necessary to address this debate. We have developed a system, in which human cardiomyocytes (CM) are differentiated from inducible pluripotent stem cells (iPSCs) derived from urine stem cells. The iPSC-CM from healthy donors and a patient with Duchenne muscular dystrophy (DMD), in which cardiomyopathy is a leading cause of mortality, have been successfully characterized both phenotypically and physiologically. In this current study, we set to systematically examine cardiac transduction efficiency of various AAV serotypes in a controlled manner in the iPSC-CM system. Green fluorescence protein (GFP) under the control of a CMV promoter is being used as a marker gene for all serotypes to be tested. Immunofluorecent staining and quantitative flow cytometry are being employed to detect expression of GFP and certain cardiomyocyte-specific marker genes. Initial experiments used 2 serotypes, AAV6 and 8, and MOI ranging from 10 to 10,000 to determine an optimal titer in the DMD-iPSC-CM. Our preliminary data showed gradual increase in GFP expression from days 3 to 7 when robust GFP expression were observed for both vectors at MOI 1,000 (Figure 1) and above.

Flow cytometry demonstrated 89% and 54% transduction efficiency of the defined cardiomyocytes by AAV6 and AAV8, respectively, at MOI 10,000. We are currently repeating the same set of studies using AAV9 and 10 to confirm the titers for AAV6 and 8. Once an optimal titer is determined, a blinded experiment will be conducted to compare transduction efficiency of all major serotypes at once in iPSC-CM from both normal and DMD patients: AAV1, 2, 5, 6, 8, 9 and 10. The knowledge gained in this study will provide much needed information for clinical translation. Further, this study will demonstrate proof –of-principle the feasibility that drug-discovery assays can be developed using iPSC-derived cardiomyocytes as a biological reagent.

Mesenchymal Progenitor Cells (MPCs) Responsible for Fat Accumulation in Dystrophic Muscle May Also Impair the Myofiber Regeneration of Muscle Progenitors in Skeletal Muscle of Dystrophic Mice

Jihee Sohn, Ying Tang, Bing Wang, Aiping Lu, Johnny Huard. Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA

Adult skeletal muscle possesses a remarkable regenerative ability which largely depends on satellite cells. However, in severe muscular dystrophies, such as Duchenne muscular dystrophy (DMD), skeletal muscle integrity is debilitated and is often replaced by a mix of fibrous tissue and white adipocytes undergoing a process termed fatty degeneration. Ectopic fat deposits in skeletal muscle alter the tissue environment and induce deregulation of muscle homeostasis. However, the cellular origin of muscle fat formation is still unclear. Based on a previously published preplate technique, we isolated two types of muscle derived cells; PDGFRa+ mesenchymal progenitor cells (MPCs) and Pax7+ myogenic muscle derived stem cells (MDSCs) from the skeletal muscle of utrophin/dystrophin double knockout (dys-/- utro-/-, dKO) mice, which is a severe animal model of DMD. Previously, we showed that compared to other muscle-derived cell populations isolated from dKO mice, MPCs display significantly increased proliferation and adipogenic potentials, both in vitro and in vivo. In the current study, we showed that not only might the MPCs be a major contributor to ectopic fat cell formation within dystrophic muscle, but also that the disease state may affect the stem cell niche, impacting the fate of the MPCs. We also propose that the interaction between MDSCs and MPCs may be important for the inhibition of myofiber regeneration. Previously, we have shown that MDSCs from dKO mice are defective in proliferation and fusion, which correlates with their muscle histopathology. We also observed significantly increased numbers of endogenous proliferating MPCs in the interstitial space of dKO skeletal muscle compared to that of WT mice, which suggests a role for these cells in muscle regeneration/degeneration. A co-culture assay using transwell inserts demonstrated that the limited myogenic differentiation potential of dKO-MDSCs was further reduced by co-culturing them with dKO-MPCs. We also observed that dKO-MPCs strongly inhibited the myogenic capacity of WT-MDSCs. WT and dKO-MPCs were investigated for their expression of IL-6 and Wnt5A, which are known to enhance terminal myogenic differentiation, and myostatin, which is a known inhibitor of myogenesis. Although we observed similar levels of myostatin expression in the WT and dKO MPCs, lower levels of IL-6 and Wnt5A expression were observed in the dKO-MPCs compared to WT-MPCs. Results from this study could provide insight into new approaches to alleviate muscle weakness and wasting in DMD patients through the inhibition of proliferation and adipogenic differentiation of MPCs in skeletal muscle.

Increased Functionality of Micro-Dystrophin by Altering Size and Rod Domain Composition

Julian N. Ramos, Stephen Hauschka, Jeffrey S. Chamberlain. Neurology, University of Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA

Recombinant adeno-associated viral (rAAV) mediated gene therapy has advanced into clinical trials for several diseases, including the muscular dystrophies. Along with the immunological challenges of rAAV administration, investigators are faced with a limited genome to package therapeutic genes and appropriate regulatory cassettes. Dystrophin, the largest known gene, whose aberrant mutations are responsible for Duchenne muscular dystrophy, has been meticulously spliced into synthetic isoforms, expressed, and assessed for both stability and functionality in striated muscle of animal models. Earlier studies have determined critical domains necessary for an effective micro-dystrophin construct. In this study, an exploration of sub-domains in the central rod domain was made. Among our goals was to find proline rich regions among the spectrin-like repeats (SRs) that could function to synergistically improve or altogether replace the internal Hinge-3 subdomain in a micro-dystrophin. Constructs with a total of five, and even six, SRs were also created and assessed in the dystrophic mdx4cv mouse model. This was possible by incorporating a refined, regulatory expression cassette based on the mouse creatine kinase promoter. These novel micro-dystrophins underwent an initial screening within individual dystrophic muscles. Those that yielded results at or above those of our μDysH3 standard proceeded to more thorough, systemic analysis. Diaphragm and gastrocnemius muscles were examined at both three and six months post-treatment of dystrophic male pups. Parameters assessed included degree of dystrophin expression, central nucleation of myofibers, specific force production, and resistance to contraction-induced injury. Four novel micro-dystrophin constructs demonstrated functionality that match or exceeded our μDysH3 standard. These constructs supported the high degree of functionality of two regions: the nNOS binding region including SRs 15 and 16 and another in SR 23. These novel micro-dystrophins may lead to increased functionality in dystrophic muscles and may enhance the prospects of gene therapy for DMD.

Systemic Gene Therapy for DMD – Advanced Preclinical/Translational Studies To Minimize Risk and Maximize Potential Benefit for Patients With Dystrophin Gene Deletions

Alock Malik, Andrew Mead, Mihail Petrov, Daniel VanBelzen, Yafeng Song, Xiangping Lu, Margaret Choi, Joe Kornegay, Hansell Stedman. Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA; Veterinary Integrative Biosciences, Texas A&M University, College Station, TX

Duchenne Muscular Dystrophy is an X-linked, severely debilitating muscle disease with an incidence of 1:3500 males. Most patients lose ambulation in the early teens and develop symptomatic respiratory insufficiency and cardiomyopathy in the late teens or early twenties. A majority of patients with DMD have sporadic deletions spanning multiple exons of the dystrophin gene. To maximize biodistribution and minimize the risk of cell-mediated immune destruction of transduced muscle cells, we have focused on the therapeutic use of dystrophin's autosomal homolog, utrophin, delivered via vectors based on AAV9. We have developed and tested in mice and dogs a family of synthetic gene constructs encoding miniaturized versions of utrophin to preserve the calponin-homologous actin-binding domains and the "WW-EF-ZZ" dystroglycan-binding domain, while maintaining the phasing of intervening spectrin-like triple helical repeats. The construct driving the highest level of expression in striated muscle was selected for extensive further testing in vivo. In blinded studies, systemic administration of the vector at 3E13/kg in dystrophin-deficient "GRMD" puppies resulted in widespread myocyte transduction with stabilization of the dystrophin/utrophin associated protein complex to wild type levels at 5 weeks post-injection. Transduction is associated with a normalization of the fiber size profile as measured histologically by minimal Feret analysis. Interferon ELISpot assays of peripheral lymphocytes using utrophin peptide libraries at 5 and 8 weeks post-AAV show no difference between uninjected control and AAV-injected dogs. In anticipation of vector dose escalation and rigorous blinded testing, we have performed a detailed characterization of the rate of loss of physiological reserve in the DMD models. In untreated dystrophic dogs, serial measures of respiratory and cardiac reserve in vivo reveal marked divergence over time from the normal control. At post-mortem, diaphragmatic morphometry shows that in the untreated dystrophic dog, the ratio of length-to-width at fixed sarcomere length is 15+/-8% of control at one year of age, providing a robust primary endpoint for investigational therapy. Cardiac mechanics, as studied in detail by isolated organ perfusion, undergo near complete loss in dystrophic dogs of both the Frank/Starling mechanism and adrenergic inotropic responsiveness by one year of age. Clinical and histological indices of myodegeneration correlate strongly with the pattern of muscle and myofiber-specific expression of myosin isoforms, especially in Myh16+ fibers from the mandibular elevators. We have most recently precision-mapped the deletion endpoints flanking the entire dystrophin coding sequence in a second canine DMD model, "GSHPMD", and have developed a plan to use this unique resource to rigorously assess the risk of deleterious autoimmunity following systemic gene therapy with AAV9-dystrophin and AAV9-utrophin.

HGF Is Critical for the Beneficial Effect of NF-κB/p65 Inhibition in Dystrophic Muscle

Jonathan D. Proto, Ying Tang, Aiping Lu, Minakshi Poddar, William Chen, Sarah Beckman, Kayla Imbrogno, Timothy Hannigan, Wendy M. Mars, Bing Wang, Johnny Huard. Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA; Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA; Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH

The ubiquitous transcription factor NF-κB has been implicated in the fatal disease Duchenne muscular dystrophy (DMD). The severity of this disease can be attenuated by NF-κB inhibition in the mdx mouse, a murine DMD model; but this approach remains problematic for treating human patients. Due to the ubiquity of this factor, an alternative, but related, target is highly desirable. We hypothesized that such a target could be found by identifying endogenous factors responsible for muscle repair. We found that muscle-derived stem cells (MDSCs) isolated from mice lacking one allele of the NF-κB subunit p65 (p65+/-) had potent anti-inflammatory properties. Not only were IL-6 and IL-10 cytokine levels in inflammatory macrophages modulated by p65+/- MDSC conditioned medium in vitro, but following transplantation to injured skeletal muscle, p65+/- MDSC engraftments induced a more rapid resolution of inflammation coupled with enhanced host regeneration. Using pharmacologic inhibitors in vitro, we found that this effect on macrophages was dependent on the activation of the hepatocyte growth factor (HGF) receptor, Met, and downstream inactivation of the kinase GSK3-β. Analysis of injured p65+/- skeletal muscle confirmed the activation of an HGF/Met/GSK3-β pathway in vivo. Furthermore, analysis of skeletal muscles from p65 haploinsufficient mdx mice (mdx;p65+/-) demonstrated that HGF up-regulation coincided with reduced inflammation and accelerated regeneration of dystrophic skeletal muscle. We determined the specific importance of HGF using a musculotropic adeno-associated viral vector (AAV9) to deliver HGF-targeting shRNA to mdx;p65+/- mice. Strikingly, HGF knockdown ablated the beneficial effect of p65 deficiency, and significantly worsened skeletal muscle inflammation and necrosis. In this investigation, we identify the critical role of HGF in DMD skeletal muscle repair. Moreover, our results support the idea that the identification of stem cell-derived factors involved in tissue repair will bring forward novel targets for the treatment of disease. HGF mimetics are currently in Phase II clinical trials to treat myocardial infarction and kidney dysfunction, suggesting this is a feasible approach to DMD treatment.

Electrotransfer of Peptide Nucleic Acids (PNA) in Normal and Dystrophic Muscle Tissue

Camilla Brolin, Takehiko Shiraishi, Pernille Højman, Thomas O. Krag, Peter E. Nielsen, Julie Gehl. Center for Experimental Drug and Gene Electrotransfer (C*EDGE), Department of Oncology, Copenhagen University Hospital Herlev, Herlev, Denmark; Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark; The Centre of Inflammation and Metabolism and the Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; Neuromuscular Research Unit, Department of Neurology Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

PNA is an artificial nucleic acid mimic, which has shown great promise as an antisense RNA interference agent, in particular regarding mRNA splicing modulation. However, the full potential of antisense PNA is still limited by poor cellular uptake and low in vivo bioavailability. The genetic disease Duchenne Muscular Dystrophy (DMD) has attracted particular attention for antisense drug discovery because it has been demonstrated that partial dystrophin restoration can be achieved by antisense agents causing skipping of the mutated exon.
Electroporation has shown potential as a PNA delivery method in cell culture. Electroporation is already used in the clinic for treatment of cutaneous metastasis (electrochemotherapy) and the clinical field is rapidly extending to new molecules and to a range of tissues with newly developed electrodes for treatment of internal tumors. Therefore, the aim of the present project was to study the effect of electroporation upon intramuscular PNA administration. In the first study, we used a PNA targeting exon 23 in the dystrophin gene in both normal mice and in mdx mice (the animal model of DMD). After optimizing voltage conditions, injection volume, PNA dose as well as examining different PNA modifications, we found statistically significant increase in PNA activity by 2-4 fold (determined by RT PCR) upon electrotransfer in NMRI mice. The effect lasted up to 4 weeks at the RNA level. In mdx mice lacking dystrophin protein, exon skipping was detected by immunostaining of newly synthesized dystrophin protein and by western blotting. Whole muscle transverse sections showed recovery of dystrophin at the sarcolemma in PNA treated regions in all 3 positions tested in mdx mice, but electroporation did not significantly increase the number of dystrophin positive fibers. Clinical use of antisense therapy for treatment of DMD patients requires systemic administration of the drug because all muscles including heart muscle are affected by the disease, and must be treated. Thus further studies on systemically administered modified PNAs are needed to elucidate if electrotransfer could eventually provide therapeutic enhancement in selected muscles.

Human Muscle-Derived Stem Cells Prevent Cardiac Dysfunction in the Aging Dystrophic Heart

William C. W. Chen, Aiping Lu, Talgat Yessenov, Xueqin Gao, Kimimasa Tobita, Arman Saparov, Johnny Huard. University of Pittsburgh, Pittsburgh; Nazarbayev University, Astana, Kazakhstan

Duchenne muscular dystrophy (DMD) is a recessive X-linked degenerative muscle disease that leads to progressive muscle weakness. The reduced level or lack of sarcolemma dystrophin in DMD patients affects not only skeletal muscles but also the myocardium, resulting in the development of dilated cardiomyopathy. Due to the extended life span of DMD patients, benefitted from new therapeutic modalities, a suitable treatment for DMD cardiac dysfunction has become increasingly important. Previously our group has demonstrated the regenerative potential of murine skeletal muscle-derived stem cells (mMDSCs) in the myocardium of mdx mice (a mouse DMD model). Here we investigate the therapeutic potential of human skeletal muscle-derived stem cells (hMDSCs) in the aging dystrophic heart where dilated cardiomyopathy gradually manifests. hMDSCs were isolated by a modified preplate technique from post-mortem human muscle biopsies. Cultured hMDSCs were transduced with lentiviral-GFP vectors and subsequently sorted by flow cytometry based on GFP expression. One million GFP+ hMDSCs were intraperitoneally injected into the lower abdomen of aged immunodeficient dystrophic mice (mdx/SCID mice, 15-16 months old). The control group received PBS injections. Cardiac function was repeatedly assessed by echocardiography (N=6/group) immediately before and at 1 and 2 months following the injection. At 2 months post-injection, LV contractile function had dramatically deteriorated in control hearts. In sharp contrast, LV contractility was notably sustained following the hMDSC injection (LVFS, p=0.006; LVFAC, p≤0.001; LVEF, p≤0.001). Significant enlargement in LV chamber dimension was also recorded in control mice at 2 month post-injection, suggesting progressive LV dilatation. In hMDSC-injected mice, markedly smaller LV chamber sizes were observed (LVEDA, p=0.011; LVESA, p≤0.001), indicating prevention of adverse cardiac remodeling. Immunohistochemical analysis (N=4/group) revealed increased CD31+ capillary structures in the myocardium of hMDSC-injected mice (60.86±8.45/HPF) when compared with control mice (52.14±4.86/HPF). Similarly, more alpha-smooth muscle actin (αSMA)-positive vessels were observed in hMDSC-injected mice (4.57±0.89/HPF) when compared with control mice (3.86±0.65/HPF), suggesting the promotion of angiogenesis and vasculogenesis in the myocardium following intraperitoneal hMDSC injection. Preliminary analysis of myocardial fibrosis by Mason Trichrome staining showed reduced fibrotic area fraction in hMDSC-treated mice (N=2/group). Additionally, a very small number of GFP+ cells (<0.1%) could be detected in the myocardium, suggesting migration of implanted hMDSCs to the diseased heart. In conclusion, our data suggest a robust therapeutic potential of intraperitoneal hMDSC implantation in preventing cardiac dysfunction of aging dystrophic hearts. Currently we are investigating the terminal cell fate of implanted hMDSCs and the mechanistic pathway(s) involved in the hMDSC-mediated therapeutic effects imparted on aged dystrophic hearts.

Next Generation of Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin ORF

Susie Jarmin, Linda Popplewell, Taeyoung Koo, George Dickson, Takis Athanasopoulos. School of Biological Sciences, Royal Holloway University of London, Egham, United Kingdom; Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom; Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, United Kingdom

Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by gene mutations to dystrophin gene leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11,055 bp). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, or other signalling cascades such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7-6kb +/- proteasomal inhibitors). We have recently developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We have recently reported that three independent AAV vectors carrying in tandem sequential exonic parts of the human DMD coding sequence can enable the expression of the full-length dystrophin protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences and through appropriate utilisation of the cellular splicing machinery. Here we report on the next generation of these AAV transplicing vectors introducing additional design features such as full codon and mRNA optimization, miRNA immunomodulation, increased safety profile and capability of pseudotyping to a range of different AAV serotypes. These methods of multiple AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases

Human Dystrophin Deficient Cardiomyocytes Derived Via Cellular Reprogramming Model Duchenne Cardiomyopathy in a Dish

Xuan Guan, David L. Mack, Julie Mathieu, Claudia M. Moreno, Stefan M. Czerniecki, Luis F. Santana, Hannele R. Baker, Martin K. Childers. Department of Physiology and Pharmacology, School of Medicine, Wake Forest University Health Sciences, Winston-Salem, NC; Department of Physiology & Biophysics, University of Washington, Seattle, WA; Department of Rehabilitation Medicine, University of Washington, Seattle, WA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; Department of Biochemistry, University of Washington, Seattle, WA

Introduction:
Human somatic cells can be converted into primitive stem cells, termed induced pluripotent stem cells (iPSCs). With the advances in differentiation protocols, various tissues, such as cardiomyocytes, can be efficiently generated from iPSCs. We recently reported that iPSC-derived cardiomyocytes faithfully preserve the disease genotype and manifest disease-associated phenotypes. Due to the genetic mutation of the membrane protein dystrophin, Duchenne muscular dystrophy (DMD) patients inevitably develop fatal cardiomyopathy. Based on cellular reprogramming technology, our goal is to establish an in vitro model system to recapitulate the Duchenne cardiomyopathy phenotype.
Methods:
Cardiomyocytes were induced from DMD iPSCs (n=2) by sequential application of GSK3 inhibitor CHIR99021 and Wnt inhibitor IWP4. Differentiated cardiomyocytes were subjected to Polymerase Chain Reaction (PCR) analysis, immunostaining, calcium imaging, patch-clamp recording, Seahorse cellular metabolism analysis, and hypotonic stress assay to assess membrane fragility.
Results:
DMD iPSCs were efficiently differentiated into contracting cardiomyocytes. Immunostaining and Western blot confirmed the absence of dystrophin protein on these cardiomyocytes. Compared to normal controls, dystrophin deficient cardiomyocytes revealed a panel of physiological abnormalities, including abnormal Ca2+ handling, fragile sarcolemma and altered energy metabolism.
Conclusions:
Cardiomyocytes can be generated from iPSCs with dystrophin gene mutations. DMD cardiomyocytes retain a dystrophin-null phenotype and manifest abnormal cellular physiology consistent with patient and animal model physiology. These data lend credibility to the idea that iPSC-cardiomyocytes recapitulate a disease phenotype that can be exploited to further explore mechanisms of Duchenne cardiomyopathy and test novel therapeutic modalities.

Induction of Local OPMD Histopathology in Common Marmoset By rAAV1 and 8-Mediated Transduction

Hironori Okada, Hidetoshi Ishibashi, Hiromi Hayashita-Kinoh, Tomoko Chiyo, Chiaki Masuda, Yuko Nitahara-Kasahara, Shin'ichi Takeda, Takashi Okada. Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Department of Neurophysiology, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan

Background: Oculopharyngeal muscular dystrophy (OPMD) caused by expansion of the poly alanine tract in Poly(A) Binding Protein Nuclear 1 (PABPN1) gene is an autosomal dominant late-onset disorder characterized by proximal muscle weakness, ptosis, and dysphagia. Although it has been reported that reduction of proteasome activity and 3' untranslated region shortening in mRNA are caused by overexpression of mutant PABPN1, it is unclear how ubiquitous PABPN1 expression causes predominant skeletal muscle pathology and why symptoms in OPMD initiate only after midlife. Therefore, animal models more genetically resembling to humans is required to understand OPMD pathology. The common marmoset ( Callithrix jacchus ) has various advantages as an experimental non-human primate (NHP) model, such as small body, short gestation, multiple birth, and early sexual maturation. Although generation of GFP transgenic common marmosets with gene-transfer to pre-implantation embryo by lentiviral vector (rLV) was reported, this method needs many donor and recipient animals for pre-implantation embryo. In contrast, adeno-associated virus vector (rAAV) is capable of safe gene-transfer and long-term transgene expression with a wide range of tissue specificity along with transduction of non-dividing cells. In order to analyze OPMD pathology, we investigated rAAV-mediated transduction of adult marmoset skeletal muscle with mutant PABPN1.
Methods: scAAV1 or 8 carrying EF1 promoter-driven expanded PABPN1 (scAAV1/8-EF1-expPABPN1) or EGFP (scAAV1/8-EF1-EGFP) was injected into adult marmoset tibialis anterior (TA) muscle of hind limb. Twitch-torque of TA muscle during transcutaneous electrical peroneal nerve stimulation was compared between both sides of hind limb at four months after scAAV1-EF1-expPABPN1 injection. TA muscles were removed more than one month after the injection for histological analysis.
Results: At four months after injection, we detected reduction of twitch-torque in TA muscle transduced with scAAV1-EF1-expPABPN1. At five months after injection, several histological features characteristic in OPMD patients, such as pyknotic nuclear clump, variable fiber sizes, central nuclei, and split fiber, were observed. At this time, EGFP expression was recognized in TA muscle transduced with scAAV1-EF1-EGFP especially around the route of injection. In contrast, robust EGFP expression was seen in whole TA muscle transduced with scAAV8-EF1-EGFP at three months after injection. Histological features observed with serotype 1 were also found in case of scAAV8-EF1-expPABPN1 at three months after injection.
Conclusion: Long-term effective transduction after local injection of the rAAV into the adult marmoset TA muscle was confirmed. Also, induction of local dysfunction and OPMD histopathology after the injection of the rAAV was successful. This strategy with rAAV transduction would be useful for pathological analysis of various diseases in marmoset.

AAV Gene Transfer Utilizing Homologous Overlap Vectors Mediates Functional Recovery of Dysferlin Deficiency

Patricia C. Sondergaard, Eric Pozsgai, Danielle Griffin, Jerry R. Mendell, Louise R. Rodino-Klapac. Center for Gene Therapy, Nationwide Childrens Hospital, Columbus, OH

There is no cure or treatment for dysferlinopathies, a group of related disorders caused by absent or mutant dysferlin (DYSF). Limb girdle muscular dystrophy type 2B (LGMD2B) is the most frequently diagnosed form of dysferlinopathy and represents one of the most common LGMDs. Loss of dysferlin leads to a progressive form of dystrophy with chronic muscle fiber loss, inflammation, fat replacement and fibrosis all leading to deteriorating muscle weakness. Dysferlin has multiple functions in muscle including vesicle trafficking, endocytosis, T-tubule formation, and most widely studied, membrane repair. Pre-clinical studies that have assessed gene replacement or surrogate gene replacement have shown that multiple strategies exhibit some efficacy in restoring membrane repair, but only those that have delivered full-length DYSF have been able to correct the underlying histopathological defects in dysferlin deficient mice. Thus, there is strong rationale to develop therapies that deliver the entire DYSF cDNA. As DYSF is too large to be accommodated with canonical packaging using AAV, we have developed a unique dual vector system using AAV to deliver and express DYSF specifically in muscle. This two vector system (AAV.DYSF.DV) packaged in the rh.74 serotype is defined by a 1 kb region of homology between the two vectors. Following delivery to muscle, this overlap serves as a substrate for recombination/repair to generate the full-length gene. In this study we compared the efficiency and safety of DYSF.DV delivery in dysferlin deficient mice as well as in non-human primates (NHPs) in preparation for clinical trials. Dysf-/- mice were treated by intramuscular injection (IM), isolated limb perfusion (ILP), and systemic injection with AAV.DYSF.DV. IM injections were performed in the flexor digitorum brevis (FDB) muscle to assess expression and restoration of membrane repair capacity using ascending vector doses (6e9-6e10vg). The FDB was extracted after 10 weeks and an ex vivo laser injury membrane repair assay was performed. DYSF.DV restored membrane repair capacity in a dose-dependent manner comparable to wild-type findings. Moreover, DYSF.DV was also efficacious when delivered through the arterial circulation to the hind limbs or systemically via the tail vein. A single systemic dose of 3e12vg of each vector resulted in widespread gene expression exceeding 50% of muscle fibers in all muscles tested; restoring functional deficits in the diaphragm. In NHPs, DYSF.DV delivery was analyzed for both gene expression and toxicity using a FLAG biomarker to ensure gene delivery. There was no evidence of local or systemic toxicity in DYSF-null mice or NHPs showing overexpression of dysferlin. Findings in mice showed clear evidence that functional dysferlin was delivered, transcribed and translated without toxicity. Studies in NHPs further confirmed expression and safety through lack of toxicity. These studies lay the foundation for clinical trial.

 β-Sarcoglycan Gene Transfer Leads to Functional Improvement in a Model of LGMD2E

Eric Pozsgai, Danielle Griffin, Kristin Heller, Mendell Jerry, Louise Rodino-Klapac. Center for Gene Therapy, Research Institute at Nationwide Childrens Hospital, Columbus, OH; Pediatrics, College of Medicine The Ohio State University, Columbus, OH; Biomedical Sciences Graduate Program, College of Medicine The Ohio State University, Columbus, OH

Limb-girdle muscular dystrophy (LGMD) type 2E results from mutations in the gene encoding β-sarcoglycan causing loss of functional protein. It is characterized by muscle weakness and progressive muscle wasting. The sarcoglycans (α, β, γ, and δ-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to myofiber death and eventual muscle weakness. To date, no effective therapy exists to treat this debilitating disease. The disease manifests primarily in skeletal muscle and therefore gene delivery to regional muscle groups could provide a functional benefit to patients. The goal of this study is to demonstrate efficacy of AAV-mediated β-sarcoglycan gene transfer in β-SG knock-out mice to provide proof-of-principle for gene replacement therapy for limb-girdle muscular dystrophy type 2E. A previous study investigating the use of AAV gene replacement therapy to treat LGMD2D by our group established proof-of-principle for AAV1.tMCK.αSG gene transfer in α-sarcoglycan KO mice which led to a successful clinical trial in LGMD2D patients (alpha-sarcoglycan deficiency). The studies conducted by our lab for AAV.αSG gene therapy provide a solid platform for this study with the goal of translating β-sarcoglycan gene therapy to LGMD2E patients. We generated a self-complementary AAVrh74 vector containing a codon optimized human β-Sarcoglycan gene (hSGCB) driven by the muscle specific tMCK promoter. To demonstrate efficacy of vector delivery we injected two doses (3x1010 and 1x1011 vg) by direct injection to the tibialis anterior muscle of β-SG KO mice. We have also treated β-SG KO mice using a clinically relevant vascular delivery model to deliver AAV.tMCK.hSGCB in two doses (1x1011 and 5x1011 vg) to the lower limb muscles. Following direct intramuscular gene transfer in β-SG KO mice, we had clear demonstration of dose dependent β-SG expression and improvement in dystrophic pathology. This included a decrease in central nucleation, reduction in inflammation, and normalization of muscle fiber size. We also showed that greater than 90% of muscle fibers expressed β-SG expression in lower limb muscles following vascular delivery at high dose following 3 months of treatment. Functional outcome measures performed on the extensor digitorum longus (EDL) muscle from these mice showed an improvement in specific force generation and protection from eccentric contraction induced injury. Delivery of a normal copy of the SGCB gene to diseased muscle will allow for the production of functional wild-type protein, resulting in restoration of the DAPC and an improvement in muscle function. This pre-clinical study is pivotal for establishing proof-of-principle for translation of AAV.SGCB gene transfer in LGMD2E patients.

Allele-Specific Silencing of a Dominant-Negative Mutation Using siRNA or LNA Antisense Oligonucleotides Alleviates the Phenotype in a Cellular Model of Ullrich Congenital Muscular Dystrophy

Véronique Bolduc, Yaqun Zou, Morten Lindow, Dayoung Ko, Susanna Obad, Carsten G. Bönnemann. Porter Neuroscience Research Center, National Institutes of Health/NINDS, Bethesda, MD; Santaris Pharma A/S, Horsholm, Denmark

Congenital muscular dystrophy type Ullrich (UCMD) is a severe progressive neuromuscular disorder of early childhood onset, presenting with generalized muscle weakness, distal joint hypermobility, proximal joint contractures and respiratory failure as the main features. At present, there are no pharmacological treatment options available for children affected with this disease. In the laboratory we aim at exploring targeted RNAi and antisense approaches as potential therapies for UCMD. Dominant and recessive mutations in the three genes coding for collagen type VI (COL6A1, COL6A2, COL6A3) underlie UCMD, with dominant negative mutations accounting for the majority of cases. Achieving allele-specific silencing of the mutant collagen VI transcript would convert this dominant-negative state into a clinically asymptomatic haploinsufficient state. We have previously demonstrated the allele-specificity and efficiency of siRNA oligos to downregulate the expression of a mutant COL6A3 transcript in vitro. We have now extended our study to Locked Nucleic Acid (LNA)-containing antisense oligonucleotides, which are short, stable, and RNaseH-competent oligonucleotides that can be delivered without any carrier. We used outcome measures such as unsaturated PCR, quantitative RT-PCR and immunoblot on treated cell lysates, and confocal microscopy on treated fixed cells, and found that both RNAi and RNase-H pathways were comparably effective. This study provides further insights into the comparative allele-specificity of these two pathways to target dominant mutations at the transcript level, with the goal of developing optimal compounds for in vivo application.

 Consequences of Temporal, Pulsing DUX4 Expression Pattern Revealed in a Novel Therapeutic Screening Platform for Facioscapulohumeral Muscular Dystrophy (FSHD)

Amanda M. Rickard, Gregory J. Block, Lisa M. Petek, Rabi Tawil, Daniel G. Miller. Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA; Neurology, University of Rochester Medical Center, Rochester, NY

Facioscapulohumeral Muscular Dystrophy (FSHD) is a dominantly inherited adult-onset myopathy with an incidence rate of approximately 1:20,000. Patients suffer from irreversible and untreatable asymmetric muscle wasting that begins in the face, shoulders, and upper arms. FSHD is caused by epigenetic transcriptional derepression at a subtelomeric macrosatellite repeat on chromosome 4, which results in aberrant expression of Double Homeobox Domain Containing Protein 4 (DUX4). DUX4 is pathologically expressed during muscle differentiation and acts as a transcription factor to activate a number of transcripts including retroviral elements and germline genes, ultimately resulting in myotube apoptosis. The expression characteristics of DUX4 are difficult to investigate, as the gene first arose in the primate lineage, the protein cannot be detected directly in biopsies, and expression levels are low but extremely toxic in cultured FSHD cells. In order to further understand FSHD biology and to develop a drug screening platform for disease treatment, we have built a fluorescent reporter for DUX4 target activation. Using this system we are able to test siRNA and small molecule therapies and to observe DUX4's pattern and consequence of expression in live myoblasts during differentiation. We've found that DUX4's temporal expression is momentary and pulsing, although downstream effects of DUX4 activation are lasting. In addition, activation of the DUX4 target reporter in live muscle cells was necessary for observation of FSHD-specific apoptosis, and reduction of DUX4 transcripts using siRNAs rescues the disease phenotype. This novel reporter system is the basis for a high-throughput siRNA screening and drug development platform that will elucidate the epigenetic and transcriptional networks governing DUX4 production and identify potential treatment options for FSHD patients.