Research that will be presented in 14th American Society of Gene Therapy - May, 18-21, 2011, Seattle, USA

1) RNAi Therapy for Dominant Limb Girdle Muscular Dystrophy Type 1A

Jian Liu, Lindsay M. Wallace, Sara E. Garwick-Coppens, Michael A. Hauser, Jerry R. Mendell, Scott Q. Harper. Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH; Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH; Department of Neurology, The Ohio State University College of Medicine, Columbus, OH; Departments of Medicine and Ophthalmology, Duke University Medical Center, Durham, NC

Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 19 disorders characterized by progressive wasting and weakness of pelvic and shoulder girdle muscles. Commonly, patients require wheelchair assistance, and individuals with some forms of LGMD may also have cardiac and respiratory muscle involvement. Modest improvements in a relatively limited set of muscles may dramatically improve patients' quality of life, but there is currently no effective treatment for LGMD. Gene therapy approaches may hold promise as future LGMD treatments. Over the last two decades, the feasibility of using gene therapy to treat muscular dystrophy in general, and LGMD in particular, has improved, but previous strategies have almost exclusively focused on replacing defective or missing genes underlying recessive disorders. These gene replacement strategies are not feasible for treating dominant muscular dystrophies, including the 7 dominant forms of LGMD (called LGMD1). Instead, patients with dominant LGMD1 would likely benefit from reduction of their pathogenic alleles. The recent emergence of RNA interference (RNAi) as a powerful tool to suppress dominant disease genes provided a mechanism to accomplish this. In this study, we developed the first RNAi-based pre-clinical treatment for LGMD1A, which is caused by dominant mutations in one allele of the myotilin (MYOT) gene. We generated several MYOT-targeted artificial microRNAs (miMYOT) that significantly reduced mutant MYOT mRNA and protein in vitro. We then delivered our lead miMYOT sequence to the T57I transgenic mouse model of LGMD1A using AAV6 vectors, and data from treated mice were just obtained at the time of abstract submission. Three months post-injection, we found that miMYOT vectors, but not controls, significantly reduced soluble mutant MYOT protein to levels undetectable by immunohistochemical staining in transduced areas and the protein aggregates that characterize LGMD1A were either absent or very small in treated muscles. We are currently quantifying these obvious histolopathological improvements, and are also determining whether our miMYOT vectors will similarly improve overall muscle weakness in T57I mice using whole muscle specific force assays. These studies represent important first steps toward translating targeted RNAi gene therapy approaches for LGMD1A, and may have broader implications. Importantly, since dominant myopathies arise from mutations in at least 37 different genes that collectively affect ∼1 in 2,400 to 3,200 individuals, our RNAi strategies could be adapted to impact a large class of dominant muscle disorders.

2) RNA Interference Improves Myopathic Phenotypes in Mice Over-Expressing Facioscapulohumeral Muscular Dystrophy Region Gene 1 (FRG1)

Lindsay M. Wallace, Sara E. Garwick-Coppens, Scott Q. Harper. Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Molecular, Cellular and Developmental Graduate Program, The Ohio State University, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH

Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of AAV-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) Region Gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1^-high mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1^-high mice. Specifically, muscles transduced with FRG1-targeted microRNAs (miFRG1) were normal in size, showed no fibrosis or fat deposition, and had no evidence of myofiber degeneration or regeneration. Moreover, grip strength testing showed that miFRG1-treated animals were as strong as wild-type mice, while control-treated or untreated animals remained significantly weaker. Our results support the feasibility of using RNAi to target other FSHD candidate genes, including DUX4, which has recently emerged as the leading pathogenic insult underlying FSHD. Moreover, this approach potentially applies to as many as 37 different gene mutations responsible for myopathies inherited as autosomal dominant disorders.

3) Systemic Delivery of RNase H-Active Antisense Oligos in a Transgenic Mouse Model of Myotonic Dystrophy Type 1

Thurman M. Wheeler, Andrew J. Leger, Sanjay K. Pandey, A. Robert MacLeod, Masayuki Nakamori, Seng H. Cheng, C. Frank Bennett, Bruce M. Wentworth, Charles A. Thornton. Department of Neurology, University of Rochester, Rochester, NY; Genzyme Corporation, Framingham, MA; Isis Pharmaceuticals, Carlsbad, CA

Objective: To test whether systemic delivery of RNase H-active antisense oligos (ASOs) can reduce or eliminate RNA toxicity in a transgenic mouse model of myotonic dystrophy type 1 (DM1). Background: DM1 is a dominantly inherited degenerative disease caused by expression of an expanded CUG repeat (CUGexp) in the 3′ UTR of the DMPK transcript. CUGexp RNA accumulates in the nucleus, sequesters poly(CUG) binding proteins, and forms nuclear inclusions. Trans-dominant effects of the mutant transcript include aberrant pre-mRNA splicing, dysregulated gene expression, myotonia, and muscular dystrophy. Human skeletal actin-long repeat (HSA-LR) transgenic mice express CUGexp RNA in the 3′ UTR of an hACTA1 transgene. In this model, the toxic RNA is retained in the nucleus and induces splicing changes similar to DM1. Antisense knockdown of pathogenic RNA would be expected to mitigate clinical features of DM1. However, skeletal muscle is less sensitive to ASO effects because distribution of ASOs to muscle tissue is low. In previous studies in rodents (n = 10), systemic ASOs failed to produce target knockdown in muscle, despite strong effects in liver. Design/methods: ASOs were 2′ methoxyethyl gapmers that were designed to work by recruiting the nuclear enzyme RNase H to the ASO-RNA heteroduplex. ASOs targeting the hACTA1 mRNA were screened in cell culture. Active ASOs then were tested by subcutaneous injection in mice (25 mg/kg biweekly for 4 weeks). Control mice were treated with saline. Treatment assignments were randomized and analysis was blinded. Control ASOs targeted Malat1, Pten, and Srb1. Results: Two hACTA1-targeting ASOs reduced transgene levels by up to 80%. Control ASOs had no effect. Serum chemistries and histopathology showed no evidence of toxicity. Knockdown of toxic RNA was associated with elimination of myotonia and correction of RNA mis-splicing in all muscles examined. These effects persisted at least 15 weeks after the final dose. By microarray analysis, > 85% of changes in gene expression were normalized or improved. Another nuclear-retained non-coding RNA, Malat1, was also sensitive to knockdown in muscle (up to 80%) using similar RNase H ASO designs. However, ASOs targeting endogenous mRNAs (Pten, Srb1) did not produce knockdown in muscle, and the distribution of ASOs to skeletal muscle in the DM1 model was not higher than in WT mice. Conclusions: Systemic delivery of RNase H-active ASOs was surprisingly effective at reducing RNA toxicity in a transgenic mouse model of DM1. Nuclear-retained transcripts may display increased sensitivity to RNase H-active ASOs and enable RNA knockdown in muscle tissue where biodistribution is low. The mechanism may relate to residence time of transcripts in the nucleus, the cellular compartment in which RNase H1 is also located. ASOs that act through the RNase H pathway may exploit the nuclear retention phenomenon to gain a therapeutic advantage in DM1.

4) Addition of Peptide Therapy To Inhibit NF-κB Activation to AAV Serotype 9 Mini-Dystrophin Gene Transfer To Treat Muscular Dystrophy in mdx Mice

Daniel P. Reay, Gabriela A. Niizawa, Jon F. Watchko, Molly Daood, Eugene Raggi, Paula R. Clemens. Neurology, University of Pittsburgh, Pittsburgh, PA; Pediatrics, Magee-Women's Research Institute, Pittsburgh, PA; Neurology Service, Department of Veteran's Affairs Medical Center, Pittsburgh, PA

Systemic gene transfer using serotype 9 adeno-associated vectors (AAV9) is promising for treatment of preclinical models of Duchenne muscular dystrophy (DMD). The ability to achieve systemic vector delivery circumvents a significant hurdle presented by the widespread distribution of skeletal muscle that is best accessed through the circulation. However, a limitation of systemic gene vector delivery is that gene transduction levels vary among muscle groups. The addition of complementary therapy could provide 1) a treatment effect independent of gene transfer and 2) facilitation of successful gene transfer. Pathological activation of the nuclear factor κB (NF-κB) signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may also inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that the addition of NF-κB inhibition in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. The NF-κB Essential Modulator (NEMO) binding domain (NBD) peptide, which is utilized in this study to inhibit NF-κB, is synthesized as a fusion peptide with a protein transduction domain of 8 lysines (8K) to facilitate intra-cellular delivery. An AAV9 vector carrying a human codon-optimized mini-dystrophin gene under control of the cytomegalovirus promoter was given as a single intraperitoneal injection (1x10¹¹ gc/pup) at age 2-3 days to 16 mdx mice (experimental mice). Starting at age 4 weeks, 8 of the experimental mice were given intraperitoneal injections of 8K-NBD peptide dosed at 10mg/kg, 3 times per week for 5 weeks. The second group of 8 experimental mice received sham intraperitoneal saline injections. Control groups (n=8/group) included normal C57BL10 and disease control untreated mdx mice. At sacrifice, ex vivo physiological studies of diaphragm force production were performed prior to histological and biochemical assays of diaphragm and limb muscle tissue. In mdx mice treated with the combination of 8K-NBD peptide and AAV9 mini-dystrophin gene delivery, the quadriceps muscle demonstrated increased levels of recombinant dystrophin expression (84% of fibers expressing, as compared to 56% with AAV9 mini-dystrophin gene delivery alone) suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of recombinant dystrophin expression. Indices of necrosis and regeneration were diminished with either AAV9 gene delivery alone or in combination with 8K-NBD treatment. In diaphragm muscle, transgene expression was sufficiently high (>95% of muscle fibers expressed dystrophin) that marked improvements in histological and physiological indices were comparable in the 2 treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer.

5) A Phase I Dose-Escalating Study of AAV1 – γ-Sarcoglycan Gene Therapy for Limb Girdle Muscular Dystrophy Type 2C

Serge Herson, Fayçal Hentati, Aude Rigolet, Norma B. Romero, Anthony Behin, France Leturcq, Pascal Laforet, Thierry Maisonobe, Rim Amouri, Hafedh Haddad, Muriel Audit, Marie-Françoise Rosier-Montus, Bernard Gjata, Carole Masurier, François M. Lemoine, Pierre Carlier, Jean-Yves Hogrel, Bruno Eymard, Lee Sweeney, Richard Mulligan, David Klatzmann, Mustapha Cheraï, Didier Caizergues, Thomas Voït, Olivier Benveniste. Internal Medicine, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; Department of Molecular Neurobiology and Neuropathology, National Institute of Neurology, Tunis, Tunisia; Institute of Myology, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; Laboratoire de Biochimie Génétique, Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Paris, France; Généthon, Evry, France; GenoSafe, Evry, France; UMR 7211, CNRS, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA; Harvard Gene Therapy Initiative, Department of Genetics, Harvard Medical School, Boston, MA

OBJECTIVE: Safety of dose-escalating AAV1 – γ-sarcoglycan gene therapy (Phase I) for Limb Girdle Muscular Dystrophy type 2C. BACKGROUND: Gamma-sarcoglycanopathy or limb girdle muscular dystrophy type 2C (LGMD 2C) is an as yet untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene (γSGC). DESIGN/METHODS: Nine non-ambulant LGMD2C patients (2 M, 7 F, age 27 y [16 to 38]), with a del525T homozygous mutation on the γSGC gene and absence of γSGC immunostaining in muscle biopsy were enrolled. They were divided into 3 groups to receive 3 escalating doses of an AAV1 vector expressing human γSGC gene under the control of the desmin promoter, by local intramuscular injection into the extensor carpi radialis muscle. The first group received a single injection of 3e+9 viral genome (vg) in 100 µl, the second a single injection of 1.5e+10 vg in 100 ul and the third group received 3 concomitant 100 µl injections at the same site (a total of 4.5e+10 vg). RESULTS: No serious side effects were observed. All patients became seropositive for AAV1 and one developed a cytotoxic response again AAV1 capsid. In biopsy specimens taken 30 days after the injections, immunohistochemistry showed γSGC expression in 5/9 patients, with 4.7 to 10.5% of positively stained fibers and detection of γSGC mRNA by RT-PCR in the 3 patients who received the highest dose. The γSGC protein became also detectable by Western blot in one patient. CONCLUSIONS: Exogenous γSGC expression can be obtained in LGMD2C patients following AAV1 gene transfer, without adverse effects, paving the way for future developments in gene therapy of hereditary muscle diseases.

6) Immune Tolerance Induction in Canine X-Linked Muscular Dystrophy with rAAV9-Microdystrophin Transduction

Hiromi Hayashita-Kinoh, Naoko Yugeta, Hironori Okada, Yuko Nitahara-Kasahara, Tomoko Chiyo, Takashi Okada, Shin'ichi Takeda. Dept. of Molecular Therapy, National Institute of Neuroscience, NCNP, 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. Also we found that DCs would trigger an immune response against the rAAV-mediated transduction. Here we used fetuses of the canine X-linked muscular dystrophy in Japan (CXMD_J) to investigate the strategy of inducing immunotolerance to the rAAV as well as the muscle transduction profiles of rAAV9-microdystrophin. Methods: For fetal transduction, pregnant CXMD_J heterozygote was anesthetized and fetuses at post-coital day 35 were injected with 1x10¹² of rAAV-CMV-microdystrophin along with 1x10¹¹ of rAAV-CAG-Luciferase into amniotic fluid with ultrasound guidance. The yolk sacs and umbilical cords were sampled at delivery, and then transduced rAAV copy numbers were estimated by qPCR. To expect systemic microdystrophin expression, we additionally injected rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks old dystrophic dog. To examine the immunotolerance to the rAAV, purified canine peripheral leukocytes were exposed to rAAV9-microdystrophin for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Temporal muscles of the rAAV-injected animals were collected by punch-biopsy at various time points for expression analysis. In addition, rAAV-injected CXMD_J and non-injected CXMD_J were compared each other to assess gait function and lameness in the hind limb. Moreover, using whole body plethysmography, echocardiography and electrocardiography, we analyzed the respiratory and cardiac functions of the rAAV-injected dogs. Results: To rAAV transduction to fetus in utero, higher amount of AAV genome in the yolk sack of the rAAV-injected dogs was detected compared to that of the rAAV-uninjected control. Expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in one of the rAAV-injected dogs, suggesting the successful induction of immune tolerance against rAAV. rAAV-derived microdystrophin expression was confirmed by qRT-PCR and immunohistochemistry in the transduced dog. Monthly analysis of the transduced dystrophic dogs demonstrated superior gait function to non-injected littermate CXMD_J. At 1 year old, respiratory function and cardiac function of the transduced affected dog was milder form than untransduced affected dog. Conclusion: Our results demonstrate that induction of oral immunotolerance against rAAV with long-term transgene expression can be achieved by direct delivery of rAAV into amniotic fluid. This strategy 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 oral immune tolerance induction after the delivery to meet clinical settings.

7) Site Directed Gene Repair of the Dystrophin Gene in Satellite Cells Mediated by PNA-ssODNs Restores Dystrophin Expression into Skeletal Muscles of a Mouse Model for Duchenne Muscular Dystrophy

Farnoosh Nik-Ahd, Refik Kayali, Carmen Bertoni. Department of Neurology, University of California Los Angeles, Los Angeles, CA

Gene editing mediated by single stranded oligodeoxynucleoitdes (ssODNs) has shown to be able to correct single point mutations and to restore dystrophin expression in several models for Duchenne muscular dystrophy (DMD). This technology takes advantages of specific repair mechanisms present in the cells that are able to recognize the presence of mismatches in genomic DNA. Oligonucleotides complementary to the target sequence but containing a mismatch at the base targeted for modification are used as templates for the correction process. Once introduced into the cell, they have been shown to anneal to the genomic DNA sequence targeted for repair and initiate the repair process leading to a single nucleotide exchange that is stably inherited throughout cell division. We have recently demonstrated that ssODNs made of peptide nucleic acid (PNA-ssODNs) are more effective in inducing single base alterations than oligonucleotides made of unmodified bases in muscle cells in culture as well as into skeletal muscle of dystrophin deficient mice. For therapeutic applications, gene correction strategies mediated by oligonucleotides are likely to require targeting not only mature myofibers, but also muscle progenitor cells also known as satellite cells (SC) that actively participate to muscle regeneration and repair. We have tested the ability of PNA-ssODNs to correct the dystrophin gene defect in SC of the mdx^5cv mouse. This strain has a point mutation in exon 10 of the dystrophin gene that creates a cryptic splice site for the dystrophin mRNA and is particularly useful to perform quantitative analysis of gene correction frequencies at both the mRNA and the genomic DNA levels. SC isolated from mdx^5cv muscles were transfected with correcting PNA-ssODNs targeting the non-coding strand of the dystrophin gene defect and transplanted into skeletal muscles of immunosuppressed dystrophin deficient mice. As control, we used PNA-ssODNs lacking the mismatch. Restoration of dystrophin expression was assessed in vitro at the mRNA and protein levels. Gene correction was demonstrated at the genomic level by direct sequencing of PCR products derived from cells transfected with targeting oligonucleotides and maintained in culture. Dystrophin was clearly detected in muscles that received mdx^5cv SC transfected with the correcting oligonucleotide but not in muscles transplanted with SC treated with control PNA-ssODNs. Expression of dystrophin was stable for up to six months after transplantation as demonstrated by immunostaining and western blot analyses. These results support the use of PNA-ssODN as a viable gene editing tool for the treatment of muscle diseases due to point mutations using cell-mediated regenerative approaches. Supported by a grant from the Muscular Dystrophy Association of the United States (MDA)

8) Long-Term Engraftment of Mesenchymal Stromal Cells That Can Differentiate To Form Myogenic Cells in Dog with Duchenne Muscular Dystrophy

Yuko Nitahara Kasahara, Hiromi Hayashita Kinoh, Hironori Okada, Jin-Hong Shin, Akiyo Nishiyama, Sachiko Ohshima Hosoyama, Michiko Wada Maeda, Akinori Nakamura, Takashi Okada, Shin'ichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. Multipotent mesenchymal stromal cells (MSCs) could be potential therapeutics, because they can be transplanted to muscle tissue and differentiated to form myogenic cells in situ. We developed the strategies for effective cell transplantation in dystrophic dog. Methods: Canine CD271-positive (+) MSCs obtained from donor bone marrow cells were enriched by immunomagnetic isolation. The dog leukocyte antigens (DLA) were analyzed to determine DLA- identical mating pairs to achieve donor or recipient of allogeneic transplantation. Cardiotoxin (CTX) was injected into tibialis anterior (TA) muscles of normal Beagle dog 5 days before MSCs injection. CD271⁺ MSCs (2x10⁶ cells) transduced with an adenoviral vector expressing MyoD (Ad-MyoD) were injected into the TA muscles of recipient canine X-linked muscular dystrophy in Japan (CXMD_J) without immunosuppression. For intra-arterial injection, MyoD-transduced CD271⁺ MSCs (5x10⁶ cells) were administered into the femoral artery with transient avascularization using a tourniquet followed by the injection of papaverin hydrochloride. The treated muscles were biopsied and analyzed histologically. Autologous CD271⁺ MSCs (2x10⁶ cells) were transduced with AAV-microdystrophin and administrated into the TA muscles of DMD dog. Results: CD271⁺ MSCs obtained from the normal Beagle dog showed greater growth expansion compared with CD271-depleted MSCs. MyoD transduction of CD271⁺ MSCs caused myogenic differentiation in vitro and myotube formation with late myogenic markers expression. Without immunosuppression, CD271⁺ MSCs in the myogenic cell lineage were transplanted into DLA-identical CXMD_J and formed clusters of muscle-like tissue at 8 and 12 weeks after the intra-muscular injection. The engraftment of CD271⁺ MSCs was also found at the site of CTX-injured muscle after the intra-arterial injection. Interestingly, CD271⁺ MSCs engraftment was improved with the pre-treatment of papaverin hydrochloride. Immunohistological analysis suggested that CD271⁺ MSCs formed muscle-like tissues and exhibited upregulation of developmental myosin heavy chain as well as dystrophin without inflammatory cell infiltration. Furthermore, CD271⁺ MSCs derived from CXMD_J also showed myotube formation in vitro by the expression of microdystrophin. We are currently conducting the transplantation of AAV-microdystrophin-transduced autologous MSCs into the CXMD_J and will discuss the effects. Conclusion: MyoD-transduced CD271⁺ MSCs enabled more efficient realization of MSCs transplantation by the intra-muscular as well as intra-arterial injection. This strategy of MSCs propagation and treatment would be promising for the future DMD cell therapy.

9) Systemic Reversal of Dominant Muscle Disease in a Mouse Model of Muscular Dystrophy

Sergia Bortolanza, Davide Gabellini, Joel R. Chamberlain. Dulbecco Telethon Institute, Seattle, WA; Division of Regenerative Medicine, San Raffaele Scientific Institute, Seattle, WA; Medicine, University of Washington, Seattle, WA

Dominantly inherited genetic disease associated with a gain-of-function mechanism poses a particular challenge for gene therapy development. A straightforward therapeutic scheme would be to eliminate the genetic product that triggers pathogenic processes. We have chosen to pursue an RNAi-based approached aimed at targeted knockdown of FRG1 mRNA in the FRG1 mouse model of facioscapulohumeral muscular dystrophy. The FRG1 mouse displays a variety of typical physical and molecular features of FSHD patients. Using the FRG1 mouse model we can harness the endogenous cellular RNAi pathway to degrade mRNA from genes expressing a toxic product in a targeted fashion. We have packaged extended short hairpin RNA (shRNA) expression cassettes targeting FRG1 mRNA into adeno-associated virus serotype 6 (AAV6) shuttles for systemic delivery in vivo. AAV6 was selected as our gene transfer vector because of its predominant muscle tropism, high efficiency transduction of muscle cells, and long-term expression of transgenes. Intravenous tail vein injection of 5 x 10¹² vector genomes of the AAV6 FRG1 shRNAs into dystrophic mice resulted in a significant improvement in muscle function as measured by treadmill running with a concomitant reversal of phenotypic changes making the mice indistinguishable from wild-type mice. We also observed improvements in histological features, including a reduced fiber size, central nucleation, adipose accumulation, and fibrosis. Accompanying molecular changes included reduction of FRG1 mRNA by 40-60% relative to saline-injected controls. Our data indicate that RNAi-mediated mRNA knockdown is a feasible approach to dominant genetic disease therapy and can be applied after onset of symptoms of a dominant muscular dystrophy in mice to reverse the course of the disorder. Application of this method to target relevant components involved in muscle disease offers a route to clinical application of RNAi for treatment of FSHD and other dominant muscle disorders.

10) Exploring the Role of Deubiquitinating Enzyme A20 for Treatment of Muscular Dystrophy in mdx Mice

Rakshita Charan, Gabriela Niizawa, Hiroyuki Nakai, Paula R. Clemens. Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Neurology Service, Department of Veteran Affairs Medical Center, Pittsburgh, PA; Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is one of the most common muscle disorders affecting about 1 in 3500 male births worldwide. It is caused by mutations in the gene dystrophin, the protein product of which is required for muscle structure and stability. Studies suggest that the lack of dystrophin protein leads to dystrophic pathology in skeletal muscle; an important pathologic mediator is nuclear factor kappa B (NF-κB), a transcription factor that regulates several genes responsible for stress responses, cell survival and various inflammatory conditions. NF-κB is pathologically activated in dystrophic muscle in DMD patients and in the mdx mouse model for DMD. NF-κB activation in mdx mice is thought to activate protein degradation and cause chronic inflammation in skeletal muscle. Furthermore, pathological activation of NF-κB downregulates myogenic regulatory factors and interferes with muscle regeneration. Attenuation of NF-κB activation in mdx mice has been shown to improve muscle stability and strength. Thus, strategies to inhibit NF-κB activation are being actively pursued as a therapeutic option for DMD. A20, a deubiquitinating enzyme, is known to attenuate NF-κB activation by deubiquitinating RIP1. We sought to characterize A20 in skeletal muscle and examine its role as a potential therapeutic target to attenuate NF-κB activation in DMD. Our studies show that siRNA blockade of A20 significantly increases NF-κB activation in cultured mdx and control C57BL/10 myotubes in vitro. Since NF-κB is known to play a role in muscle regeneration and differentiation, we studied the effect of blocking A20 on muscle differentiation in myoblasts obtained from these mice. We observe a delay in myoblast differentiation in the absence of A20 indicating a role for A20 in promotion of muscle differentiation. Further characterization of A20 localization in vivo in mdx muscle demonstrated expression predominantly in regenerating fibers; interestingly, most are fast-twitch muscle fibers. A time-profile assessment of A20 expression in mdx mice compared with control mice showed an increase in A20 protein expression during the 7-12 week time period in mice that corresponds with the ages when cycles of severe muscle degeneration and regeneration occur in mdx mice. This correlation between expression of an NF-κB inhibitor attenuating NF-κB activation and pathology of DMD is a promising observation to further explore the efficacy of A20 as a therapeutic target. We are developing adeno-associated viral vectors carrying A20 expression cassettes in order to test the therapeutic benefit of A20 over-expression in the mdx model of DMD.

11) Restoration of Dystrophin Expression in the mdx Mouse Model for Duchenne Muscular Dystrophy (DMD) Induced by RTC13

Refik Kayali, Liutao Du, Jin-Mo Ku, Gladys Completo, Olga Prikhodko, Yosuf Subat, Hailiang Hu, Michael Jung, Richard A. Gatti, Carmen Bertoni. Department of Neurology, David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA; Department of Pathology and Laboratory Medicine, David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA; Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations. We have tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Intramuscular injections of RTC13, promoted read-through of the mdx UAA stop codon more efficiently than gentamicin, PTC124 or RTC14 making it our lead drug candidate. When administered systemically, RTC13 was shown to restore dystrophin protein in different muscle groups, including diaphragm and heart. Improved muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase (CK) levels demonstrating that the compound was able to slow down muscle degeneration and turnover. No signs of toxicity were detected in mdx after prolonged administration of RTC13 demonstrating that the compound was well tolerated in mice. The levels of direct bilirubin (DBIL), blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were significantly decreased in RTC13-treated mice as compared to untreated mdx or mdx mice that received vehicle alone confirming that restoration of dystrophin expression in muscles was able to ameliorate some of the secondary pathology associated with the disease in mdx. Structure activity relationship (SAR) studies were used to optimize the molecular structure of RTC13 and to identify a derivative that meets optimal safety profiles while still maintaining maximal read-through activity. These results advance the development of RTC13 as an effective drug candidate for DMD. They also offer hope for the treatment of numerous other genetic disorders due to nonsense mutations and premature termination of protein synthesis.

12) Novel Dual AAV Vectors To Express an nNOS-Recruiting Mini-Dystrophin Gene for Duchenne Muscular Dystrophy Gene Therapy

Yadong Zhang, Dongsheng Duan. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO

Duchenne muscular dystrophy (DMD) is a lethal childhood muscle disease. We recently demonstrated that a 7kb synthetic mini-dystrophin gene that can restore neuronal nitric oxide synthase (nNOS) may yield superior protection in the murine models of DMD (J Clin Invest. 2009;119: 624-35). Adeno-associated virus (AAV) holds great promise in treating all affected muscles in the body. However, AAV application in DMD gene therapy is limited by the 5 kb maximal virion packaging capacity. We have previously developed a series of dual AAV vector strategies for doubling AAV packaging capacity. These include overlapping, trans-splicing and hybrid vector approaches. In dual AAV vectors, a fully functional expression cassette is split into two parts and each part is individually packaged in an AAV virion. Reconstitution is achieved through homologous recombination and/or AAV inverted terminal repeats-mediated viral genome concatamerization. In this study, we first developed two GFP-dystrophin fusion constructs. In one construct, GFP is fused in-frame to the 5'-end of the 7 kb mini-dystrophin gene. In another construct, GFP is fused in-frame to the 3'-end of the 7 kb mini-dystrophin gene. A series of overlapping and hybrid dual AAV vectors were engineered to express the GFP-mini-dystrophin fusion gene. Dual AAV vectors were packaged in Y445F tyrosine mutant AAV serotype-6 vectors. Initial studies in dystrophin-deficient mdx4cv mice revealed robust mini-dystrophin expression and successful recruitment of nNOS to the sarcolemma. Additional studies are currently underway to test functional rescue with these new dual AAV mini-dystrophin vectors (Supported by NIH and MDA).

13) Fukutin-Related Protein Knock-Down by Systemic AAV9-shRNA Delivery or by Transgenic Knock-In Mutation Resulted in Similar Muscle and Heart Pathology of LGMD 2I

Chi-Hsien Wang, Yiumo M. Chan, Bin Xiao, Chunping Qiao, Ru-Hang Tang, Hui Zheng, Elizabeth Keramaris-Vrantsis, Juan Li, Peijuan Lu, Qilong Lu, Xiao Xiao. Department of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel Hill, NC; McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC

Fukutin-related protein (FKRP) has been speculated as a putative glycosyltransferase. The mutations of the FKRP not only cause late onset and milder limb-girdle muscular dystrophy 2I (LGMD2I) without central nervous system defect, but also cause severe allelic diseases such as congenital muscular dystrophy 1C, Walker-Warburg Syndrome and muscle-eye-brain disease. Since FKRP knockout mice are embryonic lethal, alternative methods are explored to generate viable animal models that closely recapitulate LGMD2I clinical phenotypes. Previously, we successfully knocked down the FKRP expression in the hind limb muscles for 10 months post i.m. injection (mpj) and recapitulated pathological and histological signs of LGMD 2I. Here, we investigated two different methods, AAV9-mediated systemic delivery of shRNA to knock-down FKRP (FKRP KD) in normal mice and genitic knock-in of the FKRP L276I missense mutation (L276I KI), to see if we can establish representative LGMD 2I animal models. To evaluate both mice models, the dystrophic pathologies were monitored. Firstly, central nucleation was observed in quadriceps of the 4 months old homo FKRP L276I KI mice. For the FKRP KD mice, it would only be found at 9 mpj. Glycosylation of the α-dystroglycan was reduced in the quadriceps of both groups at 9 months. We also found abnormal electrocardiogram (ECG) in the FKRP KD group, i.e., cardio conduction abnormality (left bundle branch block, LBBB) but there was no significant change in heart functions, e.g., percent of ejection fraction (EF%): 54.2±5.9 vs control: 54.0±1.6. Differently, the homo FKRP L276I KI mice showed both LBBB and right bundle branch block (RBBB). Furthermore, heart function impairment was revealed by echocardiography (Echo), i.e. significant decreases in EF% and percentage fractional shortening (FS%) in homo FKRP L276I KI mice (EF%: 73.7±10.5 vs. control 82.3±3.6; FS%: 42.5±8.4 vs. control 49.6±3.8). The echo readings of left ventricular internal dimensions in diastole (LVIDd) and systole (LVIDs) were also significantly increased in homo FKRP L276I KI mice (LVIDd: 3.3±0.4 vs. control: 2.8±0.3; LVIDs: 1.9±0.5 vs. control: 1.5±0.2), indicating dilated left ventricle. Moreover, the LV volumes in end-systole (LV-Vol; d), end-diastole (LV-Vol; s), and mass were significantly increased in homo FKRP L276I KI mice (LV-Vol; d: 44.0±12.9 vs 30.0±6.4; LV-Vol; s: 12.6±8.0 vs 5.9±1.8; LV Mass: 91.6±14.9 vs 75.2±14.4), suggesting enlarged left ventricle. In summary, using two different methods to reduce the FKRP expression in mice, we have obtained similar LGMD2I phenotypes. But an earlier onset of myopathies and severe heart failure were found in homo FKRP L276I KI mice. These animal models should be useful in studying LGMD 2I pathology as well as development of therapeutic approaches, such as gene therapy.

14) Inhibition of CD26 Activity Enhances Engraftment of Donor Cells to Regenerating and Dystrophic Skeletal Muscle

Maura H. Parker, Carol Loretz, Rainer Storb, Stephen J. Tapscott. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA; Department of Medicine, University of Washington, Seattle, WA; Department of Neurology, University of Washington, Seattle, WA

Muscle-derived cell transplantation has the potential to effectively treat many human diseases, including muscular dystrophy. A variety of cell populations engraft into skeletal muscle of mdx mice, effectively restore dystrophin expression and reconstitute the satellite cell pool. Yet, a direct and quantitative comparison of engraftment to determine the most effective cell population is lacking. We have developed a canine-to-mouse xenotransplantation model to rapidly and quantitatively compare canine muscle cell engraftment. Specifically, we demonstrate that canine muscle derived cells engraft into regenerating mouse muscle, and engraftment is quantifiable and consistent. The canine-to-mouse model allows us to quantitatively compare cell populations and modulating factors, and establish priority for transplantation experiments using a clinically relevant immune tolerant cxmd canine model of muscular dystrophy. We used the xenotransplant model to show that canine muscle derived cells sorted for expression of CXCR4 do not display a greater level of engraftment when compared to a mixed cell population. However, pre-treating a mixed cell population with diprotin A, a positive modulator of CXCR4-SDF-1 binding, significantly enhances engraftment of donor cells to the mouse satellite cell niche. Translating these results to the immune tolerant canine, we demonstrate that injection of diprotin treated donor cells results in a significantly increased number of muscle fibers expressing dystrophin as comapred to untreated cells. Temporal regulation of CXCR4/SDF-1 binding may be an important means of expanding the effective range of engraftment after transplantation.

15) Antisense Drug Development for Skipping Human Dystrophin Exons In Vitro and In Vivo Systematically

Bo Wu, Ehsan Benrashid, Peijuan Lu, Caryn Cloer, Allen Zillmer, Mona Shaban, QiLong Lu. McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC

Antisense therapy has recently been demonstrated with great potential for frame-restoring of dystrophin mRNA in human muscle cells and in local muscles of Duchenne muscular dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs), for targeted skipping of human dystrophin exons. Identification of AOs with highest efficiency and specificity is therefore crucial for AO drug development. In this study, we applied three cell culture systems, C2C12 myoblasts containing a GFP-reporter gene expressing human dystrophin exon 50, normal human myoblasts and a culture of DMD patient-derived skin fibroblasts, to screen AOs targeting human dystrophin exon 50. The GFP reporter system was most effective and sensitive for quantitative measurement of exon skipping efficiency. The efficacy of selected AOs was further investigated with Vivo-Morpholino chemistry in the hDMD transgenic mouse carrying the full-length human dystrophin gene. Our results suggest that a combination of in vitro cell culture based selection systems and a Vivo-Morpholino based evaluation in vivo systemically, provides stringent screening to identify effective AOs targeting human dystrophin exon for drug development to treat DMD patients.

16) AAV-Based shRNA Silencing of NF-κB Ameliorates Muscle Pathologies in mdx Mice

Qing Yang, Ying Tang, Kara Imbrogno, Aiping Lu, Anmin Chen, Fengjin Guo, Freddie H. Fu, Johnny Huard, Bing Wang. Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China

Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease affecting 1 of every 3500 male births. Chronic inflammation, promoted by an up-regulated NF-kappaB (NF-κB) pathway, plays a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Higher immune alertness, due to chronic inflammation and immune effector cell infiltration, challenges recombination adeno-associated viral (AAV) vector-mediated gene replacement treatment for DMD, especially in large animal models and clinical trials. We and others found that blocking the NF-κB pathway could be a viable solution to promote muscle regeneration and to decrease necrosis in mdx mice (a murine DMD model), by using transgenic and AAV-mediated transfer (dominant-negative forms of IKKα & β) approaches, without any side effect. Based on the small interference RNA (siRNA) technology used in this study, we examined whether an AAV vector carrying small hairpin RNA (shRNA) targeting the NF-κB/p65 subunit, a major subunit of NF-κB associated with inflammation in mdx mice, inhibits NF-κB activation as well as ameliorates pathological necrosis and regeneration in the locally-treated gastrocnemius (GAS) muscle of 1 and 4 month old mdx mice. At 1 month post-treatment, NF-κB/p65 levels in locally-treated muscles were significantly decreased by the efficient transfer of specific shRNA in both of the different treated age groups. This led to remarkable decreases in necrosis and myofiber damage compared to mdx mice treated by a phosphate-buffered saline (PBS) or a control AAV vector containing a scrambled shRNA. The shRNA silencing of the NF-κB pathway diminished the pathological myofiber regeneration in young mdx mice treated at 1 month of age, but it enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age. Moreover, quantitative analysis revealed that central nucleation of the myofibers of treated young mdx muscles was reduced from 67% to 34%, but centrally-located nuclei were not significantly reversed to the peripheral position in treated old mdx mice. Our findings indicate that AAV-mediated NF-κB/p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by degrading mRNA of the NF-κB/p65 subunit.

17) Implication of nNOSμ Delocalization on the Muscle Force in Dystrophin Deficient and δ-Sarcoglycan Knockout Mice

Dejia Li, Yongping Yue, Yi Lai, Chady H. Hakin, Dongsheng Duan. Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO

The mechanism of force reduction is not completely understood in Duchenne muscular dystrophy (DMD), a dystrophin-deficient lethal disease. Nitric oxide regulates muscle force. Interestingly, neuronal nitric oxide synthase μ (nNOSμ), a major source of muscle nitric oxide, is lost from the sarcolemma in DMD muscle. We hypothesize that nNOSμ delocalization contributes to force reduction in DMD. To test this hypothesis, we generated dystrophin/nNOSμ double knockout mice. Genetic elimination of nNOSμ significantly enhanced force in dystrophin-null mice. Pharmacological inhibition of nNOS yielded similar results. To further test our hypothesis, we studied δ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy. These mice had minimal sarcolemmal nNOSμ delocalization and muscle force was less compromised. Annihilation of nNOSμ did not improve their force either. To determine whether nNOSμ delocalization itself inhibited force, we corrected muscle disease in dystrophin-null mice with micro-dystrophins that either restored or did not restore sarcolemmal nNOSμ. Similar muscle force was obtained irrespective of nNOSμ localization. Additional studies suggest that nNOSμ delocalization selectively inhibits muscle force in dystrophin-null mice via nitrosative stress. In summary, we have demonstrated for the first time that nitrosative stress elicited by nNOSμ delocalization is an important mechanism underlying force loss in DMD.

18) Evaluation of rAAV6-Microutrophin Expression Driven by a Highly Active Muscle Specific Regulatory Cassette in Dystrophic Mice and the Non-Human Primate

Guy L. Odom, Glen B. Banks, Quynh Nguyen, Eric Finn, James M. Allen, Jacqueline Wicki, Stephen D. Hauschka, Jeffrey S. Chamberlain. Neurology, University of Washington, Seattle, WA; Biochemistry, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD), the most common inherited muscle disorder of children, is caused by mutations within the dystrophin gene and results in progressive muscle wasting. Several genetic intervention strategies are under investigation, and progress towards clinical gene therapy with adeno-associated viral (AAV) vector delivered transgenes remains promising. Highly truncated dystrophin coding sequences have previously been shown to be therapeutic in small and large animal models of DMD following rAAV gene transfer. However, given the potential for deleterious cellular immune responses to dystrophin in human patients, particularly those with large deletions in the dystrophin gene, the capacity of utrophin (a dystrophin paralog) to act as a therapeutic transgene is of high interest. To this end, we provide a comparative regulatory element study evaluating microutrophin expression and functionality in the highly dystrophic mouse model (mdx/utrn-/-). We also tested microutrophin expression in a large non-human primate, macaca nemistrina. mdx/utrn-/- mice were injected intravenously with rAAV6/ microutrophin vectors, carrying either the ubiquitously active CMV or under control of a novel muscle specific promoter (MSP) containing modified versions of the M-creatine kinase enhancer and promoter. Treated mice, displayed alleviation of most of the pathophysiological abnormalities associated with muscular dystrophy and no significant differences were observed between animals treated with the two types of vectors. Importantly, the MSP vector demonstrated a clear lack of expression in non-muscle cell types both in vivo and in vitro. We have also generated rAAV6 vectors that express a macaca nemistrina derived microutrophin. Intramuscular injection of this non-human primate utrophin vector demonstrated widespread expression along the sarcolemma of monkey myofibers, where it co-localized with endogenous dystrophin. Consistent with studies in mice, no evidence of a dominant negative effect of the utrophin expression in wild type macaca nemistrina muscles was observed. These studies reveal that microutrophin can function not only as a therapeutic trangene in dystrophic models, but it can also serve as a sensitive reporter gene to develop systemic gene delivery protocols in large animal models. These rAAV/microutrophin vectors can also serve as a test system for the potential immunogenicity of microutrophin. Preliminary studies on the potential immunogenicity of this delivery system will be presented.

19) Efficient Long-Term Bodywide Expression of an AAV9-Minidystrophin in the Muscle and Heart of Young Adult GRMD Dogs after Intravascular Injection without Immune Suppression

Juan Li, Chunping Qiao, Janet Bogan, Dan Bogan, Ruhang Tang, JianBin Li, Chunlian Chen, Hui Zheng, Jennifer Dow, Joe N. Kornegay, Xiao Xiao. Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC; Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

Previously we reported that AAV9 rendered long-term bodywide expression of a human minidystrophin after intravenous injection in neonatal golden retriever muscular dystrophy (GRMD) dogs; and that AAV8 achieved long-term expression of a canine minidys in the limb muscles after isolated limb vein perfusion in young adult GRMD. Here we report that long-term (≥ 2 years) and bodywide AAV9-minidys expression was also achieved without any immune intervention in young adult GRMD dogs, either by isolated limb vein perfusion or by simple intravenous (i.v.) injection. Methods: 1) Isolated limb perfusion. Three GRMD dogs received 1 x 1013 v.g./kg vector. Dog Jelly (2 mon old; 6.3 kg) was given AAV9-CMV-opti-hMinidys (codon-optimized human minidys); while Jasper and Peridot (4 mon old; 12.2 kg and 12.5 kg) were given AAV9-CMV-cMinidys (canine minidys). The vectors were injected via leg vein with a tourniquet placed at hindlimb proximal extremity for 10 minutes. Muscle biopsies were taken at 2, 7, 12 & 24 months and analyzed. 2) Intra-venous injection. Dog Duncan (2.5 mon old; 6.5 kg) was given a simple i.v injection of AAV9-MS-opti-hMinidys (muscle-specific promoter) at the dose of 4 x 1013 v.g./ kg. Biopsy and necropsy were done at 4 and 12 months. Results: 1) In the limb perfusion study, all 3 dogs had long-term, widespread minidys expression in all muscle biopsies with improved histology. The most important finding was that both injected and non-injected limb had minidys expression. Up to 70% of myofibers were minidys positive in certain muscle groups without discernable T-cell infiltration. Both legs showed similar degree of expression. The human minidys persisted for ≥ 2 years (last biopsy) in Jelly, now 4 years of age. But in Jasper and Peridot, injected with a canine minidys gene without codon-optimization, stronger expression was always seen in the vector-injected legs in the 22 months time course. No minidys was detected in the heart. 2) In the intravenous injection study, dog Duncan showed robust and uniform minidys expression in the majority of the skeletal muscles for 1 year (duration of the study). The most important finding is the efficient, MS promoter-mediated cardiac expression, which was previously found very weak or undetectable in the heart with the CMV promoter. Conclusions: 1) AAV9 achieved systemic minidys gene delivery after isolated limb perfusion, possibly due to incomplete tourniquet blockade; 2) A simple i.v. injection of AAV9-MS-opti-hMinidys achieved robust and long-term bodywide expression; 3) Efficient cardiacexpression after i.v. injection is most likely due to the use of muscle-specific promoter; 4) Long-term expression of both human or canine minidys under the control of different promoters suggest the lack of or minimal cellular immunity. The mechanisms remain elusive and warrant further studies.

20) Effective Limb Transduction and Phenotypic Correction after Injection of rAAV8-U7 snRNA in GRMD Dogs

Caroline Le Guiner, Marie Montus, Laurent Servais, Luis Garcia, Yves Fromes, Jean-Yves Hogrel, Pierre Carlier, Yan Cherel, Philippe Moullier, Thomas Voit, The AFM-Sponsored Duchenne Consortium. Genethon, Evry, France; INSERM UMR 649, Nantes, France; Institut de Myologie, Paris, France; INRA UMR 703, Nantes, France

In Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in in-frame mRNA transcripts that are translated into shorter but functionally active dystrophin. The goal of our project was to determine in GRMD, the effective dose of our therapeutic product defined as a recombinant Adeno-Associated Virus serotype 8 (rAAV8) expressing a modified U7 snRNA specific for the skipping of exons 5 to 10 of the GRMD dystrophin transcript. The mode of delivery was the locoregional high-pressure intravenous (IV) injection of a forelimb. Several groups of GRMD dogs were exposed to different rAAV8-U7snRNA doses. Each dog was followed ∼3 months after injection. The primary outcomes were the restoration of dystrophin expression and the improvement of the tissue pathology in the injected limb compared to the controlateral limb. The secondary outcomes were the muscle strength correction, the biodistribution and shedding patterns as well as the immune response against rAAV8 capsid and dystrophin. Our preliminary results suggest a dose effect of our therapeutic rAAV. Injection of 2,5E13vg/kg and of 5E12vg/kg of our vector was able to restore 50 to 80% of Dystrophin expression in the injected limb. This expression of a semi-functional dystrophin resulted in improvement of tissue morphology as well as of several functional and MRI parameters. No tissue inflammation occurred following the procedure. We built a unique network of laboratories with complementary skills to deliver a GLP-compliant set of preclinical data to further define the regulatory toxicology studies. The organization of our network and the results obtained in our GRMD dogs study will be presented. This project is supported by AFM (Association Française contre les Myopathies) and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by research and innovation aid from the French public agency, OSEO.

21) Stem Cells Isolated from p65 Deficient Mice Improved the Histopathology of Skeletal Muscle in Dystrophic Mice

Aiping Lu, Ying Tang, Minakshi Poddar, Bing Wang, Denis C. Guttridge, Paul D. Robbins, Johnny Huard. Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, OH; Microbiogy and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is a deadly genetic disease mainly characterized by progressive weakening of the skeletal, cardiac and diaphragmatic muscles. It is critical to find a successful therapy that will improve the histopathology of the muscles of DMD patients and restore their normal function. Recent studies have demonstrated that blocking p65, a subunit of NF-κB, enhances muscle regeneration in injured and diseased skeletal muscle which suggests that the NF-kB signaling pathway is a contributing factor to the dystrophic pathology in DMD patients. Previously we demonstrated that muscle derived stem cells (MDSCs) isolated from the skeletal muscles of heterozygote P65 knock-out (P65+/-) mice showed better muscle regeneration in vitro and in vivo compared to the MDSCs isolated from wild-type (WT) mice. We also demonstrated that the transplantation of P65+/- MDSCs could reduce inflammation. Based on these results we performed a set of experiments to determine if these P65+/- MDSCs could alleviate the pathology associated with DMD more efficiently than wild-type MDSCs. When the p65+/- MDSCs were injected intraperitoneally (IP) into dystrophin/utrophin deficient (dys-/-:utro-/-, dKO) mice, a reliable mouse model of DMD, we found that the histopathology improvement in various skeletal muscles including the gastrocnemius, diaphragm, gluteus maximus and tibialis anterior muscles by H&E staining. Many centrally nucleated muscle fibers (new regenerated fibers) were found in the p65+/- MDSC injection group. Mouse IgG staining showed that there were less necrotic muscle fibers in the mice injected with p65+/- MDSCs compared to the PBS treated and un-treated muscle. An antibody against embryonic muscle heavy chain (E-MyHC) was used to evaluate newly regenerated and pathological muscle fibers and another antibody against F4/80 (a macrophage marker) was used to analyze the extent of inflammation in the muscle tissues. Those results showed less inflammation and pathological muscle fibers within the muscle injected with p65+/- MDSCs compared to untreated and PBS treated muscles. Trichrome staining showed there were less fibrosis and fewer necrotic muscle fibers in the mice injected with p65+/- MDSCs compared to PBS treated and the untreated muscles. Our findings indicated that IP transplantation of p65+/- MDSCs can decrease macrophage infiltration, fibrosis formation, and necrosis, while improving muscle regeneration in 4 to 6 week old dKO mice. We also attempted to inject WT MDSCs isolated from normal animals; however, the results did not show a similar benefit in the histopathology of the dKO mice. These results suggest that combining stem cell therapy with anti-NF-κB therapy is a potential therapeutic approach for the treatment of DMD. Additional experiments are required to determine whether the benefit observed with NF-κB inhibition via stem cell therapy is superior than anti-NF-κB gene therapy.

22) Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse

Tomohiro Suga, En Kimura, Yuka Morioka, Masahito Ikawa, Sheng Li, Katsuhisa Uchino, Yuji Uchida, Satoshi Yamashita, Yasushi Maeda, Jeffrey S. Chamberlain, Makoto Uchino. Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; Research Institute for Microbial Diseases, Osaka University, Suita, Japan; Department of Neurology, University of Washington School of Medicine, Seattle; Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, however it has not been studied in striate muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in type I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of type I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.

23) Losartan Enhances the Success of Myoblast Transplantation

Raouia Fakhfakh, Yann Lamarre, Daniel Skuk, Jacques P. Tremblay. Human Genetics Unit, Chul-Chuq, Quebec, QC, Canada

Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor β (TGF-β) is a negative regulator of skeletal-muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that down-regulates TGF-β expression. In vitro, blocking TGF-β activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in Tibialis anterior cross-sections 1 month post-transplantation revealed more human dystrophin-positive myofibers in these mice than in non-treated dystrophic mice. Thus blocking the TGF-β signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation and changing the expression of myogenic regulator factors.

24) Interference of Myostatin and TGF-beta Signaling by Antisense-Mediated Exon Skipping in ALK4⁄5 Receptors

Dwi U. Kemaladewi, Sandra H. van Heiningen, Annemieke Aartsma-Rus, Gert-Jan B. van Ommen, Peter ten Dijke, Peter A. C. 't Hoen, Willem M. H. Hoogaars. Human Genetics, Leiden University Medical Center, Leiden, Netherlands; Molecular and Cell Biology, Leiden University Medical Center, Leiden, Netherlands

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, which is characterized by the loss of muscle fibers and replacement by fibrotic tissue due to the lack of the dystrophin protein. Myostatin and Transforming Growth Factor (TGF)-beta play important roles in regulation of muscle differentiation and fibrosis, and are emerging as attractive therapeutic targets for DMD treatment. Both cytokines signal primarily via the overlapping Smad2⁄3-dependent signaling pathways. In this study we targeted myostatin⁄TGF-beta type 1 receptors Acvr1b (ALK4) and Tgfbr1 (ALK5) using antisense oligoribonucleotides (AON) targeting regions in the pre-mRNA encoding ligand binding and⁄or kinase domains of the receptors. Transfection of ALK4 or ALK5 AONs resulted in skipping of the targeted exon, ∼50% downregulation of the full length transcript and enhanced myoblast differentiation. In addition, local administration into mdx mice showed considerable downregulation of full length ALK4 (∼80%) or ALK5 (∼50%), leading to ∼50% increase of myogenic genes expression and ∼40 % decrease of fibrotic markers expression. Furthermore, we used these AONs to study how TGF-beta and myostatin signalings are regulated in different cells origins. We showed an exclusive requirement of ALK4 for myostatin signaling in myoblasts, but preference for ALK5 in the other cell types, which suggested that at least two co-receptors are involved in this cell-type specific regulation of myostatin signaling. In summary, we were able to use this specific genetic approach to selectively disrupt myostatin or TGF-beta signaling, dissect the overlapping pathways and revealed a novel muscle specific mechanism of myostatin signaling. Systemic administrations are currently ongoing to further assess the therapeutic benefits of these AONs and to determine the differential effect of ALK4 and ALK5 knockdown on the dystrophic pathology of the mdx mice.

25) PMO Delivery Systems with Bubble Liposomes and Ultrasound Exposure into Skeletal Muscles of the mdx Mice

Yoichi Negishi, Yuko Ishii, Shoko Sekine, Yoko Endo-Takahashi, Ryo Suzuki, Kazuo Maruyama, Yukihiko Aramaki. Department of Drug and Gene Delivery Systems, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan; Department of Biopharmaceutics, School of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa, Japan

Background: Duchenne muscular dystrophy (DMD) is genetic disorders causing by mutations in the DMD gene that lead to an absence of functional protein. Previous reports have shown that a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin induce dystrophin expression in dystrophin-deficient mdx mice. However, efficient delivery method of PMO into the mdx mice still needs to be developed. Ultrasound (US) in combination with microbubbles has recently been acquired much attention in the safe method of gene or antisense oligonucleotides (AOs) delivery. However, microbubbles have problems with size, stability, and targeting function. Liposomes have been known as drug, antigen, and gene delivery carriers. To solve the above-mentioned issues of microbubbles, we have previously developed the polyethyleneglycol (PEG)-modified liposomes entrapping echo-contrast gas, “Bubble liposomes” (BLs), which can function as a novel gene delivery tool by applying them with US exposure. In this study, to assess the feasibility and the effectiveness of BLs for a PMO delivery system into the mdx mice, we tried to deliver a PMO, which is designed to skip the mutated exon 23 from the mRNA of murine dystrophin, into skeletal muscles of the mdx mice by the combination of BLs and US exposure. Methods: A solution of BLs and PMO for mouse exon 23 was injected into tibialis (TA) muscle of mdx mice and US exposure (1 MHz, 2 W/cm², 60 sec., duty cycle 50 %) was immediately applied at the injection site. Two weeks after the treatment, exon 23 skipping levels were analysed by RT-PCR. The number of dystrophin-positive fibers after the treatment was also analysed by immunohistochemical analysis. Results: Mice were euthanized 2 weeks after the injection, the injected muscles were isolated and analyzed by RT-PCR and by immunohistochemistry. In the combination of BLs and ultrasound exposure, we found that the PMO significantly restored the dystrophin mRNA expression and dystrophin-positive fibers by skipping the mutated exon 23 in the injection site of mdx mice, compared to the PMO injection alone. Conclusions: These results suggest that PMO delivery into the muscle of mdx mice can be improved by the combination of BLs and US exposure. This US-mediated BLs technique may provide an effective noninvasive method for DMD therapy in the clinical field. Acknowledgments: This study was supported by Industrial Technology Research Grant Program (04A05010) in '04 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.

26) Exploring the Potential of Plasma Pheresis To Circumvent Pre-Existing Antibodies to AAV8 on Transgene Expression Following Targeted Vascular Delivery

Louis G. Chicoine, Louise R. Rodino-Klapac, K. Reed Clark, Chrystal L. Montgomery, Thomas J. Preston, William G. Bremer, Katherine J. Campbell, Zarife Sahenk, Paul T. Martin, Christopher M. Walker, Jerry R. Mendell. Pediatrics, The Ohio State University/Nationwide Children's Hospital, Columbus, OH; Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH; Center for Vaccines and Immunity, Research Institute at Nationwide Children's Hospital, Columbus, OH; Neurology, The Ohio State University/Nationwide Children's Hospital, Columbus, OH

Gene therapy for muscle disease has taken several positive steps forward. We recently reported sustained AAV mediated alpha-sarcoglycan (SGCA) gene expression for as long as 6 months in 5 of 6 limb girdle muscular dystrophy (LGMD2D) treated patients (Mendell et al. 2010). The one patient that did not express SGCA was found to have high AAV neutralizing antibodies prior to gene transfer. With vascular gene therapy to treat multiple muscles as the next logical step clinically, impediments may lie with the immune response and the presence of pre-existing antibodies to various AAV serotypes. We tested the hypothesis that pre-existing antibodies to AAV8 would attenuate transgene expression following vascular delivery of two potentially therapeutic transgenes AAV8.MCK.micro-dystrophin.Flag and AAV8.MCK.Galgt2 in a large study of 72 non-human primates. Prior to gene transfer, all animals were pre-screened for binding antibodies to AAV8. Those animals with a titer of <1:100 were considered negative, while others (>1:100) were considered positive. Animals were stratified into positive and negative groups with scheduled sacrifice times at 3 and 6 months (n=9 per group). Vascular delivery to the gastrocnemius muscle of an isolated limb (Rodino-Klapac et al. 2010) was performed on each animal where 2 x 10¹² vg/kg of the appropriate vector was given. None of the animals suffered noticeable edema or adverse effects from the procedure. Three or six months after transfer, the macaques were euthanized, and the gastrocnemius muscle was harvested. Samples of the proximal, central and distal muscle were stained with anti-FLAG or CT2 antibody. The contralateral gastrocnemius served as a negative control. Gene expression was visualized in all subjects and subjects without pre-existing AAV8 antibodies demonstrated significantly higher transgene expression than subjects with pre-existing antibodies (62% vs 33% muscle fibers transduced, P ≤ 0.001). Regression analysis confirmed a direct inverse correlation between the AAV8 antibody titer and the percent gene expression. From these data we conclude that the presence of pre-existing antibodies to the vector will attenuate transgene expression. Plasma pheresis studies appeared to optimize transgene expression and this procedure would potentially be used to increase the number of patients amenable to gene therapy and make some patients candidates for retreatment when necessary.

27) Low-Level Expression of a Near-Full Length Dystrophin Improves Muscle Force and Lifespan in a Strain of Severely Affected Dystrophin/Utrophin Double Knockout Mice

Dejia Li, Yongping Yue, Dongsheng Duan. Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, Columbia, MO

Utrophin/dystrophin double knockout mdx (u-dko) mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that minimal level dystrophin expression can improve the clinical outcome of u-dko mice. It has been shown that mdx3cv (3cv) mice express a near-full length dystrophin protein at ∼ 5% of the normal level. We crossed utrophin-null mutation (all utrophin isoforms are inactivated) to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

28) AAV9-Mediated FKRP Gene Therapy Restores the Expression of Functional Glycosylation of α-DG

Lei Xu, Pei Lu, Chi-Hsien Wang, Jianbin Li, Elizabeth Keramaris, Xiao Xiao, Qi Long Lu. McColl-Lockwood Laboratory for Muscular Dystrophy Research, Department of Neurology, Cannon Research Center, Carolinas Medical Center, Carolinas Healthcare System, Charlotte, NC; Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC

Mutations in FKRP gene are one of the most common causes of muscular dystrophies, accounting for more than 19% of all the reported Limb Girdle muscular dystrophies (LGMD). Currently there is no effective treatment available. Gene replacement therapy is one of the most promising therapeutic strategy. In this study we examined the effects of AAV-FKRP in the P448L^KI mice via local and systemic delivery. Mutant mice (12 months) were administrated with 5x10⁹ v.g of AAV9-CB-FKRP to the tibialis anterior (TA) muscle and 1x10¹² v.g via introperitoneal injection (i.p.). Muscles were examined 4 weeks after injections by immunohistochemistry and western blot with antibodies to FKRP and glycosylated epitope of functional α-dystroglycan. Our results showed FKRP expression in all muscles including heart, diaphragm and other skeletal muscles in the mice treated by systemic delivery of AAV9-CB-FKRP. Consistent with our previous observation, the induced FKRP was mainly localized to Golgi apparatus within muscle fibers. Expression of FKRP consequently restored functional glycosylation of α-DG as judged by immunohistochemistry with the IIH6 antibody in all muscles examined including the cardiac muscle. Expression of the FKRP transgene and induction of glycosylation of α-DG and its binding to laminin were confirmed in all the skeletal and cardiac muscles on Western blots. Our results thus further confirmed that loss of FKRP function is the cause of hypoglycosylation of α-DG and LGMD2I.

29) Mechanism of Uptaking Morpholino into Dystrophin-Deficient Muscle Fibers

Yoshitsugu Aoki, Tetsuya Nagata, Toshifumi Yokota, Shinichi Takeda. Department of Molecular Therapy, National Institute of Neuroscience, National Center for Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC

Introduction: A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AO) such as phosphorodiamidate morpholino oligomers (PMO). As we already demonstrated using exon 52 deficient mdx (mdx52) mice, short term systemic treatment of PMO targeting exon 51 induced highly efficient truncated but functional dystrophin in body-wide skeletal muscles (Mol Ther.2010;18:1995-2005). This result is encouraging for the ongoing multinational systemic exon-51 skipping clinical trial. However, the optimal therapeutic dose of PMO in DMD patients have not been established. Surprisingly, the mechanism of PMO entry into dystrophin-deficient fibers remain to be completely elucidated. Purpose: To verify the mechanism of PMO entry into dystrophin-deficient fibers using mdx52 mice. Methods: The combination of two AOs targeting the 5' and the 3' splice site was used for skipping exon 51. To research the relation between PMO entry and dystrophin deficient membrane, a total of 80-640 mg/kg of PMO was injected into the tail vein of mdx52 and C57BL/6J mice aged 5 wks singly. Muscles were isolated 2 weeks after the injection and analyzed by RT-PCR. Then, to determine the relationship between PMO entry and muscle regeneration, PMO (10 μg) was injected into each tibialis anterior (TA) muscle of mdx52 mice aged 3 to 32 wks with oral administration of BrdU (0.8 mg/ml for 7 days). Muscles were obtained 2 weeks after the injection and analyzed by RT-PCR, immunohistochemistry and western blotting. Results: Specific exon 51-skipping and dystrophin expression were induced in a dose-related manner in mdx52 mice, while on the other hand, no exon 51-skipping was detected in C57BL/6J mice even at 640 mg/kg. Dystrophin positive fibers were detected with the greatest numbers when PMO was injected into TA muscle of mdx52 mice aged 5 wks when the ratio of centrally nucleated fibers were highly increased. Dystrophin positive fibers were classified into the two groups: BrdU-labeled regenerated and -unlabeled degenerated fibers in mdx52 mice.

Conclusion: We suppose that there are two types of PMO entry into dystrophin-deficient fibers. Interpretation: BrdU-labeled regenerated fibers could uptake PMO regardless of dystrophin-deficiency and BrdU-unlabeled degenerated fibers could be 'fibers with leaky membrane'. Our study has a great potential to exploit an opportunity to optimize antisense therapy using PMO and to apply the treatment to other neuromuscular diseases.
 

30) Blocking the NF-κB Pathway Enhances Mini-Dystrophin Efficiency in Dystrophin/Utrophin Deficient Mice

Qing Yang, Ying Tang, Anmin Chen, Fengjin Guo, Freddie H. Fu, Johnny Huard, Bing Wang. Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China

Chronic inflammation, promoted by an up-regulation of the NF-kappaB (NF-kB) pathway plays a key role in the progressive muscle wasting and degeneration of Duchenne muscular dystrophy (DMD) patients. Higher immune alertness in dystrophic muscle, due to chronic inflammation and immune effector cell infiltration, challenges recombinant adeno-associated viral (rAAV) vector-directed DMD gene therapy in dystrophin/utrophin deficient (dys-/-:utro-/-, dKO) mice, a reliable mouse model of DMD. In our previous study, AAV-mediated systemic delivery of mini-dystrophin for the treatment of the dKO mice was effective at partially ameliorating the muscle pathology and increasing function in these mice, yet it was ineffective at reversing the course of muscle wasting due to the dystrophic muscle fibrosis, necrosis, and wasting which occurred in this chronically inflamed environment; therefore, in this study we combined mini-dystrophin replacement and anti-inflammation. We accomplished this by using two AAV vectors; one carried a mini-dystrophin gene and the other carried a short hairpin RNA (shRNA) to block the NF-κB pathway. The goal was to investigate if blocking inflammation could enhance mini-dystrophin efficiency. A single intraperitoneal (i.p.) injection was performed in the dKO neonates using a combination of the two AAV9 vectors which included AAV9-NF-κB/p65-shRNA_ZsGreen (7 x 10¹¹ v.g. particles/injection) and a low dose of the AAV9-CMV-hDysΔ3849 vector (5 x 10¹⁰ v.g. particles/injection). An injection of the low dose AAV9-mini-dystrophin vector (5 x 10¹⁰ v.g. particles/injection) served as the control. Mice were sacrificed 10 weeks after AAV vector injection for muscle histology analysis. The ZsGreen monitored the shRNA efficiency of AAV delivery in skeletal and cardiac muscles (Green). By monitoring the ZsGreen expression, we found that the shRNA was delivered to the skeletal, cardiac and diaphragmatic muscles very efficiently. The efficient transduction of the diaphragm and cardiac muscles was of particular interest because these are the muscles whose failure causes the pre-mature death of DMD patients. A higher level of mini-dystrophin gene expression was observed in the skeletal and cardiac muscles of the dKO mice treated with both the mini-dystrophin and NF-κB/p65-shRNA compared to the mice treated with mini-dystrophin alone. In this study, we found that enhanced mini-dystrophin expression could be achieved by combining the dystrophin gene replacement with shRNA silencing to block the NF-κB pathway using two AAV vectors. However, the simultaneous injection of two rAAV vectors was complex and would be inefficient for clinical application. This prompts us to develop a single AAV vector which will carry both the mini-dystrophin and NF-κB/p65-shRNA cassettes. This approach may offer a more synergistic effect via the combination of gene replacement and anti-inflammation in a single-step procedure.

31) Noninvasive Monitoring of Whole Body Muscle Function in Dystrophin-Deficient Dogs

Jin-Hong Shin, Brian B. Greer, Chady H. Hakim, Zhihai He, Dongsheng Duan. Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; Electrical and Computer Engineering, University of Missouri, Columbia, MO

Dystrophin-deficient dogs are highly valuable tools for evaluating gene/cell therapies to treat Duchenne muscular dystrophy (DMD). Unfortunately, robust muscle functional end points are poorly defined in the canine DMD models. Here, we described a set of non-invasive methods for whole body muscle function analysis in normal and affected dogs. Briefly, the gait and voluntary activity were recorded using high-resolution digital video recorders. Gait recoding was repeated three times on three different days for each dog. Voluntary activity monitoring was performed under low-lux red lighting between 7pm and 5am for each dog. The stride length, stride speed, range of the motion and overall activity were analyzed using a customer-developed program. Three normal and four affected dogs of a litter were examined. Affected dogs showed significantly reduced stride length (affected, 35.7 ± 8.5 cm; normal, 80.2 ± 6.1 cm; p <0.02) and stride speed (affected, 61.7 ± 16.4 cm/sec; normal, 156.5 ± 23.8 cm/sec; p <0.03). The range of the motion in forelimb (palm, forearm and wrist) and hind limb (sole, low leg and ankle) was also significantly decreased in affected dogs. The affected dogs also showed significantly less movement during overnight monitoring. In summary, our results suggest that whole body muscle function was significantly compromised in affected dogs. Non-invasive video recording may represent a convenient and reliable method to monitor disease progression and gene/cell therapy effect in canine models of DMD.

32) Recombinase Strategies To Make and Modify iPS Cells

Marisa Karow, Christopher L. Chavez, Alfonso P. Farruggio, Jonathan M. Gesinger, Joseph C. Wu, Yanru Chen-Tsai, Michele P. Calos. Stanford University School of Medicine, Stanford, CA

Induced pluripotent stem cells (iPSC) have revolutionized the stem cell field. iPSC are most often produced by using retroviruses, but the resulting cells are ill-suited for clinical application. Several alternative strategies to make iPSC have been developed, but none encompass within them a simple methodology to facilitate the precise insertion of a therapeutic gene for gene therapy approaches. Here we report practical strategies to create and genetically modify murine iPSC that rely on plasmid DNA and the sequential application of three site-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette at preferred locations in the genome, producing iPSC demonstrated to be pluripotent by a full range of in vitro and in vivo criteria, including teratoma formation and generation of chimeric mice. The reprogramming plasmid includes the target attP site for a second site-specific integrase, which can be utilized for precise insertion of a therapeutic gene into the target site. Cre recombinase can be employed for clean excision of the entire reprogramming cassette and most plasmid sequences. We demonstrated the efficient operation of this type of strategy in accessible cell types, including fibroblasts and adipose stem cells. Furthermore, to illustrate a clinically relevant example, we generated iPSC from the A/J limb girdle muscular dystrophy mouse model, introduced the therapeutic dysferlin gene, and deleted the reprogramming genes. This simple strategy produces pluripotent cells that are corrected for a genetic defect and have the potential to be used in a clinical setting.

33) Sustained Expression of Canine Micro-Dystrophin and Amelioration of Muscle Function in Dystrophic Dogs Following Large Scale AAV-Mediated Treatment

Zejing Wang, Rainer Storb, Donghoon Lee, Christine Halbert, Martine K. Childers, Glen Banks, James Allen, Eric Finn, Martin Kushmeric, Dusty Miller, Jeffery S. Chamberlain, Stephen J. Tapscott. Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Medicine, University of Washington, Seattle, WA; Radiology, University of Washington, Seattle, WA; Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Neurology and Regenerative Medicine, Wake Forest University, Charlotte, NC; Neurology, University of Washington, Seattle, WA

Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown promise both in preclinical studies and clinical trials for a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD). We and others have shown that dogs and humans mount immune responses against all tested AAV serotypes, which compromised vector delivery and prevented sustained therapeutical gene expression. We have developed a transient immunosuppressive regiment consisting of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF), which was effective in preventing immune responses to AAV and enhanced AAV6-mediated gene delivery to canine skeletal muscle. Here, we demonstrate that long term expression for 2 years of a species – specific, functional canine-micro-dys can be achieved in large scale in skeletal muscles of DMD dogs with ATG/CSP/MMF. Moreover, for the first time we achieved amelioration of muscle histology and function as a result of sustained expression of the therapeutic gene as demonstrated both by non invasive magnetic resonance imaging (MRI) and kinametic gait analysis. In order to identify an even more effective and less toxic immunosuppressive regimen, we have begun studies using molecules that block T-cell costimulation. Further, we are also examining AAV vectors made by a modified production method that minimizes aberrant packaging of the capsid gene to further reduce immunogenicity in dogs. In conclusion, the finding of long term dystrophin expression in DMD dogs given a short course of standard immunosuppression opens the possibility of translating these strategies to a human DMD trial.

34) Inactivation of Dystrophin Expression Via Cre-loxP Mediated Gene Excision

Julian N. Ramos, Andrea Arnett, Brian Schultz, Jeffrey S. Chamberlain. Molecular & Cellular Biology, University of Washington, Seattle, WA; Neurology, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is a recessive X-linked disease in which the dystrophin gene is aberrantly expressed or not at all. Several potential therapies have demonstrated efficacy in animal models and have proceeded to human clinical trails. Among these strategies is to express truncated dystrophins, or micro-dystrophins, in the targeted striated muscle. Although micro-dystrophin (µDys) constructs have demonstrated a high degree of functionality in dystrophic models, it is uncertain how stable these truncated proteins are in adult muscles. To address this question, we generated a double transgenic floxed-µDys/Cre-ERTM/mdx4cv line expressing micro-dystrophin in skeletal muscle. Primary myoblast cultures administered 4-hydroxytamoxifen had <25% µDys-expressing cells by 7 days after the end of the regimen. To ascertain the persistence of the truncated protein in vivo, Cre-mediated recombination was induced with tamoxifen delivered intraperitoneally. At 3 weeks post-treatment, multiple hind limb muscles exhibited less than <50% expression. In order to accurately gauge the protein's stability, the EDL muscles of floxed-µDys/ mdx4cv mice were injected with rAAV-CMV-Cre-NLS. This produced a transient knockdown of dystrophin expression where significant loss was determined by physiological and morphological aspects, in addition to dystrophin labeling. These results suggest that a micro-dystrophin with a modular design similar to one described herein has a maximal half-life of 7 days in vivo.

35) The Polyproline Site in Hinge 2 of Microutrophin Does Not Lead to Structural Abnormalities like That for Microdystrophin

Glen B. Banks, Guy L. Odom, Eric E. Finn, Jeffrey S. Chamberlain. Department of Neurology, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in dystrophin. A potential therapeutic option for DMD is delivery of rationally designed truncated (micro) dystrophins using recombinant adeno-associated virus serotype 6 (rAAV6). However, there remains a possibility that immune responses against dystrophin could develop in some patients that would lead to elimination of transduced myofibers. Gene delivery of utrophin, a paralogue of dystrophin, may be able to circumvent this T-cell mediated immune response. The truncated utrophin (microutrophin^{ΔR4-R21/ΔCT}) that we previously designed was based on that of microdystrophin^{ΔR4-R23/ΔCT}. We had previously demonstrated in mice that microdystrophins carrying a polyproline site in hinge 2 could lead to the development of structural abnormalities in limb muscles, including small myofibers, myotendinous strain injury, ringed fibers and abnormal neuromuscular synapses. We improved the microdystrophin design by substituting hinge 2 for hinge 3 (PLoS Genetics, 2010). Here we examined whether the hinge structure of microutrophin^{ΔR4-R21/ΔCT} had a similar impact on transduced myofibers of dystrophic mice. We found that the polyproline site was conserved in hinge 2 of microutrophin^{ΔR4-R21/ΔCT}. However, microutrophin^{ΔR4-R21/ΔCT} could mitigate muscle degeneration in mdx mice without leading to myotendinous strain injury or ringed fibers. Microutrophin^{ΔR4-R21/ΔCT} was able to prevent synapse fragmentation, but was unable to completely restore the synaptic folds. Microutrophin^{ΔR4-R21/ΔCT} also led to muscle fiber hypertrophy and pronounced sarcolemmal festoons. Thus, the polyproline site did not adversely influence the function of microutrophin^{ΔR4-R21/ΔCT} like that for microdystrophin^{ΔR4-R23/ΔCT}.