Annual Meeting of American Society of Gene & Cell Therapy - May 17-20, Washington, USA

1) Laminin-111: A Highly Potent Therapeutic Agent To Treat Duchenne Muscular Dystrophy

Sebastien Goudenege, Yann Lamarre, Nicolas Dumont, Joel Rousseau, Skuk Daniel, Jacques P. Tremblay.  Unité de recherche de recherche en Génétique Humaine, Université Laval, Quebec City, QC, Canada; Unité CRML, Université Laval, Quebec City, QC, Canada.

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments and myoblast transplantation is considered as a potential approach to repair damaged skeletal muscles. Duchenne muscular dystrophy is a genetic disease that usually begins in childhood and results in the complete loss of dystrophin and the dystrophin glycoprotein complex (DGC) from muscles. The loss of dystrophin and the consequent absence of the DGC in the sarcolemma appear to be key players in the development of muscular dystrophy. Indeed the lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. The most studied isoform, laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. As tissues mature, it disappears and is replaced by other forms of laminin. In this study, we show that intramuscular injection of laminin-111 reduced the damage to sarcolemma, as indicated by a reduction Evans blue dye positive fibers. As a consequence of better muscle state, the inflammatory reaction characterized by macrophage infiltration was drastically reduced. These changes induced by laminin-111 not only increased resistance in dystrophin-deficient muscle but also increased the muscle strength (44 %). Moreover, we also demonstrated that the co-injection of laminin-111 with myoblasts significantly improved this cell transplantation in RAG-mdx mice: i.e., the total number of human dystrophin fibers resulting from the fusion of the transplanted myoblasts with the dystrophin negative fibers was significantly increased (33 %). To explain the improvement of success in transplantation, we showed that laminin-111 increase proliferation (28 %) and drastically migration (743 %) in vitro. These results strongly suggest that the simple injection of laminin-111 protein may a simple and safe therapy to prevent repetitive cycles of injury, fibrosis and loss of muscle function in DMD and to improve the success in myoblast transplantation.

2) Safety and Feasibility of Transvenous Limb Perfusion with Saline in Human Muscular Dystrophy

William Powers, Zheng Fan, Keith Kocis, Robert Valley, Manisha Chopra, James Howard, Jr, Joseph Muenzer.  Department of Neurology, University of North Carolina School of Medicine, Chapel Hill, NC; Department of Anesthesiology, University of North Carolina School of Medicine, Chapel Hill, NC; Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, NC; Wellstone Muscular Dystrophy Cooperative Research Center, University of North Carolina, Chapel Hill, NC; Gene Therapy Center, University of North Carolina, Chapel Hill, NC.

High-pressure retrograde transvenous limb perfusion has been successfully used to deliver plasmid DNA and AAV-minidystrophin transgenes into skeletal muscle in experimental animals. Translating this promising technique to humans with muscular dystrophy requires addressing multiple safety and logistical aspects including analgesia, vascular access and substantially larger infusion volumes. We are conducting an IRB-approved dose escalation study of transvenous single limb perfusion with 0.9% saline in adults with Becker and limb-girdle muscular dystrophies starting with infusions of 5% of limb volume. We are monitoring the following parameters to determine safety: (1) systemic cardiovascular function (2) limb tissue compartmental pressures (3) limb tissue oximetry (4) Doppler ultrasonography for local venous and arterial damage (5) electrodiagnostic studies for local nerve damage (6) local muscle function (7) serum and urine muscle enzymes and (8) renal function. To date, four subjects have been studied with written informed consent (Table). Limb volume was determined by water immersion. An 18 or 20 g intravenous catheter was inserted into the distal lesser saphenous vein. A Zimmer ATS 2000 single cuff tourniquet was placed just above the knee. Infusion of normal saline was carried out with a Belmont FMS 2000 Rapid Infuser. Cardiac function was continously monitored by echocardiography while observing for any intracardiac microcavitations indicative of saline leakage during the infusion. In subject 1, analgesia and anxiolysis was provided with fentanyl and midazolam. In subjects 2-4, a combination of fentanyl, midazolam and propofol was administered. No subject complained of any post procedure pain other than due to needle punctures. No adverse safety events occurred in any of the monitored parameters. Conclusion: We have demonstrated that high-pressure retrograde transvenous limb perfusion with saline up to 15% of limb volume at these infusion parameters is safe and feasible. We will escalate the volume to a maximum of 40% of limb volume to determine the maximum safe perfusion parameters and then document the effectiveness of different infusion parameters to produce entry of fluid into muscle by T2 MRI. These studies will serve as a basis for future gene therapy clinical trials.

      Infusion Parameters
Age Diagnosis Tourniquet Pressure (mm Hg) Max Infusion Line Pressure (mm Hg) Volume (% limb) Volume (mL) Flow Rate (mL/min)
31 LGMD2A 310 290 5 245 60
35 LGMD2A 310 300 5 200 67
38 LGMD 310 300 10 700 70
27 Becker MD 310 295 15 975 80

3) Induction of Oral Immunotolerance to rAAV9-Microdystrophin in Canine X-Linked Muscular Dystrophy

Hiromi H. Kinoh, Naoko Yugeta, Hironori Okada, Yuko N. Kasahara, 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. Also we found that DCs would trigger an immune response against the rAAV-mediated transduction. Here we used neonates and fetuses of the canine X-linked muscular dystrophy in Japan (CXMDJ) to investigate the strategy of inducing immunotolerance to the rAAV as well as the muscle transduction profiles of rAAV9-microdystrophin.
Methods: Normal canine (1 day after birth) dogs were injected with the 1x1013 of rAAV9-CAG-Luciferase into the jugular vein. Two or four weeks after the rAAV injection, expression of rAAV-derived luciferase in the skeletal muscle and cardiac muscle was analyzed. For fetal transduction, pregnant CXMDJ 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 examine the immunotolerance to the rAAV, purified canine peripheral leukocytes were exposed to rAAV9-microdystrophin for 4 hours, and then IFN-β expression was analyzed using RT-qPCR. To expect systemic microdystrophin expression, we additionally injected rAAV9-CMV-microdystrophin into the jugular vein of 6 weeks old dystrophic dog. Temporal muscles of the rAAV-injected animals were collected by punch-biopsy at various time points 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: In the case of transduction of neonate, transgene expression was not significant by immunohistochemistry. In transduction of fetus, 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-β 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 RT-qPCR and immunohistochemistry in the transduced dog. Monthly analysis of the transduced dystrophic dogs demonstrated superior gait function to non-injected littermate CXMDJ.
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.

4) A New Therapeutic Approach for Duchenne Muscular Dystrophy: Restoration of the Dystrophin Reading Frame with a Meganuclease

Jacques P. Tremblay, Pierre Chapdelaine, Christophe Pichavant, Joel Rousseau, Frédéric Pâques.  Unité de Génétique Humaine, CRCHUQ, Québec, QC, Canada; Cellectis Genome Surgery, Cellectis S.A., Romainville, France.

Mutations in Duchenne muscular dystrophy are either inducing a nonsense codon or a frame shift. Meganucleases can be engineered to induce double strand break at specific DNA sequences. These breaks are repaired by Non Homologous End Joining (NHEJ), which results in insertions or deletions (Indels) of a few base pairs. To verify whether meganucleases could be used to restore the normal reading frame of a dystrophin gene with a frame shift mutation, we have inserted a 29 bp sequence containing a target sequence for a meganuclease. The co-transfection in 293FT cells of the target micro-dystrophin plasmid and of the appropriate meganuclease restored the micro-dystrophin expression. Meganucleases also restored micro-dystrophin expression in myoblasts and in muscle fibers in vivo. The mutation of the targeted micro-dystrophin sequence was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that meganucleases engineered to target appropriate sequences in dystrophin gene would be able to restore the normal reading frame of that gene in DMD patients with out of frame deletion. Meganucleases targeting a sequence including or near nonsense mutation could also be used to delete it.

5) Cell Therapeutic Approach to Duchenne Muscular Dystrophy Using Myogenic Differentiation of Multipotent Mesenchymal Stromal Cells

Yuko N. Kasahara, Hiromi H. Kinoh, Hironori Okada, Jin-Hong Shin, Akiyo Nishiyama, Sachiko O. Hosoyama, Michiko W. Maeda, Akinori Nakamura, Takashi Okada, Shin'ichi Takeda.  Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.

Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality. We studied gene and cell therapeutic approach to DMD in dogs using myogenic differentiation of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) to establish an efficient protocol. Because of safety and utility, MSCs would make a larger contribution to clinical benefit than iPS cells. Since current methods of myogenic differentiation of MSCs are generally inefficient, we employed MyoD, the master switch protein for myogenic differentiation, to convert MSCs into myogenic cells. We also investigated the strategies for cell expansion, and delivery route 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- matched mating pairs to achieve donor or recipient of allogeneic transplantation. Cardiotoxin (CTX) was injected into tibialis anterior (TA) and extensor carpi ulnaris (ECU) muscles 5 days before MSCs injection. MSCs transduced with adenoviral vector expressing MyoD (Ad-MyoD) were injected into the CTX- injured TA (2x106 cells) and ECU muscles (1x106 cells) of recipient normal dog with oral administration of cyclosporine and mycophenolate mofetil. We also injected 1 or 2x106 of MSCs into DMD dog. For intra-arterial administration, MyoD-transduced MSCs (5x106 cells) were administered into femoral artery with transient avascularization using a tourniquet. The treated muscles were biopsied and analyzed histologically. Results: CD271-enriched MSCs obtained from normal dog showed 20-fold higher growth expansion compared with CD271-depleted MSCs. MyoD transduction of the CD271-enriched MSCs revealed in vitro myogenic differentiation and myotube formation. MSCs transplanted to the DLA- matched recipient dog were observed as several clusters in the injected muscles 4 weeks after the intramuscular injection. The engraftment of MSCs was also successful with the immunosuppressant-free transplantation into DMD dog by the intramuscular injection. Furthermore, extensive engraftment of MSCs was detected at the site of the CTX-injured muscle 2 weeks after the intra-arterial injection. Immunohistological analysis suggested that most of MSCs formed muscle-like tissues with the upregulation of developmental myosin heavy chain. Conclusion: MyoD-transduced MSCs enabled more efficient realization of MSCs transplantation by the intramuscular as well as intra-arterial injection. Although further study is required for effective differentiation in vivo, this strategy of MSCs propagation and treatment would be promising for the future DMD cell therapy.

6) Gentamicin Treatment of Duchenne Muscular Dystrophy Reinforces the Potential for Mutation Suppression Therapy

Vinod Malik, Louise R. Rodino-Klapac, Laurence Viollet, Cheryl Wall, Wendy King, Roula Al-Dahhak, Sarah Lewis, Christopher J. Shilling, Janaiah Kota, John Hayes, John D. Mahan, Katherine J. Campbell, Brenda Banwell, Majed Dasouki, Victoria Watts, Kumaraswamy Sivakumar, Ricardo Bien-Willner, Kevin M. Flanigan, Zarife Sahenk, Richard J. Barohn, Christopher M. Walker, Jerry R. Mendell.  Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH; Pacific University, Forest Grove, OR; The Ohio State University, Columbus, OH; vaccines & Immunity, The Research Institute at Nationwide Childrens Hospital, Columbus; University of Toronto, Toronto, ON, Canada; University of Kansas, Kansas City, KS; Neuromuscular Research Center, Scottsdale, AZ.

Duchenne Muscular Dystrophy (DMD) is a severe X-linked genetic muscle disease caused by nonsense mutations of the dystrophin gene in about 15% of cases. Pre-clinical studies in mdx mice demonstrate that gentamicin suppression of premature termination codons allows readthrough, thereby translating the full length protein. Clinical attempts to reproduce these findings in DMD patients have been inconsistent. In the current clinical study we have attempted to address several issues: 1) the biopotency of gentamicin comparing stop codon DMD subjects with patients harboring frameshift mutations; 2) the feasibility of long-term gentamicin administration considering that readthrough would be an ongoing requirement; 3) the percent dystrophin expression required to provide clinically meaningful outcomes; 4) the potential immunogenicity of newly expressed dystrophin epitopes induced by stop codon readthrough. Four DMD cohorts received gentamicin. In a short-term 14-day study, Cohort 1 (n = 10) stop codon DMD patients and Cohort 2 (n = 8) frameshift DMD subjects were treated with daily gentamicin 7.5mg/kg/day. The serum creatine kinase (CK) dropped by 50% in the stop codon patients (11,320 ± 2575 U/L to 5429 ± 1317 U/L, p < 0.007) but not in the frameshift subjects. Based on these findings an unprecedented six month delivery study was implemented with weekly or biweekly gentamicin (7.5mg/kg) potentially permitting an accumulating dystrophin pool to reach therapeutic levels. After 6-months of gentamicin, dystrophin levels significantly increased (p = 0.027) reaching levels 13% to 15% of normal accompanied by a drop in serum CK, stabilization of muscle strength indicating a shift in natural history rate of decline, and a slight increase in forced vital capacity. It appeared that stable transcripts escaping nonsense mediated decay or resulting from alternative splicing predicted the greatest increase of dystrophin following gentamicin. Novel immunogenic epitopes were found in post-treatment biopsies by antigen specific IFN-g ELISpots. Taken together our results confirm that aminoglycoside antibiotics suppresss nonsense mutations and support on-going efforts to achieve drug-induced mutation suppression of stop codons. Immunogenic epitopes resulting from readthrough emphasize the importance of monitoring T cell immunity during clinical gene manipulation trials.

7) Long Term Antisense Treatment in Dystrophic Mouse Models for Duchenne Muscular Dystrophy

Annemieke Aartsma-Rus, Christa L. de Winter, Hans A. Heemskerk, Judith C. van Deutekom, Gert-Jan B. van Ommen.  Human Genetics, Leiden University Medical Center, Leiden, Netherlands; Proesensa Therapeutics, Leiden, Netherlands.

Antisense-mediated reading frame restoration is presently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD). In this approach, antisense oligoribonucleotides (AONs) induce specific exon skipping during pre-mRNA splicing of mutated dystrophin transcripts. This is aimed to restore the disrupted open reading frame and allow synthesis of internally deleted, partly functional Becker-like dystrophin proteins. The approach is theoretically applicable to over 70% of all patients, with exon 51 skipping being applicable to the largest group of patients (13% of all mutations). Proof of concept has been achieved in cultured muscle cells from patients carrying different mutation types, in the mdx mouse model, and recently in DMD patients in clinical trials after local or subcutaneous (systemic) treatment of PRO051, a 2'-O-methyl phosphorothioate modified AON. In each case AON treatment resulted in skipping of the targeted exon and dystrophin restoration in the absence of adverse effects. A subsequent trial where patients are treated systemically has recently been completed successfully and a 6-months followup trial using the most effective dosage is underway by Prosensa.
Due to AON turnover, repeated treatment is necessary. Therefore, long term safety and efficacy of subcutaneous 2'O-methyl phosphorothioate AON treatment was tested in mouse models with varying levels of severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx +/-; intermediate phenotype). Mice were treated with weekly SC injections of 200 mg/kg for up to 6 months. This was well tolerated during treatment and liver and kidney weights and serum parameters were similar for 4, 8, 12 and 24 week treated mice compared to saline treated controls at the end of treatment. In both models treatment resulted in significantly improved serum creatine kinase (marker for muscle quality) and rotarod running time (marker for muscle function) compared to the controls. In the more severely affected mdx +/- mice the therapeutic effect was larger. Time course experiments revealed that exon skipping levels increased for the first 12 weeks, but remained constant after that. Protein analysis revealed a similar pattern.
These results indicated that long term subcutaneous treatment with 2'-O-methyl phosphorothioate AONs is safe and efficient in dystrophic mouse models, which is encouraging for future long term trials in patients, which are currently planned by Prosensa Therapeutics.

8) Lentiviral Vector Mediated Delivery of Full-Length Dystrophin for Gene Therapy of Muscular Dystrophy

En Kimura, Katsuhisa Uchio, Tomohiro Suga, tatsuya Koide, Yuji Uchida, Yasushi Maeda, Satoshi Yamoshita, Jeffrey S. Chamberlain, Makoto Uchino.  Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; Pharmacology, Sojo University, Kumamoto, Japan; Neurology, University of Washington School of Medicine, Seattle, WA.

Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder, and there is currently no effective treatment. DMD causes respiratory and/or cardiac failure and results in death at about 20 years of age. Lentiviral vectors are an efficient gene delivery tool for skeletal muscle fibers as well as myogenic progenitor cells in vivo and ex vivo. The integration ability of lentiviral vectors is a huge advantage for targeting DMD myogenic cells, which require the missing gene product, dystrophin, to be expressed permanently, which can not be achieved with non-integrating vectors. We have shown that stable transduction of myogenic stem cells in vivo using lentiviral vectors could be of benefit for treating dystrophic muscles in mdx mice. Injection of micro-dystrophin expressing lentiviral vectors into neonatal mdx muscles resulted in widespread and stable expression of micro-dystrophin for at least two years, and led to a marked amelioration of dystrophic pathology.
As the carrying capacity of lentiviral vector has been considered to be limited to ∼9 kb, it can package truncated versions of dystrophin gene, such as the micro- or mini-dystrophins. Although some groups have shown that larger constructs can be packaged, little data is available on the titers that can be obtained for fully packed particles. Recently, several modifications of lentiviral vector production methods have been successfully employed to generate relatively higher titers of vector stocks. Consequently, we have explored the ability to package the full-length dystrophin cDNA in a VSVG-pseudotyped lentiviral vector. Although the functional titer was still relatively low compared with smaller dystrophin cDNAs, it was able to deliver the full-length transgene into mdx myoblasts. These genetically corrected mdx myoblasts successfully expressed the full-length 427 kDa of dystrophin protein, which could also be provided to dystrophin deficient myotubes in vitro by cell fusion in co-cultures. After intramuscular transplantation, these genetically modified myogenic cells supplied full-length dystrophin protein to mdx muscle fibers. Our candidate strategy with lentiviral vectors carrying full-length dystrophin cDNAs could be useful for ex vivo cell therapies.

9) Transient Expression of a Therapeutic Dystrophin Transgene in Duchenne Muscular Dystrophy Revealed by T Cell Mediated Immunity

Jerry R. Mendell, Katherine Campbell, Louise R. Rodino-Klapac, Zarife Sahenk, Christopher Shilling, Sarah Lewis, Dawn Bowles, Steven Gray, Chengwin Li, Gloria Galloway, Vinod Malik, Brian Coley, K. Reed Clark, Juan Li, Xiao Xiao, Jade Samulski, Scott McPhee, R. Jude Samulski, Christopher M. Walker.  Research Institute at Nationwide Childrens Hospital, Columbus, OH; University of North Carolina, Chapel Hill, NC.

Duchenne muscular dystrophy (DMD) is an X-linked genetically inherited disease of childhood characterized by progressive muscle weakness resulting from mutations in the dystrophin gene. Recombinant adeno-associated virus (rAAV) vectors can package miniaturized versions of the dystrophin gene less than half of the 11 kb coding sequence. Sustained expression of these smaller dystrophin proteins in rAAV transduces dystrophic muscle of mdx mice providing partial restoration of muscle force generation paving the way for a clinical trial. The minidystrophin transgene used in this study encoded the actin binding domain, 5 rod spectrin repeats (R1, 2, 22, 23, and 24), 3 hinge domains (H1, 3 and 4), and the cysteine-rich domain driven by the human cytomegalovirus early promoter. Vector genomes were packaged in a hybrid AAV2 capsid with 5 amino acid substitutions designed to minimize recognition by serum neutralizing antibodies. Six subjects with frame-shifting deletions in the dystrophin gene were enrolled in a phase I pilot clinical gene therapy trial. Vector was injected into one biceps muscle. Four hours prior to gene transfer, subjects received intravenous methylprednisolone (2.0 mg/kg) to reduce potential inflammation caused by needle manipulation. Subjects 001, 002, and 003 received low dose vector (6x1011 vg) while subjects 004, 005, and 006 received a dose 5-fold higher (3x1012 vg). No adverse events were observed. Treated and control contralateral biceps muscles were sampled at day 42 (subjects 001, 003, 004, 006) or day 90 (subjects 002, 005) to assess for vector genomes and minidystrophin protein. Vector DNA ranged from 0.01 to 2.56 genome copies per diploid genome in the treated but not the control side in study subjects. Expression of dystrophin protein was not sustained after gene transfer. T cell immunity against non-self and self dystrophin epitopes was detected in a subset of participants. In one subject, CD4+ and CD8+ T cells were targeted to non-self dystrophin epitopes encoded by the transgene in a domain of a large frame-shifting endogenous dystrophin gene deletion. In two subjects, dystrophin specific T cells were detected in a pre-treatment ELISpot IFN-γ assay that targeted dystrophin self-epitopes expressed on revertant muscle fibers. In one of these individuals the frequency of self-reactive T cells increased rapidly after expression of the dystrophin transgene in skeletal muscle. This study demonstrates for the first time the potential for cellular immune response to therapeutic transgene products in humans treated with rAAV vectors. Recall of auto-reactive T cells also has important clinical significance for strategies to increase expression of functional dystrophin in DMD and should be considered in design and monitoring of experimental therapies for this disease. ( ref. no. NCT00428935).

10) Optimization of AAV Vectors for Improved Therapeutic Protein Expression in the Canine Models for Duchenne Muscular Dystrophy

Alock Malik, Marilyn Mitchell, Andrew Mead, Mike Petrov, Hansell Stedman.  University of Pennsylvania, Philadelphia, PA.

A major bottleneck in the development of effective gene therapy for Duchenne Muscular Dystrophy is the design of vectors that yield the highest possible expression of dystrophin or utrophin in the target tissues. The magnitude of this challenge has come into clearest focus during the transition from small to large animal models in the preclinical setting. We recently initiated studies of limb-wide, vascular gene transfer in the dystrophic dog using AAV vectors based on internally deleted versions of the wild type canine dystrophin and utrophin cDNA sequences. Early in the course of these studies, the need for higher levels of protein expression normalized to the administered vector dose prompted a parallel effort to further optimize the entire vector expression cassette. Iterative improvements were made through the analysis and serial modification of plasmids encoding identical proteins using distinct coding sequence and transcriptional cassettes. Systematic screening, first in HEK 293 cells and then in mdx mouse muscle allowed us to determine the single best expression cassette from a pool of plasmids with different promoters (ubiquitous, muscle specific and hybrid-synthetic promoters), introns, poladenylational signals, transcriptional enhancer elements, and peptide coding sequences. The best combination of these improvements thus far has provided at least a 10-fold higher level of recombinant protein expression than our first generation cassette, with further improvements under additional analysis. Selected versions of these transcriptional cassettes are being further characterized in the context of myotrophic AAV vectors following administration to mdx mice and GRMD dogs. 10-fold or greater improvements due to the optimization process should greatly accelerate the pace of therapeutic trials in the dystrophic dogs.

11) Rescue from Respiratory Dysfunction by Transduction of Full-Length Dystrophin to Diaphragm Via the Peritoneal Cavity in Muscular Dystrophy Mouse

Ishizaki Masatoshi, Yasushi Maeda, En Kimura, Makoto Uchino.  Neurology, Kumamoto University, Kumamoto, Japan.

Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder with no effective therapy so far. DMD causes respiratory or cardiac failure as well as the muscle waste. Among the various symptoms the respiratory insufficiency is a major mortal cause of DMD patients at about 20 years of age. So, the improvement of respiratory function will make the patient's life longer.
Here we first report a sensitive procedure using wholebody plethysmography to monitor respiratory parameters detected in the utrophin/dystrophin double knockout mouse (dko mouse), showing quite similar systemic symptoms to human DMD including restrictive ventilatory impairment.
Furthermore, we show that high efficient dystrophin-transduction to dko's diaphragm achieved by simple intraperitoneal injection of a helper-dependent adenovirus vector (HDAdv) containing the full-length dystrophin expression cassette (HDAdv-mFLmyc-dys). In spite of dystrophin expression only in the diaphragm could result in the rescue from the ventilatory impairment (increased tidal volume and improvement of compensatory hyperpnea). Our result suggests that DMD patient's mortal ventilatory impairment may be improved by technically easy means of the intraperitoneal injection of HDAdv.
Method and Results We have constructed helper-dependent adenovirus vector (HDAdv) contained the murine full-length dystrophin expression cassette (HDAdv-mFLmyc-dys). Each 7-day-old dko mice were injected with the HDAdv-mFLmyc-dys by the intraperitoneal injection. In the diaphragm, the transgene was widely expressed and prevented the dystrophic changes pathologically in injected dko mice. Furthermore, We showed that respiratory function of injected dko mice recovered by using wholebody plethysmography. Conclusion These total assessment system, including wholebody plethysmography, may be useful to evaluate the therapeutic approaches for the neuro-muscular disease models. Moreover, therapeutic gene transfer with HDAdv may ameliorate respiratory insufficiency of DMD patients in future.

12) First Generation AAVmicroutrophin Vector Infused into the Isolated Pelvic Limb of a Canine Model for Duchenne Muscular Dystrophy

Mihail Petrov, Marilyn Mitchell, Alock Malik, Andy Mead, Frederick Balzer, Leonard Su, Jacqueline Farag, Benjamin Kozyak, Kapil Gopal, Charles Bridges, Janet Bogan, Martin Childers, Joe Kornegay, Hansell Stedman.  Surgery, University of Pennsylvania, Philadelphia, PA; University of North Carolina, Chapel Hill, NC; Wake Forest Univ., Winston Salem, NC.

Germline and somatic gene transfer of internally deleted dystrophin and utrophin coding sequences into dystrophic mice has provided evidence for phenotypic amelioration. Myotrophic AAV vectors have been shown to be amenable to a vascular route of administration, suggesting that partial phenotypic correction should be feasible in a large animal disease model. To this end, we initially injected the isolated pelvic limbs of dogs hemi- or homozygous for the GRMD mutation (dystrophin intron 6 splice acceptor site) with a “first generation” AAV6 vector containing a constitutive promoter/enhancer driving transcription of a “microutrophin” cassette based on the wild type cDNA sequence. The afferent, transvenular extravasation route of administration used recapitulates that previously shown to transduce essentially 100% of the skeletal muscle fibers in the mature canine leg (Su, Gopal, et al, 2005). A proportion of the dogs underwent transient single agent immunosuppression using a protocol previously shown to prevent inhibitory antibody formation in a canine model for hemophilia B (Arruda, Stedman, et al, 2005). Dogs were injected with vector at one of three doses. Follow up studies for force transduction were performed by a group of investigators blinded as to the AAV dose, identity of the injected limb, and the presence or absence of prior immunosuppression. In a group of dogs receiving the highest dose of AAV.utrophin-1 (10E13.5vg/kg), the ratio of torque developed by the treated vs. untreated limb is 1.07+/-.2. Five of six dogs in this group showed flexion strength improvement on the treated side. If one disallows the data from the one confounding dog on the grounds that a minor technical problem occurred (electrode migration during force transduction, as suspected from the discrepancy between serial measurements in this dog) the average ratio among remaining dogs is 1.15+/-.08. Importantly, with this combination of treatment and immunosuppressive regimen the muscles did not become weaker, as one might have expected with an AAV-induced myositis. Interestingly, data on other non-immunosuppressed groups of dogs suggest that the vector might have caused a subclinical myositis. Among dystrophic dogs receiving even lower doses of AAV.utrophin-1 without cyclophosphamide, the ratio of treated to untreated limb torque was 0.94+/-0.05. Moreover, among non-dystrophic dogs receiving the unrelated AAV.F.IX (containing a “self” transgene) without immunosuppression, the ratio of treated to untreated limb strength was 0.85+/-0.06. In conjunction with emerging trends in AAV-based clinical investigation, these observations heighten the impetus to formally address the prevention of cellular immune response directed against input AAV capsid antigens.

13) Cell Therapy of Muscular Dystrophy with Engineered Cd133+ Cells

Andrea Farini, Mirella Meregalli, Daniele Parolini, Marzia Belicchi, Simona Maciotta, Paola Razini, Joao da Silva Bizario, Luis Garcia, Nereo Bresolin, Yvan Torrente.  Dept of Neurological Sciences, Fondazione IRCCS Policlinico of Milan, Università degli Studi, Dino Ferrari Center, Milan, Italy; AADM/UNAERP, San Paolo, Brazil; Facultè de Medecine Pierre et Marie Curie, UMR S 787, INSERM/UPMC, Institut de Myologie, Paris, France.

DMD is a genetic disease caused by mutations in dystrophin gene. Forced exclusion (skipping) of a single or multiple exons can restore the reading frame, giving rise to a shorter, but functional dystrophin protein. We selected the GRMD dog, that shares with DMD patient progressive clinical signs and severe myopathy with contractures and premature death. We isolated CD133+ cells from skeletal muscle biopsies of GRMD dogs and we transduced them with lentiviral vectors constructed to convey antisense oligonucleotides able to eliminate the mRNA segment from exon 6 to 8. Under appropriate sedation, the dogs received arterial systemic injections through a catheter introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia: cells were released mainly in the two large arteries under fluoroscopic guidance in order to provide the whole body musculature. Serial injections of the engineered CD133+ cells do not stimulate an immunoreaction in the treated dogs. The delivery of these cells results in a partial recovery of stiffness and ambulation disability of the treated dogs. The muscle biopsies of the transplanted dogs showed clusters of dystrophin positives fibers. This approach should offer a preclinical evidence for future therapies based on autologous transplantation.

14) Systemic Delivery of Antisense Morpholino Corrects RNA Mis-Splicing and Reduces Myotonia in a Transgenic Mouse Model of Myotonic Dystrophy Type 1

Thurman M. Wheeler, Yong-Fu Li, Krzysztof Sobczak, Paul A. Morcos, Charles A. Thornton.  Department of Neurology, University of Rochester, Rochester, NY; Gene Tools, LLC, Philomath, OR.

Objective: To test whether morpholinos modified for systemic delivery can reverse symptoms in a transgenic mouse model of myotonic dystrophy type 1 (dystrophia myotonica type 1; DM1). Background: DM1, the most common muscular dystrophy in adults, is a dominantly inherited disorder characterized by expression of a toxic RNA that leads directly to symptoms. DM1 is caused by expression of an expanded CUG (CUGexp) repeat in the DM protein kinase mRNA. Nuclear foci of CUGexp RNA sequester RNA binding proteins in the muscleblind-like (MBNL) family, leading to abnormal alternative splicing of select genes. Mis-splicing of the muscle chloride channel, ClC-1, leads to myotonia in DM1. Intramuscular injection of antisense morpholino targeting the CUGexp RNA can displace Mbnl1 protein and eliminate myotonia in a transgenic mouse model of DM1 (Wheeler, 2009). However, muscle uptake of unmodified morpholino after systemic delivery is low. Morpholinos coupled to an octa-guanidine dendrimer (Vivo Morpholinos) are designed for enhanced tissue uptake after systemic delivery. Design/methods: 200 μg of Vivo Morpholino (Gene Tools) was injected into the tail vein of HSALR transgenic mice twice weekly for two months. Two dendrimer-modified morpholinos were tested. One morpholino was a CAG repeat, previously shown to displace Mbnl1 protein from CUGexp RNA in mice. The sequence of the second morpholino previously demonstrated selective suppression of exon 7a of ClC-1 (ClC-1 antisense) in mice (Wheeler, 2007). Control mice were injected with vehicle alone (saline). Treatment assignments were randomized. Injections and electromyography were blinded. Results: In mice treated with the CAG repeat oligo, RNA mis-splicing of several transcripts was improved and myotonia was reduced in all 3 hindlimb muscles tested. Treatment with ClC-1 antisense completely reversed ClC-1 mis-splicing and eliminated myotonia in all 3 hindlimb muscles tested. ClC-1 protein was restored to the muscle surface membrane in both groups of morpholino-treated mice. Identical dosing for one month produced a partial effect. Intravenous injection of saline had no effect. Conclusions: By using a dendrimer-modified morpholino, whole-body correction of RNA mis-splicing and reduction of myotonia can be achieved in a transgenic mouse model of DM1. These results support the feasibility and effectiveness of systemic delivery of antisense morpholino as treatment for DM1.

15) Gene Transfer-Driven Inhibition of Cardiac Ankyrin Repeat Protein as a Potential Therapy for the Correction of Muscular Dystrophies

Nathalie F. Danièle, Laurence Suel-Petat, Isabelle Richard.  Research, Généthon, Evry, France.

Muscular dystrophies (MD) are a group of genetic disorders characterised by progressive muscle degeneration and weakness, with a broad range of genetic and phenotypic variations. Up until now, the treatments, which are only palliative, aim at alleviating the symptoms. The research of new treatments, mainly orientated towards gene therapy, is largely impeded by the fact that each therapy has to be tailored for the targeted disease. In order to find a therapeutic target eligible for the correction of the largest possible number of MD, we previously screened the expression of pivotal proteins in several major MD and demonstrated that Cardiac Ankyrin Repeat Protein (CARP), a transcription regulator, is constitutively up-regulated in every muscular dystrophy considered, including the most common form Duchenne Muscular Dystrophy. Whether this up-regulation is beneficial or detrimental for the muscle tissue remains unknown, although the anti-proliferative function of CARP is consistent with an aggravation of the phenotype. In the present work, we assess the down-regulation of CARP as a potential therapy in the alpha-sarcoglycan deficient mouse model. We demonstrate that CARP over-expression occurs in both muscle fibres and mononucleated cells in these animals. In order to target each of these tissues, two different CARP-specific Sh-RNA sequences, previously validated in cellular models, were vectorized into rAAV-[2/1] or SIN-lentiviral vectors. The vectors are injected directly in the TA muscle of young animals (3-weeks or 3-days old, depending on the nature of the vector) and the treatments efficiencies are assessed after 1 and 3 months of expression. Altogether, the analyses of these protocols will be useful to ascertain whether CARP is a suitable therapeutic target for the treatment of MD.

16) Multiple Exon-Skipping Using Cell-Penetrating Morpholinos for Dystrophic Dogs

Toshifumi Yokota, Takashi Saito, Nobuyuki Urasawa, Tetsuya Nagata, Akinori Nakamura, Ryszard Kole, Peter Sazani, Terence Partridge, Shin'ichi Takeda, Eric Hoffman.  Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC; Department of Molecular Therapy, National Institute of Neuroscience, Kodaira, Tokyo, Japan; AVI BioPharma, Inc., Bothell, WA.

Duchenne muscular dystrophy (DMD), the most common and fatal X-linked myopathy, and its milder form, Becker muscular dystrophy (BMD), are caused by mutations in the DMD gene. Exon skipping using antisense oligonucleotides is currently one of the most promising molecular therapies for DMD. The exon skipping approach leads to the production of internally deleted, in-frame mRNA transcripts that are translated into truncated dystrophin protein that has missing amino acids, but retains some functions like Becker dystrophy. Previously we reported first successful exon-skipping treatment in body-wide skeletal muscles in Canine X-linked muscular dystrophy (CXMD) using a cocktail of phosphorodiamidate morpholino oligomers (PMOs, morpholinos) targeting exon 6 and exon 8 of dystrophin mRNA in vivo (multiple exon-skipping) (Ann Neurol 2009;65:667–676; Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs). However, unmodified morpholinos exhibit inefficient delivery to the heart leading to dystrophin induction with barely detectable in the cardiac muscle. Here, we sought to recover the expression of dystrophin in cardiac muscles in dystrophic dogs using morpholinos conjugated to short arginine-rich cell-penetrating peptides (PPMOs) and morpholinos conjugated with octa-guanidine dendrimer (vivo-morpholinos), and demonstrated that the delivery moieties significantly improved dystrophin production in both skeletal and cardiac muscles. The efficacies of vivo-Morpholinos and PPMOs are similar after local injections in dystrophic dogs. Single intravenous or cardiac catheter injection with PPMOs at 12 mg/kg restored dystrophin expression in cardiac muscles as well as body-wide skeletal muscles. No obvious toxicity was detected by blood tests and histology. Our results show the potential of PMO conjugates as therapeutic agents for DMD and many other diseases by targeting mRNAs.

17) Validation of Intravascular Administration of Adeno-Associated Vvirus for Muscular Dystrophies in a Non-Human Primate Model

Aravind Asokan, Julia Hegge, Joshua C. Grieger, Chengwen Li, Swati Yadav, Jade J. Samulski , Xiao Xiao, Juan Li, Jon Wolff, R. Jude Samulski, Scott W. J. McPhee.  University of North Carolina, Chapel Hill, NC; Mirus BioCorporation, Madison, WI; Asklepios BioPharmaceutical Inc, Chapel Hill, NC.

Duchenne muscular dystrophy (DMD) results from mutations in the dystrophin gene, and is characterized by severe skeletal muscle deterioration in the first decades of life. Effective genetic interventions will likely require systemic intravascular delivery of the therapeutic to target this widespread and large volume of target tissue. We are developing Adeno Associated Virus (AAV) mediated minidystrophin gene delivery and have previously conducted a Phase I trial of intramuscular administration in patients with DMD. The next step in clinical development is the progression to regional limb delivery via an intravascular route of administration. The Hydrodynamic Intravascular Infusion technique was initially developed to mediate efficient naked DNA transfer to an individual limb. This is achieved by transient restriction from systemic circulation using a tourniquet secured around the upper limb to isolate the vasculature. A large volume is then rapidly delivered via a line into a distal superficial vein. We have adapted this technique to evaluate regional delivery of multiple AAV serotypes including 2.5, 8, 9 and the novel capsid 2i8 to the lower limb. We tested reporter gene delivery via the saphenous vein in Rhesus macaques and quantified luciferase expression levels in a large number of lower hind limb muscle groups. We studied infusion volumes ranging up to 35% of limb volume, and the procedure was well tolerated by all animals. With AAV 2i8 we observed the highest luciferase expression levels after low volume (<0.1mL/mL limb volume) delivery. AAV2i8 vector is a novel capsid with a reengineered receptor footprint to mediate systemic muscle transduction in conjunction with detargeting of the liver. These results provide preclinical feasibility to support advancing regional AAV minidystrophin gene delivery to the limbs of patients with DMD.

18) AAV9-Mediated Catalase Expression in Mitochondria Improves Exercise Performance in mdx Mice

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

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle disease caused by dystrophin deficiency. The precise mechanisms by which absence of dystrophin results in the muscular dystrophy remain not fully understood. It has been suggested that increased oxidative stress may contribute to myofiber necrosis and degeneration. Here we tested whether reducing mitochondrial free radical could ameliorate muscle disease in the mdx model of DMD. The human catalase gene was engineered with the mitochondria-leading sequence for targeted expression in the mitochondria (Schriner et al Science 308:1909, 2005). The mitochondrial catalase gene was packaged in adeno-associated virus serotype-9 (AAV9) and delivered to neonatal mdx mice. Histopathology and muscle force were examined three months later. We did not detect morphology improvement in muscle section, neither was serum creatine kinase level reduced. However, the eccentric contraction-induced damaged was mitigated in the extensor digitorium longus muscle. When challenged with downhill treadmill running, AAV-infected mice significant outperformed untreated mice. Taken together, our results suggest that the mitochondria-derived reactive oxygen species may contribute to muscle weakness in mdx mice. Strategies to reduce oxidative stress in mitochondria may represent a viable approach to treat muscular dystrophy.

19) Genetic Evidence for a Structural Role of α–dystrobrevin-3 within the Dystrophin-Glycoprotein Complex

Guy L. Odom, Glen B. Banks, Dewayne Townsend, Marvin E. Adams, Joseph M. Metzger, Stanely C. Froehner, Jeffrey S. Chamberlain.  Neurology, University of Washington, Seattle, WA; Integrative Biology & Physiology, University of Minnesota, Minneapolis, MN; Physiology and Biophysics, University of Washington, Seattle, WA.

α-Dystrobrevin (αDB) is an integral component of the dystrophin-glycoprotein complex (DGC) at the sarcolemma of myofibers. Mice lacking αDB1-3 (adbn-/-) in striated muscle present with several phenotypic changes consistent with dystrophy including myopathy, prominent neuromuscular and myotendinous junction defects, and a mild cardiomyopathy. Decreasing amounts of αDB from the sarcolemma contributes to the severity of disease in several muscular dystrophies including Duchenne muscular dystrophy (DMD). Both αDB1 and αDB2 transgenes restore the skeletal muscle abnormalities in the adbn-/- to varying degrees because of subtle differences in their location within the sarcolemma and varying functional (phosphorylation/protein-interaction) domains contained in each protein(Grady et al., 2003; Grady et al., 1999; Grady et al., 2000). Because αDB3 is the shorter isoform which lacks these functional domains, including being unable to bind directly to dystrophin, it has previously been assumed to play only a negligible role in the DGC. Here, overall cardiac functional workload was assessed in wild type and mutant mice using a dobutamine stress test protocol followed by measurement of hemodynamic properties. We measured 18 ECG parameters at rest, and at 5 time points. None of these parameters showed significant differences, suggesting a relatively normal electrical activity. We next analyzed the role of αDB3 in skeletal muscle by systemically delivering αDB3 to adbn-/- mice with recombinant adeno-associated virus serotype 6. The results demonstrated αDB3 to be an important DGC component within skeletal muscle, showing localization to the sarcolemma despite being unable to bind dystrophin, and a robust interaction with ankyrin as demonstrated by in vitro co-immunoprecipitation, suggesting a cooperative role toward the stabilization of myofibers. Overall, expression in vivo of αDB3 resulted in the mitigation of prominent dystrophic abnormalities including the prevention of muscle degeneration, synapse fragmentation, and shallow folds within the myotendinous junction. These results suggest an important structural role of αDB3 whose absence can contribute to sarcolemma fragility in DMD and other muscular dystrophies.

20) Characterization of A20 as an Inhibitor of NF-κB Activation in Dystrophic Mice

Rakshita Charan, Paula R. Clemens.  Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Department of Veterans Affairs Medical Center, Veterans Affairs, Pittsburgh, PA.

Of all muscular dystrophies, Duchenne muscular dystrophy (DMD) is the most common affecting about 1 in 3500 male births worldwide. The disease 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 structural support in dystrophin-deficient muscle fibers may be responsible for muscle pathology in progressive muscular dystrophy. It is also known that nuclear factor-kappa B (NF-κB), which is a nuclear transcription factor, is up regulated in dystrophic muscle in DMD patients as well as in the mouse model for DMD (mdx). NF-κB regulates several genes responsible for stress responses, cell survival and various inflammatory conditions. Thus, the up regulation of this transcription factor is thought to activate protein degradation and cause chronic inflammation in skeletal muscle. Furthermore, NF-κB downregulates myogenic regulatory factors and this process likely interferes with muscle regeneration. Attenuating NF-κB activation in these dystrophic mice has been shown to improve muscle stability and strength. Strategies of inhibition of NF-κB activation are being actively pursued as a therapeutic option for DMD. One of the NF-κB pathway attenuators, A20 is a deubiquitinating enzyme, known to inhibit NF-κB activation by deubiquitinating RIP1; ubiquitination of RIP1 is essential for NF-κB activation.
Our aim is to characterize A20 in skeletal muscle and establish its role as a potential therapeutic target as attenuator of the NF-κB pathway activation in DMD. We show that blocking of A20 using A20siRNA increases NF-κB activation in mdx as well as control C57BL/10 mice myotubes. We further characterized localization of A20 in muscle and established that A20 is expressed predominantly in fast-twitch muscle fibers. Interestingly, we also observed that in mdx muscle, A20 is over-expressed in regenerating fibers. To study the localization of A20 through the life of the mdx mouse, we did a time-profile assessment of A20 expression and compared it with control mice. We see an increase in A20 protein expression during the 7-10 week time period in mice, which is correlative of the ages when severe degeneration and regeneration cycles take place in mdx mice. This is the first observation of a correlation between expression of an NF-κB inhibitor and pathology of DMD.
Our studies support the utility of therapeutic manipulation of A20 to promote NF-κB inactivation in dystrophic muscle fibers and provide a potential therapy for DMD.

21) Impact of the Host Environment on the Control of Gene Transfer-Induced Immune Responses with miRNA142.3p-Regulated Vectors

Florence Boisgérault, David-Alexandre Gross, Maxime Ferrand, Sylvie Darocha, Jérôme Poupiot, Marc Bartoli, Isabelle Richard, Anne Galy.  UMR951, Genethon, Evry, France; CNRS UMR8587, Genethon, Evry, France.

Recombinant AAV2/1 vectors have emerged as strong candidates for gene transfer into skeletal muscle. However their efficacy is partially limited by the development of anti- transgene immune responses which affect long-term muscular reconstruction in the context of muscular dystrophies. In a murine model of limb-girdle muscular dystrophy type 2D (Sgca-null), administration of rAAV2/1 expressing the alpha-sarcoglycan (sgca) transgene under control of the ubiquitous CMV promoter results in a transient expression of the protein in skeletal muscle whereas an efficient and more stable expression was obtained using a muscle-specific promoter. To understand how transgene expression regulates the development of immune responses, a reporter (Sgca-HY) transgene was modified by the introduction of two epitopes of the murine male HY antigen, and used to follow transgene-specific CD4+ and CD8+ T cell responses in mice of the C57BL/6 background. With this system, a single i.m. injection of the rAAV-CMV-sgca-HY vector in C57BL/6 or in Sgca-null mice induced a strong cellular CD4 and CD8-mediated immune response resulting in an extensive mononuclear cell infiltrate in the injected muscle associated with muscular destruction and loss of gene-modified cells. To determine the impact of direct antigenic presentation on immune responses, the transgene was de-targeted from hematopoietic cells by inserting miRNA142.3 target sequences into the expression cassette. This strategy abrogated the development of effector anti-transgene CD4+ and CD8+ cellular immune responses in C57Bl/6 mice, enabling long-term transgene persistence. This approach was also tested in the pathological sgca-null model but was not as effective as in normal mice. Following rAAV-mediated gene transfer in sgca-null mice, transgene expression was improved but not maintained over time. The mir142.3p-regulated vector prevented the development of transgene-specific CD8+ T cell responses but failed to control CD4+ T cell reactivity. These data suggest that miRNA142.3p-regulated expression systems are useful to reduce the initial immunogenicity of the transgene after rAAV-mediated gene delivery into normal skeletal muscle but may not be entirely efficient to control antigen presentation to CD4+ T cells in an inflammatory context. This suggests that controlling anti-transgene CD4+ T cell responses following rAAV2/1-mediated gene delivery is essential to establish long-term transgene persistence.

22) Feasibility and Effectiveness of Exon 51 skipping in Human-Like mdx Mutation

Yoshitsugu Aoki, Toshifumi Yokota, Takashi Saito, Akinori Nakamura, Tetsuya Nagata, Hitoshi Okazawa, Shin'ichi Takeda.  Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; Medical Research Institute, Tokyo Medical and Dental School University Graduate School, Bunkyo-ku, Tokyo, Japan; Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC.

Background: Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin at the sarcolemma. Systemic delivery of anti-sense oligonucleotide (AO) is a promising approach to therapy for Duchenne muscular dystrophy (DMD). Current clinical trials for DMD involve exon 51-skipping but are unaccompanied by animal model studies of recovery of skeletal muscle function with this skip. Here, we use the exon 52-deletion mdx mouse to screen AO sequences for skipping exon 51, making assessment of molecular and functional efficacy. Methods: We tested a series of phosphorodiamidate morpholino oligomers (PMOs) singly and in combination against exon 51 of the mouse DMD gene, and screened for molecular efficacy by intramuscular injection and then intravenously injected an optimized PMO combination (160 mg/kg/each, once weekly for 7 weeks) into mdx52 mice. The efficiency and efficacy of exon 51 skipping were tested at the mRNA, protein, histological, and functional levels. Results: The highest splicing efficiency of an individual sequence (50-55%) was that targeting the 3' splice site (+10-15bp). But, yet more effective (75%), was a two-AO combination targeting both 5' and 3' splice sites. This, on systemic delivery, induced 10-40% of wild-type dystrophin expression in all muscles, accompanied by amelioration of dystrophic pathology and improvement of skeletal muscle function. Blood tests and histological examination of liver and kidney indicated no evidence of toxicity. Interpretation: This demonstration of efficient exon 51-skipping leading to clear improvement in skeletal muscle function, supports the pre-clinical rationale for current human trials. However, since the greatest efficacy was associated with targeting of dual sequences unrelated to those in the current human trials, further testing and selection of target sequences is indicated.

23) Sustained Alpha-Sarcoglycan Gene Expression in LGMD2D Following Gene Transfer

Jerry R. Mendell, Louise R. Rodino-Klapac, Xiomara Rosales-Quintero, Brian D. Coley, Gloria Galloway, Sarah Lewis, Vinod Malik, Christopher J. Shilling, Barry J. Byrne, Thomas Conlon, Katherine J. Campbell, William G. Bremer, Christopher M. Walker, Zarife Sahenk, K. Reed Clark.  Center for Gene Therapy, Research Institute at Nationwide Children's Hospital, Columbus, OH; Pediatrics, University of Florida College of Medicine, Gainesville, FL; Center for Vaccines and Immunology, The Research Institute at Nationwide Children's Hospital, Columbus, OH.

Limb-girdle muscular dystrophy (LGMD) type 2D is defined as a deficiency of a-sarcoglycan (α-SG), a sarcolemmal transmembrane protein contributing to membrane stability. There are few if any treatment options for this disease. We previously reported a successful trial of SGCA gene transfer in three patients with persistence of gene expression for at least three months. The number of α -SG positive fibers reached 57%, 69%, and 62% and western blot showed a 4 to 5 fold increase in each subject with the maximum restoration of α-SG in any block 76% of normal control muscle. Persistent gene expression was complemented with restoration of the full sarcoglycan complex and an increase in mean fiber diameter in one subject analyzed at 3 months. To evaluate whether long term gene expression was achievable, a second cohort of 3 patients was evaluated at 6 months. As in the first cohort, a double-blind, randomized controlled trial was initiated in LGMD2D subjects using rAAV1 with full length human SGCA under control of a truncated muscle creatine kinase (tMCK) promoter. The extensor digitorum brevis (EDB) muscle received 3.25 X 1011 vector genomes on one side, while the control side was given saline. All analyses were done prior to breaking the blind. The entire EDB muscle was removed from both sides at 6 months in all three subjects. Transgene specific PCR analysis demonstrated expression on only one side in each subject which corresponded with gene expression. Subject 1 increased α -SG by 2 fold on the side of gene transfer reaching wild-type levels. This was accompanied by an increase in mean muscle fiber diameter, 28.2 ± 11.1 to 52.2 ± 13.1. Subject 2 demonstrated robust gene expression, again equivalent to wild-type levels on the side of gene transfer. MHCI and CD4, CD8 mononuclear cells were upregulated on the side of gene transfer in subjects 1 and 2. ELISpots for antigen specific production of IFN-γ secretion were monitored beginning pre-gene transfer. Subject 1 had a transient minimal response to AAV1 capsid pools at days 14 and 28. Patient 2 showed no response to α-SG or AAV1 capsid peptide pools. In contrast to the first two patients, Subject 3 had pre-existing immunity to AAV1 (neutralizing antibodies) and an ELISpot IFN-γ response within the first week to AAV following gene transfer suggesting a memory response. Levels of gene expression were also reduced in this subject compared to others. Overall the promising results from this study with gene expression persisting for 6 months lays the foundation for potentially achieving long-term sustained correction of the underlying gene defect in muscular dystrophy. However, the influence of pre-existing immunity to AAV may affect initial muscle fiber transduction or sustained gene expression.

24) ncRNAs Originating from the Dystrophin Gene as Biomarker for Assessing Antisense Therapy

Matteo Bovolenta, Annarita Armaroli, Marcella Neri, Simona Brioschi, Marina Fabris, Chiara Scotton, Sofia Falzarano, Paola Rimessi, Emanuele Valli, Giovanni Perini, Luciano Merlini, Francesca Gualandi, Alessandra Ferlini.  Department of Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy; Department of Biology and Evolution, University of Bologna, Bologna, Italy.

The DMD gene is the largest in the human genome, it spans 2.2 Mb and is made of 79 exons and 7 isoforms, all finely regulated and expressed in specific tissues. Mutations in this gene lead to three distinct phenotypes: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy and X-linked dilated cardiomyopathy. Therapeutic approaches are now becoming reality in DMD, nevertheless, clinical outcome measures may not always be sensitive enough to detect small changes in disease progression/regression and after short treatment periods. It is therefore imperative to identify enrichment endpoints, as biomarkers, able to document benefits of the treatment at early stages and at the individual level. Recently, genome-wide approaches to monitor transcription have revealed a notable number of non coding RNAs (ncRNAs) with many regulatory functions. To address this issue we designed a novel gene-specific Gene Expression tiling array covering the full DMD gene and used it to search for non-coding transcripts in polyA+ RNAs from human brain, heart, skeletal muscle and skin. The search identified 13 sense and 2 antisense oriented transcripts, originating from dystrophin introns/UTR regions. Six of these ncRNAs (four sense polyA+ ncRNAs, located adjacent to dystrophin known promoter regions, and the 2 antisense) were validated through Northern blotting and fully characterized by RACE PCR and sequencing. Their length ranges from 1800 to 2800 bp, 5 were unspliced, but one was spliced in several isoforms. Compartmentalisation studies demonstrated that all the six ncRNAs are located in the nucleus. None of these has an open reading frame, suggesting that they belong to the long non-coding RNA category. These ncRNAs are highly represented in human heart, skeletal muscle and brain, known tissues of elective dystrophin expression. CGH-DMD analysis in 5 DMD patients with dystrophin exons 45-50 deletion (thus eligible for exon 51 skipping antisense therapy) allowed us to define the deletion breakpoints therefore predicting the loss/maintenance at the genomic level of the ncRNAs region. One sense oriented ncRNA, which localises within intron 44 and the corresponding genomic region was deleted in only one DMD boy, was consistently missing in his myogenic cells. Further studies are in progress in order to profile the expression of these ncRNAs in patients' myogenic cells before and after AONs treatment for exploring if these transcripts may be used as muscle transcriptomic biomarkers for monitoring the impact of novel treatment in dystrophinopathies.


25) Expression of Micro-Dystrophin and Full-Length Dystrophin in Mouse and Dog Muscles

Christophe Pichavant, Pierre Chapdelaine, Joe N. Kornegay, João C. S. Bizario, Xiao Xiao, Jacques P. Tremblay.  CHUL, Quebec, QC, Canada; University of North Carolina School of Medicine, Chapel Hill; Muscular Dystrophy Research Center, Ribeirão Preto-SP, Brazil.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin in the muscle. Since the phenotype of the dystrophic dog is closer to the human phenotype than the mdx mouse, we have studied the introduction of the dog dystrophin in dystrophic dog muscles. To achieve that, two different isoforms of the dystrophin were used in our experiments: the dog micro-dystrophin (µdys) and the full-length dog dystrophin (FLDYS). Muscle precursor cells (MPCs) were transduced with a lentivirus coding for the µDys in fusion with a V5 tag (µDys.V5) and transplanted in immunodeficient mouse and dog muscles. A non viral gene therapy, the electrotransfer, was also used to introduce the plasmids coding for the µDys.V5 cDNA and for the FLDYS cDNA. These plasmids were electrotransferred in immunodeficient mouse, dog and dystrophic dog muscles.
Concerning the use of the µDys.V5 in mouse muscles, the percentage of V5 positive fibres was more important in the muscles treated by ex vivo gene therapy than those electrotransferred with the plasmid. Dog muscles were also transplanted with dog MPCs transduced with the µDys.V5 lentivirus. No V5 expression was detected in the treated muscles one month following the graft. However, accumulations of CD8+ cells were observed in these muscles. A biopsy of another muscle transplanted with the same transduced cells has been also studied at two months and as expected, no transgene was found and also no CD8+ cell. Dog muscles were also electrotransferred with the µDys.V5 plasmid and two weeks later, fibres expressing this dystrophin were co-localised or not with CD8+ cells. To introduce the FLDYS into muscles, only the electrotransfer technique was used since the plasmid containing this transgene was large (∼17 kb). Our results showed that the electrotransfer of this large plasmid into mouse muscles allowed the FLDYS expression in the treated muscles. The electrotransfer of FLDYS in a dystrophic dog muscle led also to the expression of dystrophin. However, accumulations of mononuclear cells were observed in this muscle, these lymphocyte infiltrations were co-localised or not with the myofibers expressing the FLDYS.
In conclusion, the introductions of the µDys or of the FLDYS by ex vivo gene therapy or by electrotransfer are potential approaches to restore the dystrophin in DMD patients. The next step will be to improve the efficiency of the delivery methods and also to verify whether the specific immune response producing the rejection of the myofibers is due to our transgene, to our bacterial backbone sequence or to other components.

26) Screening of Nature Compounds for Enhancing Antisense Oligonucleotide-Induced Exon Skipping of Dystrophin Gene In Vitro and In Vivo

Bo Wu, Caryn Cloer, Peijuan Lu, Mingxing Wang, Wei Li, Kazuo Koike, Qi Long Lu.  McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC; Department of Pharmaceutical Sciences, Toho University, Narashino, Chiba, Japan.

Antisense oligonucleotide-mediated exon skipping therapy has demonstrated great potential for treating Duchenne muscular dystrophy (DMD). Phosphorodiamidate morpholino oligomer (PMO) is the most widely used and being applied in clinical trials. However unmodified PMO has limited efficacy due to low potency and ineffciency in systemic delivery, especially failure to restore dystrophin in heart. Recently our and others' groups have found that modifications of PMO with cell-penetrating peptides and non-peptide dendrimeric octa-guanidine significantly improve the delivery of PMO and achieved short-term rescue of dystrophin expression in body-wide muscles including the cardiac muscle with improved pathology and partial restoration of muscle functions. However, the safety of the long-term use of positively charged polymers remains to be investigated. Here we developed a drug screening system using C2C12 myoblasts and myotubes expressing a reporter GFP with its reading frame disrupted by the insertion of a targeted dystrophin exon to screen and identify nature compounds capable of enhancing the exon skipping effect by specific antisense oligomers. We identified a series of nature compounds (5) capable of increasing exon skipping efficiency when used in combination with PMO in vitro. Three compounds were able to enhance PMO-induced exon skipping two fold in TA muscles of dystrophic mdx mouse by local injection when compared with unmodified PMO. Onecompound significantly increased systemic effect of PMO in the cardiac and skeletal muscles with reduction of serum creatine kinase levels. Nature compounds could be explored for enhancing specific exon skipping with antisense oligomers for DMD therapy.

27) External, Non-Invasive Monitoring of Progressive Cardiorespiratory Dysfunction in a Canine Model of DMD

Andrew Mead, Alock Malik, Mihail Petrov, Martin Childers, Janet Bogan, Joseph Kornegay, Hansell Stedman.  University of Pennsylvania, Philadelphia, PA; Wake Forest University, Winston-Salem, NC; University of North Carolina, Chapel Hill, NC.

The GRMD (Golden Retriever Muscular Dystrophy) model for Duchenne Muscular Dystrophy (DMD) mimics human disease progression with regard to both histopathology and locomotive function. However, unlike DMD, the GRMD model exhibits a high degree of variability in both the severity and speed of progression, even among littermates. This aspect of the model poses challenges for assessing efficacy in pre-clinical therapeutic trials of gene therapy, and requires further improvement in quantitative measures of disease progression. Electrical stimulation of distal limb muscles has provided the most reliable quantitative data, however this requires serial episodes of general anesthesia, with attendant risks of cardiopulmonary complication in the dystrophic dogs. Moreover, the data are limited to locomotive muscle function. Most non-invasive means of assessment, such as running times, are unreliable due to their volitional nature, and fail to dissect the cardiac, respiratory and locomotive components of disease progression. Here we investigate the use of a non-invasive device that records EKG, 3D accelerometry, and 2-band plethysmography in a wireless package that has negligible effect on animal behavior, the “Lifeshirt” (Vivometrics). In particular we simultaneously examine cardiac and respiratory function, two physiological parameters relevant to longevity in DMD. 1. We hypothesize that the progressive loss of ventilatory reserve caused by degeneration of the diaphragm will manifest, in the GRMD model, as a progressive respiratory paradox, (i.e. asynchrony in the chest wall and abdominal wall excursion) during and immediately after mild exertion. The degree of paradox will correlate with trans-diaphragmatic pressure development during stimulated spontaneous breathing and phrenic nerve stimulation at the time of necropsy. 2. Progressive heart failure, also a hallmark of DMD in humans, will manifest first as parasympathetic withdrawal measured by depressed respiratory sinus arrhythmia in affected dogs as compared to unaffected littermates. Decreased RSA would be expected to correlate with impaired cardiac contractility as measured by more direct means, e.g. Langendorff isolated perfused heart studies performed post mortem.
Data obtained using the "Lifeshirt"system on GRMD pups reveals that by 6 months of age there are early indicators of respiratory and cardiac dysfunction. In preliminary studies of 11 dystrophic and 7 normal dogs under 8 months of age RSA was significantly reduced in the affected group (p<.006), and respiratory phase angle was higher (p<.05). Affected pups were also marked by the complete absence of diaphragm-intensive panting behavior. These differences between normal and affected animals, and among affected animals at different stages of disease progression increased markedly after moderate voluntary exercise. Standardization of these approaches will enhance the GRMD model's role in translational studies of gene therapy for cardiac and respiratory muscle disease in DMD.

28) Splice Site Strength and Nonsense-Associated Exon Skipping in the DMD Gene

Kevin M. Flanigan, Diane M. Dunn, Jerry R. Mendell, Alan Pestronk, Julaine M. Florence, United Dystrophinopathy Project Consortium, Robert B. Weiss.  Center for Gene Therapy, Nationwide Children's Research Institute, Columbus, OH; Department of Neurology, Washington University, St. Louis, MO; Department of Human Genetics, University of Utah, Salt Lake City, UT.

Understanding the molecular pathogenesis of DMD gene mutations is more compelling than ever considering evolving treatment strategies that include gene replacement therapy, exon skipping, and nonsense mutation readthrough. Nonsense mutations can be associated with a spectrum of phenotypes: Duchenne Muscular Dystrophy (DMD) with loss of walking by age 12, Becker Muscular Dystrophy (BMD) with continued walking after age 15, and an intermediate (IMD) phenotype with loss of walking between ages 12 and 15. In some cases, a nonsense mutation has been shown to ablate an exon splice enhancer (ESE) or create an exon splice suppressor (ESS) motif, resulting in altered mRNA splicing with a resultant milder phenotype. To assess the generality of this mechanism, we evaluated 166 unique nonsense mutations found in 210 patients with well-established phenotypes. Among mutation sites in which mutation-induced exon skipping would be predicted to result in an out-of-frame transcript, only 5% resulted in BMD or IMD (n=4) and 95% resulted in DMD (n=77). In contrast, among mutations in an in-frame flanking exon context, 18% (n=15) resulted in BMD or IMD, and 82% (n=70) resulted in DMD. Thus, in BMD or IMD the exonic sequence context is typically in-frame (15/19) (P=.014 by Fisher's exact test), although the presence of an in-frame exon context is not by itself predictive of BMD. We calculated the splice site strengths of in- versus out-of-frame exons using maximum entropy (MaxEnt), multiple dependence decomposition (MDD), first order Markov model (MM), and a weight matrix model (WMM). On average, in-frame exons have weaker splice site (ss) signals than out-of-frame exons, most notably in the 5'ss strength (MaxEnt p=0.006; MDD p=0.065; MM p=0.029; WMM p=0.049). The subset of in-frame exons that are enriched for BMD nonsense mutations have on average weaker 3'ss strength compared to the other in-frame exons (MaxEnt p=0.015; MDD p=0.015; MM p=0.053; WMM p=0.015). Thus, exons with in-frame BMD nonsense mutations appear to have the weakest splice site signals of all the DMD exons, as measured by the mean difference between consensus values for 3'ss and 5'ss strengths (MaxEnt: 13.21 vs. 15.96 for in-frame BMD vs. in-frame DMD, and 13.21 vs. 16.93 for in-frame BMD vs. out-of-frame exons). In contrast, BMD mutations are not more frequently associated with either the ablation of ESE motifs or the creation of an ESS site than are DMD mutations (Mann-Whitney-Wilcoxon rank sum test, p=0.7). Our data suggest that in BMD, ESE ablation or ESS creation signals are not the sole determinant of nonsense mutation-associated exon skipping, but rather that the effects of these exonic signal alterations are manifest on a background of weak intrinsic splice site signals within a restricted set of exons. An improved understanding of DMD splice site metrics may shed light on the design of antisense oligonucleotides, and the interpretation of results from upcoming clinical trials of exon skipping.

29) The Polyproline Site in Hinge 2 Influences the Functional Capacity of Truncated Dystrophins

Glen B. Banks, Luke M. Judge, James M. Allen, Jeffrey S. Chamberlain.  Department of Neurology, University of Washington, Seattle, WA.

Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy (BMD). The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophinΔR4-R23/ΔCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophinΔR4-R23/ΔCT led to small myofibers (12% smaller than wild-type), Achilles myotendinous disruption, ringed fibers and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophinΔR4-R23/ΔCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area and maintain the junctions. We conclude that the rigid α-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.

30) Restoration of Wild-Type DMD mRNA in an Immortalized Human Fibroblast Line from an Exon 8-9 Duplicated DMD Patient

Ling Zhao, Gillian Butler-Browne, Vincent Mouly, Soraya Chaouch, Kamel Mamchaoui, Steve D. Wilton, Kevin M. Flanigan.  Human Genetics, University of Utah, Salt Lake City, UT; UPMC Université Paris and Institut de Myologie, Paris, France; University of Western Australia, Perth, Australia; Center for Gene Therapy, Nationwide Children's Research Institute, Columbus, OH.

Both 2'O-methyl and morpholino antisense oligonucleotides are currently in trials as a method of altering pre-mRNA splicing in patients with Duchenne Muscular Dystrophy (DMD). The goal is to convert out-of-frame DMD mutations into in-frame transcripts, resulting in a milder Becker Muscular Dystrophy phenotype. DMD duplications account for around 5% of all patient mutations. Skipping of only the duplicate exons would restore a wild-type DMD transcript, and be predicted to have an even greater clinical response than that expected from restoring the reading frame around a genomic deletion to make a BMD-like dystrophin isoform. To study skipping of duplicated exons, we developed immortalized fibroblast clonal cell lines derived from a patient with a tandem duplication of exons 8 and 9 in the DMD gene (F43401-ht-MyoD) and from a normal control (WT-ht-MyoD). Cells were immortalized by stable lentiviral transfection with the human telomerase gene, followed by a tet-inducible MyoD construct; similar cells have been demonstrated to express dystrophin protein following transplantation into immunodeficient mice (Chaouch S, Hum Gene Ther. 2009 Jul;20(7):784-90.). The patient cells demonstrated growth curves similar to wild type. Induction of MyoD expression with doxycycline (2 ug/ml) resulted in the transdifferentiation of F43401-ht-MyoD fibroblasts into a myogenic lineage, with 95% of cells expressing desmin at day 4. Five days after doxycycline treatment, cells were treated with 2'O-methyl modified AONs directed toward either exon 8, exon 9, or both exons 8 and 9 at four concentrations (25, 50, 100, and 300 nM), and harvested 5 days later for RNA extraction and rtPCR analysis using primers spanning exons 7 through 10. Treatment with exon 8 AONs resulted in significant skipping of both exons 8 and 9, with a maximum effect at 100 nM resulting in 86% wild type transcript. AONs directed to either exon 9 or exons 8 and 9 in combination showed no significant wild type mRNA. However, significant dystrophin protein expression was not detected in either wild-type or AON-treated cells maintained up to 12 days post-transdifferentiation. Diminished DMD mRNA levels in these lines in comparison to primary myoblasts may explain this paucity of dystrophin expression. Nevertheless, such clones have several potential benefits as tools for exploring exon skipping or other therapies. In particular, the robust inducible transdifferentiation avoids variability between experiments in viral myoD transfection, and thus may facilitate comparative studies of AON chemistries and sequences. Our data confirm that skipping of both exons 8 and 9 occurs with the use of only a single exon 8 AON, as previously found in normal and DMD primary cell lines, and suggest that skipping of selected DMD exonic duplications may be titratable in a fashion that allows restoration of wild type DMD mRNA.

31) Pre-Injection of Polymers Can Increase the In Vivo Transduction Efficiency of Adeno-Associated Virus Type 2

Gilles Moulay, Sylvie Boutin, Carole Masurier, Daniel Scherman, Antoine Kichler.  Genethon, BP60, Evry Cedex, France; CNRS UMR 8151 – U1022 Inserm, Unité de Pharmacologie Chimique et Génétique et d'Imagerie, Université Paris Descartes, Chimie Paristech, Paris Cedex, France.

Background The success of muscular dystrophy gene therapy requires widespread and stable gene delivery. In this context, adeno-associated virus (AAV) has attracted great attention as an optimal vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy large amounts of recombinant vector are essential. Injection of very high doses of viral vectors not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome both problems would be to find strategies able to increase the transduction efficiency in vivo. In the present work, we investigated whether polymers can act as adjuvants to increase the efficiency of AAV-2 vector after systemic injection. Methods Our strategy consists in the injection of cationic or anionic polymers before intravenous administration of Balb/c mice with AAV-2 encoding the reporter gene mSeAP (a murine secreted alkaline phosphatase). The transduction efficacy was followed by quantifying the mSeAP in serum at different time points. Vector biodistribution was evaluated in different tissues by quantification of the mSeAP protein as well as by real time PCR of the recombinant viral genomes. Results The pre-injection of a cationic polymer resulted in a 4 to 12-fold increase of seric mSeAP levels. Histochemical analysis showed the appearance of mSeAP positive muscle fibers with this strategy when no staining was visible in control AAV-2 injected mice. The PCR data confirmed an overall increased tissue transduction by AAV-2. Pre-injection of an anionic polymer resulted in a 2-fold increase of mSeAP expression in serum. Interestingly, while this latter increase is moderate this strategy permitted to significantly reduce the neutralizing antibody titer raised against the AAV-2 capsid. Conclusion Our results show that strategies of pre-injection of polymers can be used either to improve the overall transduction of systemically administered AAV-2 or to reduce the humoral response against the capsid proteins.

32) Successful Expression of a R16/17 Micro-Dystrophin Gene in Dystrophic Dogs with a Tyrosine-Mutant AAV Vector and a Brief Course of Immune Suppression

Jin-Hong Shin, Yi Lai, Yongping Yue, Arun Srivastava, Bruce F. Smith, Dongsheng Duan.  Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; Division of Cellular Biology and Molecular Therapy, University of Florida, Gainesville, FL; Department of Pathobiology, Auburn University, Auburn, AL.

Adeno-associated virus (AAV)-mediated micro-dystrophin expression is a promising strategy to treat Duchenne muscular dystrophy (DMD). Two recent reports demonstrated micro-dystrophin expression in dog models of DMD with traditional AAV serotypes. While their results are encouraging, there are several limitations. First, the micro-dystrophin used in these studies cannot restore neuronal nitric oxide synthase (nNOS) to the sarcolemma. Second, the transduction efficiency of traditional AAV serotypes may not meet the clinical need. Third, a complicated immune suppression scheme was used to prevent immune rejection. To further improve AAV micro-dystrophin gene therapy, we tested a novel R16/17 microgene, a tyrosine-mutant AAV-6 vector and an abbreviated immune suppression protocol in the golden retriever muscular dystrophy (GRMD) model. In contrast to the previously published microgenes, the R16/17 microgene recovers membrane-associated nNOS expression and enhances exercise performance. Recent studies also suggest that mutating surface tyrosine residues can significantly boost AAV transduction efficiency. We delivered 2.85 x 1012 v.g. particles of the tyrosine-mutant AAV-6 canine R16/17 microgene vectors to the forelimb muscles of three GRMD dogs. Immune suppression with oral cyclosporin and mycophenolate mofetil started at one week before injection and continued for five weeks. Injection sites were marked with a tattoo dye. Muscle samples were obtained at 3, 7 and 11 weeks after injection. Robust micro-dystrophin expression was observed in all the time points. No scene of extensive inflammatory cell infiltration was observed at any time point. Taken together, our results suggest that tyrosine-mutant AAV-6-mediated R16/17 microgene expression may represent a viable approach for DMD gene therapy. Further, a relatively brief course of immune suppression may suffice the need of pre-clinical studies in the dog model.

33) Reverse Perfusion Affords Efficient Whole-Body Gene Transfer to Muscle in the Large Animal

Frederick Balzer, Jacqueline Farag, Alock Malik, Mihail Petrov, Andrew Mead, Benjamin Kozyak, Kapil Gopal, Marilyn Mitchell, Connie Choi, Jessie Chen, David Low, Charles Yarnall, Charles Bridges, Hansell Stedman.  Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA.

* contributed equally to this work Proof of concept studies in small animal models of severe muscular dystrophy have shown survival benefit following intraperitoneal or intravenous injection of myotrophic adeno-associated virus (AAV) vectors of serotypes 1, 6 and 8. Before translating these discoveries into clinical therapy, a major limitation must be addressed: the vascular systems of larger terrestrial vertebrates are substantially less permeable than their homologues in mice, in order to manage the hydrostatic pressure gradients associated with gravitation, acceleration and changes in posture. This presents a problem of access, since only after binding to serotype-specific receptors on the cell surface are AAV particles internalized and transported to the nucleus. This is especially relevant to the muscular dystrophies, in which diagnosis is usually made after the young patient has achieved an age of three years and a standing height of three feet. To approximate this scenario, limb-wide, vector-mediated gene transfer to striated myocytes in the mature large animal model has relied upon transient alterations in endothelial permeability utilizing approaches that require isolation from the systemic circulation. Rapidly applied post-capillary venular pressures of 50 to 100 torr are needed to achieve extravasation of vector in the normothermic limb, supporting the hypothesis that vector transport occurs primarily by way of reversibly induced, pressure-dependent gaps in this anatomically unique portion of the endothelial sheet. We further hypothesized that, by carefully manipulating the circulation and body temperature to protect critical organ systems from acute injury, one could safely elevate peripheral venous pressure to the range of 80 torr during vector infusion and achieve efficient, body-wide transport of vector from vascular space to muscle interstitium in the maturing large animal.
This required development of a novel technology for atraumatic balloon occlusion of the full length of the aorta and vena cavae, in order to isolate the viscera and restrict pressure and vector to the more peripheral muscle compartments during a period of protective deep hypothermia and cardiac arrest. We now report the evolution of this approach to optimize safety while realizing an average 300-fold enhancement in the efficiency of body-wide AAV1-mediated gene transfer to striated muscle in the adolescent dog.

34) Combining Gene and Stem Cell Therapy in the Treatment of Dysferlinophaties

Daniele Parolini, Claire Navarro, Andrea Farini, Mirella Meregalli, Marzia Belicchi, Paola Razini, M. Krahn, Louis Garcia, Nicolas Lévis, Yvan Torrente.  Dept. of Neurological Sciences, Università degli Studi di Milano, Milan, Italy; Laboratoire de Genetique Moleculaire, Hopital d'Enfants de la Timone, INSERM U910 Genetique Medicale et Genomique Fonctionnelle, Marseille, France; UMRS787, INSERM/UPMC, Institut de Myologie, Faculté de Médecine Pierre et Marie Curie, Paris, France.

Mutations in gene encoding dysferlin are involved in two main muscular dystrophies: Miyoshi myopathy and Limb-Girdle Muscular Dystrophy 2B. Both diseases are characterized by progressive weakness and skeletal muscle wasting. Dysferlin is expressed in skeletal and cardiac muscles, where its main function is membrane repair. So far, no treatment is available and development of effective therapies remains a big challenge. New hopes are coming from stem cell and gene therapies. Based on work developed in DMD, we investigated the feasibility of stem cell engineering by exon-skipping in dysferlinopathies. We focused our efforts on a patient carrying a deletion in exon 22 found at heterozygous composite state with a large deletion (Δ25-29) predicted to be in-frame. According to the absence of protein observed, we supposed a destabilization and/or degradation of this predicted in-frame truncated mRNA and/or protein. In this study both exons 22 and 23 needed to be removed to restore the ORF and allow production of a truncated functional protein. As proof of principle of the exon-skipping feasibility, we firstly tested if the skipped dysferlin product can be correctly expressed. To achieve this aim, constructs encoding deleted forms of dysferlin were transfected into HEK and 3T3 cells. The truncated dysferlin Δ22-23, mimicking the skipped allele of our patient, was detected in WB, correctly addressed to the membrane and functional. Interestingly, also the protein Δ25-29 was produced, suggesting that the absence of dysferlin in our patient is most probably due to mRNA instability. Finally, a third deleted form was produced carrying the deletion Δ22-29, suggesting that all these exons can be removed without affect dysferlin expression. We then designed antisense oligonucleotides (AONs) able to target acceptor or donor splice sites as well as ESE sequences of exons 22 and 23. Their ability to skip exons of interest was tested on human normal myoblasts and circulating CD133 stem cells isolated from the patient. Unfortunately, very low skipping efficiency was observed and results were not reproducible. To overcome exon skipping difficulties, we developed a strategy based on complete dysferlin delivery by lentivirus vector in blood-derived CD133 stem cells isolated from the same and unskippable patients. The produced vector allowed dysferlin expression both in vitro, in human fibroblasts and mononucleated cells from our patients, and in vivo after intramuscular injection of transduced CD133 stem cells in the scid/blAJ mouse model. We strongly believe that the combination of gene and stem cell therapy represent a useful tool for new therapeutic approaches in dysferlinopathies. Moreover, lentivirus carrying complete dysferlin can be useful to bypass all unskippable mutations, as those located in the transmembrane domain or other essential parts of the gene.

35) Late Gestation Intrauterine Gene Transfer of AAV9 in Non-Human Primates Preferentially Transduces the Central Nervous System, Liver, Skeletal and Cardiac Musculature

C. N. Mattar, N. Johana, A. Fisk, A. Biswas, J. Lu, M. H. Tan, L. G. Tan, A. Rahim, S. Waddington, M. Choolani, J. Chan.  Experimental Fetal Medicine Group, National University of Singapore, Singapore, Singapore; University of Queensland, Brisbane, Australia; DSO National Laboratories, Singapore, Singapore; University College, London, United Kingdom; Reproductive Medicine, KK Women's and Children's Hospital, Singapore, Singapore.

Monogeneic diseases resulting in irreversible brain damage are potential candidates for intrauterine gene transfer. IUGT mediates access to specific organ compartments not easily targeted in adult recipients and may circumvent a nullifying immune response to the transgene and vector. AAV9 targets neonatal neuronal tissues and adult glia when administered systemically, suggesting different environmental influences affecting vector tropism during development and post-natal life. Here we examined vector biodistribution and transduction efficacy, transgene expression and immune response to AAV2/9 following late gestation IUGT in non-human primates. Materials and methods
M fasicularis females were time-mated and 1x1013 vector genomes of scAAV2/9-CMV-eGFP were injected via the fetal intrahepatic vein under ultrasound guidance. Maternal viraemia was determined with qPCR following serial blood sampling. Selective maternal tissue sampling was performed after delivery of infants. At predetermined time-points comprehensive necropsies were performed on injected offspring and transgene expression assayed through stereoscopic bioimaging, immunohistochemistry and molecular analyses. Results
Two fetuses received 3.3 to 4.0x1013 vg/kg (birth weight) of scAAV9-GFP at 0.9G. Low-level maternal viraemia was detected between 5 minutes and 48 hours following IUGT with peak levels at 1.8 and 2.6 vectors/µl plasma. Offspring were delivered one week later. Despite an increased anti-AAV9 antibody response in mothers and infants persisting up to 3 months following IUGT, GFP expression was prominent in the maternal ovarian cortex, mononuclear cells and skin of both mother and infant sampled at delivery. Varied levels of maternal tissue transduction were demonstrated at delivery with levels ranging from >300 vg/dg in skeletal muscle to >1800 vg/dg in mononuclear cells. Stereoscopic fluorescence microscopy at necropsy demonstrated prominent GFP expression in skeletal muscles, diaphragm, dorsal root ganglia, myocardium and renal cortex. Multiorgan vector distribution was noted with a wide range of transduction levels, from <2 copies per cell in the intestines to several-fold higher in the liver. Immunohistochemistry confirmed the expression of GFP in the central nervous system, renal cortex, liver, seminiferous tubules and myocardium. Conclusions
A single dose of AAV9 delivered systemically in late gestation resulted in widespread vector biodistribution with robust transgene expression in skeletal muscle, diaphragm, myocardium, kidney, central nervous system and dorsal root ganglia. Pre-existing maternal immunity to AAV9 did not neutralize transgene expression in injected offspring. Thus AAV9 may be a useful vector with which to target such genetic diseases as spinal muscular atrophy, cystic fibrosis and Duchenne muscular dystrophy.

36) Monitoring and Suppression of Immune Responses to AAV-Mediated Gene Therapy in Canine Muscle

Zejing Wang, Rainer Storb, Donghoon Lee, Martin Kushmeric, Baocheng Chu, Jeffery Chamberlain, Stainly Riddell, Stephen Tapscott.  Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Radiology, University of Washington, Seattle, WA; Neurology, University of Washington, Seattle, WA; Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA; Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA.

The use of Adeno-associated viral (AAV) vectors as a gene delivery vehicle has shown promise both in preclinical studies and clinical trials for a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD). We previously demonstrated that direct intramuscular injections of rAAV2 or rAAV6 in wild type and DMD dogs resulted in robust T-cell responses to viral capsid proteins. Others have shown that cellular immunity to AAV capsid proteins coincided with liver toxicity and elimination of transgene expression in a trial of human hemophilia B. It has been suggested that activation of T cells after AAV injection in mice was due to binding of the virion to heparan sulfate proteoglycans (HSPG), which facilitates its uptake by dendritic cells. Both AAV2 and AAV6 contain HSPG binding motifs. Hence, we compared the immunogenicity of capsids from AAV6 to those from AAV1, which is a serotype without a heparin-binding domain, and AAV1-E531K which has an introduced heparin-binding site. We found that immune responses were elicited independent of HSPG binding. We also confirmed the utility of ELISpot assays for detecting such responses with cells derived from peripheral blood. AAV vectors are currently being used in human trials for intramuscular delivery of transgenes. An important component of human clinical trials will be non-invasive monitoring of inflammation at sites of gene delivery. We tested whether magnetic resonance imaging (MRI) could be used for non-invasive detection of inflammatory reactions to rAAV vectors in muscle. Studies on dogs injected with different vectors (rAAV6, rAAV1 and rAAV1-E531K) showed that MRI accurately detected local inflammatory responses following intramuscular rAAV injection. We previously demonstrated that immune response to AAV vectors in DMD dogs could be averted by a brief course of immunosuppression with anti-thymocyte globulin, cyclosporine, and mycophenolate mofetil, which resulted in long-term and robust expression of transgenes in the skeletal muscle. In order to reduce toxicities associated with these immunosuppressants, we have initiated studies of alternative, efficient but less toxic regimens through T-cell co-stimulation blockade with or without rapamycin. In conclusion, our studies suggested that heparin-binding ability of a given AAV serotype did not alter induction of T-cell responses after intramuscular injection in dogs. Further, the studies validated the utility of ELISpot assays and MRI for monitoring immune and inflammatory responses to rAAV vectors in dogs. Therefore, these assays should be part of future human clinical trials of AAV gene therapy to monitor immune responses. Finally, immunosuppression is probably required when applying AAV-mediated gene therapies to treat human patients.

37) Irradiation of Adult mdx Mice Prior to Full-Length Murine Dystrophin cDNA Transfer Results in a Delayed and Diminished Anti-Dystrophin Host Immune Response

Saman Eghtesad, Heng Zheng, Hiroyuki Nakai, Michael W. Epperly, Paula R. Clemens.  Neurology, University of Pittsburgh, Pittsburgh, PA; Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; Neurology Service, Veterans Affairs Medical Center, Pittsburgh, PA.

Duchenne muscular dystrophy (DMD) is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, and for which dystrophin gene transfer could be an effective treatment. The host immune response to dystrophin protein, however, is an obstacle to successful therapeutic gene expression. Understanding the host immune response induced by dystrophin will facilitate discovery of strategies to prolong expression of recombinant dystrophin protein in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse prior to intramuscular high-capacity adenoviral-mediated gene transfer of a full-length, murine, dystrophin cDNA, we temporally removed the host immune system. A 600rad dose removed peripheral immune cells before gene transfer and was followed by self-reconstitution of immunity. A 900rad dose removed central and peripheral immune cells and was followed by adoptive transfer of wild-type syngeneic whole bone marrow after gene transfer. Control mice were either left untreated or received gene transfer without irradiation. We observed a delayed anti-dystrophin humoral response that correlated with a decreased, but not complete loss of dystrophin expression in both 600rad and 900rad irradiated, vector-treated mice. Non-irradiated, vector-treated mice showed a complete loss of muscle dystrophin expression that correlated with an earlier anti-dystrophin humoral response and necrotic muscle fibers surrounded with infiltrating CD4+ and CD8+ T cells. Although the levels of infiltrating T cells in muscle of vector-treated mdx mice were similar whether or not the mice were irradiated, the pattern of T cell infiltration was more dispersed in those mice that were irradiated and did not appear to be focally attacking individual muscle fibers. With 900rad, but not 600rad, irradiation, we observed a significantly increased number of infiltrating regulatory T cells in vector-treated muscles. In conclusion, by studying antigen-specific humoral responses to dystrophin and cellular infiltration in muscle in the setting of dystrophin gene transfer and temporal removal of the host immune system, our data support that dystrophin gene transfer induces an anti-dystrophin immune reaction that is comprised of both humoral and cell-mediated responses. Immunity induced in the dystrophic host, in response to dystrophin gene transfer, is associated with loss of dystrophin-transduced fibers. This immunity is delayed, but not eliminated by temporal removal of the host central or peripheral immune response. Therefore, immunity induced by recombinant dystrophin expression will have significant implications for dystrophin gene therapy for DMD.

38) Identifying Genes and Signaling Pathways Regulating the Quiescent State in Muscle Stem (Satellite) Cells

Kristy Boyle, Jeffrey S. Chamberlain.  Department of Neurology, The University of Washington School of Medicine, Seattle, WA.

Duchenne muscular dystrophy (DMD) is an X-linked degenerative disease affecting approximately 1/3,500 newborn males and is caused by mutations in the dystrophin (dmd) gene. Loss of dystrophin causes membrane instability of skeletal muscle fibers resulting in degeneration and subsequent cycles of regeneration within the musculature. Regeneration is mediated by the resident stem cells, termed satellite cells, which are located between the external lamina and the sarcolemma of adult skeletal muscle fibers. Satellite cells are generally quiescent however, in response to muscle disease, injury, exercise and growth they become activated, proliferate, and differentiate into post-mitotic myofibers. As endogenous muscle stem cells, satellite cells are an attractive population for cell-mediated therapy of DMD however the mechanisms governing the quiescence, activation and self-renewal of these cells remain to be fully elucidated. Quiescence is a poorly understood cell state that appears to be under active control and is not simply a state of inactive transcription. Understanding the mechanisms that control satellite cell quiescence may provide key insights into muscle regeneration and enhance the ability to manipulate these processes in order to further understand the potential for satellite cells to be used as a cell therapy. To investigate this, we isolated satellite cells from the hind limb muscles of WT mice by collagenase/dispase digestion and contaminating CD45+/Mac-1+/Sca-1+ cells were subsequently removed by MACS cell separation. The resulting population was approximately 80% pure as shown by immunostaining, and cell cycle analysis revealed that over 80% of the resulting myogenic cell population was in G0, indicating a high proportion of quiescent cells in the isolated population. A previously reported microarray study identified a collection of approximately 500 genes expressed more highly in quiescent compared to activated satellite cells, which included three Kruppel-like factors (Klf4, 9, and 15), and the Oncostatin M receptor (OSMR) (1). By RT-PCR we confirmed that the expression of all three Klf genes was increased in quiescent satellite cells and that expression subsequently decreased during a time-course of activation in culture. Furthermore, this decrease in expression correlated with increasing expression of MyoD, which is upregulated in activated satellite cells, therefore indicating a potential role for the Klf genes in the regulation of satellite cell quiescence. Lastly, exogenous administration of OSM to satellite cells in vitro resulted in retained expression of the Klf genes over a three day activation period and a concomitant decrease in MyoD expression, indicating a potential link between OSM signaling and Klf gene expression. (1) Fukada et al. (2007) Stem cells. 25(10):2448-59

39) Gene and Cell Therapy Approaches Tto Treating the Dysferlinopathies

Bradley A. Williams, Nilah Monnier, Douglas E. Albrecht, Nupur Garg, Laura E. Rufibach, Esther Hwang, Angela Salerno, Plavi Mittal.  Jain Foundation Inc., Bellevue, WA.

The dysferlinopathies, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi myopathy, are a group of inherited diseases caused by mutations in the gene encoding dysferlin. These diseases are characterized by progressive skeletal muscle wasting, typically beginning in the late teenage years. Our foundation supports targeted research projects that address fundamental roadblocks and challenges in the use of gene and cell therapy to treat the dysferlinopathies. Gene therapy is complicated by the fact that the dysferlin gene is large (roughly 6 kb, above the packaging capacity of AAV vectors) and that the structural subdomains important for its function have not been characterized. We are supporting the development of a dual-vector AAV strategy in which the two halves of the dysferlin gene are delivered independently to muscle fibers, where they are reassembled by trans-splicing into the full-length gene. We are also supporting the development of non-viral methods to deliver and integrate the full-length dysferlin gene into cells, including transfection of full-length dysferlin and phiC31 integrase into autologous patient-derived muscle stem cells. In addition, we are supporting structural studies of the dysferlin protein, which may suggest combinations of subdomains with the potential to form a functional mini-dysferlin small enough to permit packaging into a single AAV vector. In the muscle stem cell therapy field, we have found that a major challenge is the lack of standardized methods for comparing and contrasting the stem cell populations being studied in different laboratories, and we are actively promoting efforts to develop these standards. In addition, we are supporting the development of methods to increase the regeneration capacity of existing muscle progenitor cells in dysferlinopathy patients, as well as supporting basic studies on the role of dysferlin in myogenic cells. We believe that pursuing all of these avenues of research in parallel has the greatest potential to realize our goal of an approved therapy for dysferlinopathy patients in the foreseeable future.

40) In Vivo Reprogramming into the Myogenic Lineage and Improving Engraftment in the mdx Mouse Model of DMD

Lindsey A. Muir, Jeffrey S. Chamberlain.  Neurology, University of Washington, Seattle, WA.

Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. Direct lineage specific reprogramming of patient-derived adult stem cells is a possible approach for cell therapy aimed at regeneration of muscle. Use of primary fibroblasts for dystrophin delivery in the mdx4cv mouse model of DMD offers potential advantages over conventional myoblasts in relative ease of retrieval, expansion, and culturing, and may be a promising alternative for use in autologous cell transplantation. We have shown that primary murine fibroblasts transduced by lentivirus with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)] and transplanted into dystrophic muscles enables tamoxifen-dependent regeneration of myofibers that express micro- and mini-dystrophin. The success of future translational studies will depend on reproducible methods for cell delivery and achieving maximal engraftment, which eventually must be explicitly delineated for a clinical setting. In order to explore whether delivery of reprogrammed fibroblasts improves function in dystrophic muscle, we are investigating methods for maximizing engraftment, in terms of both cell survival and contribution to muscle fibers. We are currently using reprogrammed murine dermal fibroblasts and a whole muscle mononuclear population to test delivery methods (volume and number), use of chemical and toxin-based injury protocols, and myoblast and fibroblast mitogens. We are additionally investigating the role of the host immune system in engraftment by evaluating differences between immune-competent and immune-deficient mdx hosts.

41) AAV Microgene Transfer Reveals the Cellular Motif for Dystrophin-Mediated Sarcolemmal Neuronal Nitric Oxide Synthase (nNOS) Localization

Yi Lai, Yongping Yue, Dongsheng Duan.  Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO.

Duchenne muscular dystrophy (DMD) is a severe inherited muscle disease caused by dystrophin deficiency. Over the last few years, several highly abbreviated micro-dystrophin genes have been developed for DMD gene therapy. Though promising, these microgenes remain suboptimal. An important function of dystrophin is to anchor neuronal nitric oxide synthase (nNOS) to the sarcolemma. We recently found that dystrophin spectrin-like repeats 16 and 17 (R16/17) are crucial for membrane-associated nNOS localization. However, the underlying mechanism is not clear. A better understanding of the nNOS recruiting motif in dystrophin may guide us develop more effective microgene therapy. Each dystrophin spectrin-like repeat contains three α-helices. Here we tested the hypothesis that R16/17-mediated nNOS anchoring depends on the correct α-helix phasing and composition. To determine the impact of the α-helix phasing, we sequentially deleted one of the six α-helices of R16/17 in the ΔR2-15/ΔR18-23/ΔC microgene. Adeno-associated virus (AAV) was used to introduce these microgenes to dystrophin-null mdx mice. The original ΔR2-15/ΔR18-23/ΔC microgene efficiently recruited nNOS to the membrane. However, none of the modified microgenes restored sarcolemmal nNOS. To determine the contribution of α-helix composition, we swapped each α-helix of R16/17 with the corresponding α-helix of repeat 18 and performed a yeast-two-hybrid assay. Except for the first α-helix of repeat 17, replacing other α-helices of R16/17 did not compromise nNOS interaction. To confirm the in vitro finding, we engineered these chimeric repeats into the ΔR2-15/ΔR18-23/ΔC microgene and performed AAV gene transfer study. In contrast to the yeast-two-hybrid result, in vivo screening suggest that the second, third α-helix of repeat 16 and the first α-helix of repeat 17 are all required for anchoring nNOS. In summary, our results suggest that the correct phasing and composition are critical for dystrophin-mediated sarcolemmal nNOS localization. An intact R16/17 is essential for micro-dystrophin based DMD gene threrapy.

42) High Force Myosin Correlated with the Early Degeneration of Jaw Closing Muscle in the GRMD Model of DMD

Andrew F. Mead, Alock Malik, Mihail Petrov, Martin Childers, Marilyn Mitchell, Janet Bogan, Joe Kornegay, Brittani Nickens, Hansell Stedman.  University of Pennsylvania, Philadelphia, PA; Wake Forest University, Winston-Salem, NC; University of North Carolina, Chapel Hill, NC.

Duchenne Muscular Dystrophy (DMD) is a disease caused by the deficiency of the cytoskeletal protein dystrophin, and characterized by the progressive degeneration of muscle. Dystrophin's normal physiological roles contribute to membrane stability during forceful contraction and to sarcoplasmic calcium regulation. In the maintenance of a colony of dystrophin null dogs it was noted that essentially all animals exhibited progressive trismus within the first year. This symptom complicates the clinical management of many dogs, often requiring hand feeding of pureed solids to support skeletal growth and to avoid accelerated general clinical deterioration. Trismus has not been reported in the constellation of disease-related problems affecting patients with DMD. We hypothesized that the discrepancy relates to another genetic difference between the species: among carnivores and primates, only humans lack the powerful masticatory muscle (MYH16) myosin because of a frameshifting deletion unique to the hominid lineage. Recent studies have shown that the MYH16+ muscle fibers of carnivores develop significantly higher specific force than all other muscle fibers from the same animals. We further hypothesized that the combined presence of MYH16 and absence of dystrophin in a single muscle fiber would further increase the susceptibility to sarcolemmal damage during forceful contracture. Histological examination of the masticatory and locomotive muscles in the GRMD model using a novel, epitope-directed antibody against a unique surface loop of the MYH16 myosin shows that fibers in the temporalis that express MYH16 are preferentially susceptible to necrosis. Further studies are underway to precisely correlate the relationships between histological indices of myonecrosis/apoptosis and the expression of isoforms of myosin and several proteins involved in calcium signaling and homeostasis

43) AAV-Mediated SERCA2a Expression Improves the Electrocardiographic Profile of Aged mdx Mice

Jin-Hong Shin, Brian P. Bostick, Yongping Yue, Roger J. Hajjar, Dongsheng Duan.  Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; Cardiovascular Research Institute, Mount Sinai School of Medicine, New York, NY.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. Dilated cardiomyopathy and subsequent heart failure is a major cause of death in DMD. Recent studies suggest that AAV-mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression holds great promise for the treatment of heart failure. Here we hypothesize that AAV-mediated SERCA2a expression may ameliorate cardiomyopathy in DMD. To test this hypothesis, we injected AAV9-SERCA2a into 12-month-old female mdx mice (n=5) via the tail vein. Mdx mice are the most commonly used animal model for DMD. These mice develop cardiomyopathy characteristic of DMD when they reach 20 months of age. Eight months after AAV9-SERCA2a administration, we compared electrocardiographic changes of SERCA2a-treated mice with age- and sex-matched untreated mdx (n=9) and normal C57BL/10 (n=7) mice. We observed significant improvement in several ECG parameters (P<0.01, compared to those of untreated mdx mice). The tachycardia seen in mdx mice (655 ± 11 bpm) was reversed after SERCA2a treatment (541 ± 18 bpm). Untreated mdx mice showed characteristic PR interval reduction (normal, 40.7 ± 1.4 msec; mdx, 35.1 ± 0.8 msec) and QT interval prolongation (normal, 20.8 ± 0.4 msec; mdx, 27.8 ± 0.8 msec). These indices were fully normalized in SERCA2a-treated mice (PR interval, 41.4 ± 1.5 msec; QT interval, 20.6 ± 1.2 msec). The cardiomyopathy index was also completely normalized (normal, 0.78 ± 0.07; untreated mdx, 1.11 ± 0.05; SERCA2a-treated mdx, 0.69 ± 0.06). Interestingly, QRS duration and Q wave amplitude were not altered by SERCA2a therapy. In summary, our results suggest that AAV-mediated SERCA2a expression improves the electrocardiographic profile in aged mdx mice. Future hemodynamic studies are warranted to further corroborate the therapeutic benefit of SERCA2a in end-stage dystrophic heart disease.

44) Increased Muscle Regeneration in Hindlimb and Diaphragm of mdx Mice Treated with AAV9 Mini-Dystrophin and Octalysine-NEMO Binding Domain Peptide

Daniel P. Reay, Geno Raggi, Bing Wang, Xiao Xiao, Paul D. Robbins, Paula R. Clemens.  Neurology, University of Pittsburgh, Pittsburgh, PA; Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; Department of Veteran's Affairs, Pittsburgh, PA; School of Pharmacy, University of North Carolina, Chapel Hill, NC.

The loss of a functional, membrane-localized dystrophin protein is the primary cause of Duchenne muscular dystrophy (DMD). The muscle inflammatory infiltrates and failure of muscle regeneration that are secondary to dystrophin loss have recently been shown to be due, in part, to activation of the nuclear factor κB (NF-κB). Dystrophic muscle shows substantially increased nuclear accumulation of NF-κB, which is well known to regulate cytokines, other inflammatory molecules and myogenic proteins. Toward the development of therapy for DMD, we designed an experiment to test the combination of dystrophin gene replacement and inhibition of NF-κB activation. For dystrophin gene replacement therapy, we utilized an AAV9 vector to systemically deliver a mini-dystrophin transgene to 3 day old neonatal dystrophin-deficient mdx mice. Beginning at 4 weeks of age, mice were then treated with tri-weekly injections of octalysine-NEMO binding domain peptide (8K-NBD), to inhibit NF-κB activation. Mice treated with either AAV9 mini-dystrophin vector alone or AAV9 mini-dystrophin vector plus 8K-NBD peptide exhibited mini-dystrophin expression and reduced necrosis in the quadriceps and diaphragm, but mice treated with the AAV9 mini-dystrophin vector plus 8K-NBD peptide additionally demonstrated increased levels of muscle regeneration in quadriceps and diaphragm. EMSA analysis for NF-κB confirmed decreased levels of nuclear NF-κB in 8K-NBD-treated mdx mice. Our data suggest that high levels of mini-dystrophin gene transfer are required to demonstrate a reduction in the nuclear accumulation of NF-κB in muscle of mdx mice treated with vector alone. Overall, treatment of mdx mice with AAV9 mini-dystrophin gene replacement therapy in combination with NBD peptide/NF-κB inhibitory therapy may provide critical insight into potential treatments for DMD.

45) A Comparison of Adeno-Associated Virus Mediated Gene Transfer into Healthy and Dystrophin-Deficient Skeletal Muscles

Gilles Moulay, Carole Masurier, Pascal Bigey, Daniel Scherman, Antoine Kichler.  Genethon, BP60, Evry Cedex, France; Unité de Pharmacologie Chimique et Génétique et d'Imagerie, CNRS UMR 8151 – U1022 Inserm, Université Paris Descartes, Chimie Paristech, Paris Cedex, France.

Skeletal muscle is an attractive gene therapy target for several reasons: it is easily accessible and constitutes about 30% of the normal adult body mass; it has an abundant blood vascular supply, thus providing an efficient transport system for the secretion of proteins into the circulation; and it provides a very permissive physiological environment for adeno-associated virus (AAV) mediated gene transfer. However, gene therapy in pathologic, in particular dystrophic muscle may be more difficult than in healthy tissue due to degenerative-regenerative processes as well as to the inflammatory context.
In the present work, we followed the expression levels of a secreted inhibitor of the pro-inflammatory TNF cytokine in healthy and dystrophic muscles after AAV-2/6 mediated transduction. Methods
AAV-2/6 vectors encoding chimeric proteins inhibiting TNF-a were injected into either mdx mice (a mouse model of Duchenne muscular dystrophy in a C57Bl/10 background) or in healthy C57Bl/10 mice. The chimeric proteins that were used are the human and murine tumor necrosis factor soluble receptor I fused with the murine heavy immunoglobulin chain (h and mTNFR-Is/mIgG1, respectively). We conducted an AAV-2/6 dose-response study and we determined the expression kinetic of the transgenic proteins by ELISA. Further, we followed the antibody response against the transgene and studied the expression pattern in the injected muscles by immunostaining. Results
Our two main findings are that: 1- when injecting an equivalent dose, the transduction efficiency by AAV-2/6 is reduced in dystrophic muscles as compared to the healthy ones; 2- the humoral response against the transgenic protein is stronger in the mdx mouse.
Taken together, our results underscore that the inflammatory context of dystrophic muscles has to be taken into consideration when designing gene therapy approaches. Not only the levels of expression are lower in the dystrophic muscle but the inflammatory context favours immune response against the transgene.

46) Sarcolemmal nNOS Anchoring Reveals a Functional Difference between Dystrophin and Utrophin

Yongping Yue, Dejia Li, Luke Judge, Akshay Bareja, Yi Lai, Kay E. Davies, Jeffrey S. Chamberlain, Dongsheng Duan.  Department of Molecular Microbiology and Immunology, The University of Missouri, Columbia, MO; Department of Neurology, The University of Washington, Seattle, WA; Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin help maintain sarcolemmal stability by linking the extracellular matrix and the cytoskeleton. Further, dystrophin recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Reduced sarcolemmal integrity has been considered as a major pathogenic mechanism in DMD. Interestingly, recent studies suggest that failure to anchor nNOS to the membrane also contributes to muscle fatigue. Over the last two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence homology, structural similarity and functional properties with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. The full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic over-expression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Our results suggest that full-length utrophin does not anchor nNOS to the sarcolemma. This finding may have important implications in developing utrophin-based DMD therapies.

47) Detailed Physical Mapping of Deletion Breakpoints in the GSHP Dog Model for DMD

Alock Malik, Acong Xu, Jesse Chen, Andrew Mead, Martin Childers, Janet Bogan, Scott Schatzberg, Joe Kornegay, Hansell Stedman.  University of Pennsylvania, Philadelphia, PA; Wake Forest University, Winston-Salem, NC; University of North Carolina, Chapel Hill; University of Georgia, Athens.

The German Short Haired Pointer model for Duchenne muscular dystrophy (GSHPMD) may provide a unique opportunity to study the immunologically relevant aspects of gene therapy for DMD as it might provide an unmitigated dystrophin null environment unlike the other known animal models for DMD (e.g. mdx, GRMD, in which point mutations result in dystrophin deficiency with the potential for reversion or read-through by alternative splicing). Although earlier studies revealed the GSHPMD mutation to be a visible deletion within the p21 region of the canine X chromosome, the precise boundaries of this deletion and their relationship to the flanking genes are not yet known. In order to localize the deletion breakpoints a PCR based strategy was used. A 9Mbp region flanking the canine dystrophin gene was targeted to strategically design various primers to amplify the region syntenic to the human Xp21 region. Based on the PCR amplification profile the deletion boundaries were quickly resolved to within a few thousand base pairs. Our data suggest that the deletion covers approximately 3Mbp upstream of the 5'UTR and ends very close to the 3'UTR knocking out the entire dystrophin gene and most likely affecting at least one flanking gene, BCMP1, as well. We anticipate sequencing across the deletion breakpoint in the coming weeks, with the opportunity to define the most probable genomic mechanism resulting in the deletion. Accurate description of the breakpoints of the deletion will help in early screening and carrier detection in GSHPMD colony, and will allow a precise depiction of the exons removed. The GSHPMD model may play a key translational role in the future development of therapies for DMD because of the anticipated complete absence of any dystrophin open reading frame and hence utility as a surrogate, from the standpoint of potential immunological response to recombinant dystrophin, for all DMD gene deletions found in the human gene pool. This model may also represent a unique opportunity to define the physiological role of the highly conserved “brain cell membrane protein 1” (BCMP1), a highly expressed, brain-specific, putative four-transmembrane protein previously suggested to be a candidate gene for X-linked mental retardation.

48) Pre-Existing Antibodies to AAV8 Attenuates Micro-Dystrophin Expression Following Targeted Vascular Delivery

Louise R. Rodino-Klapac, Chrystal Montgomery, William Bremer, Nancy Davis, Kimberly Shontz, Vinod Malik, Katherine Campbell, Thomas J. Preston, Zarife Sahenk, K. Reed Clark, Brian D. Coley, Christopher M. Walker, Jerry R. Mendell, Louis G. Chicoine.  Center For Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH; Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, OH; Heart Center, Nationwide Children's Hospital, Columbus, OH.

Duchenne muscular dystrophy (DMD) is the most common muscle disease of childhood and is caused by mutations within the dystrophin gene. Thus it is potentially amenable to gene therapy. Several gene replacement strategies are under investigation and progress towards clinical gene therapy with adeno-associated virus (AAV) delivered transgenes remains promising. An impediment to successful vascular gene therapy is the presence of pre-existing antibodies that cross-react with various AAV serotypes. We tested the hypothesis that pre-existing antibodies to AAV8 would attenuate transgene expression following vascular delivery of a rAAV8.micro-dystrophin vector to the gastrocnemius of non-human primates. Rhesus macaques (n=6) were stratified into two groups based on AAV8 binding antibody sero-status (plus or minus). Animals were intubated and placed under isoflurane anesthesia and the femoral artery was isolated and cannulated with the goal of targeting the gastrocnemius muscle for vascular delivery of vector. Prior to vector administration, a prevector flush of saline (2.5 ml/kg) was given over 1 minute. This was immediately followed by occluding blood flow to the extremity using proximal and distal tourniquets to compartmentalize the gastrocnemius. rAAV8.MCK.micro-dys.FLAG, was infused over 1 minute at a dose of 2 × 1012 vg/kg in 2.5 ml/kg of Tris buffered saline. The extremity remained isolated from the circulation for 10 minutes. A postvector flush (2.5 ml/kg) was infused over 1 minute and then the tourniquets were released. Bleeding was controlled by direct pressure and the wound was surgically closed. None of the animals suffered noticeable edema or adverse effects from the procedure. Three months after transfer, the macaques were euthanized, and the gastrocnemius muscle harvested. Samples of proximal, central and distal muscle were stained with anti-FLAG antibody and the percentages of muscle fibers expressing the transgene were measured. The contralateral gastrocnemius served as a negative control. Efficient micro-dystrophin expression was visualized in all six subjects. However, subjects without pre-existing AAV8 antibodies demonstrated more transgene expression compared to subjects with pre-existing antibodies (% positive muscle fibers: Proximal 71.3 ± 3.5; Central 82.0 ± 4.0; Distal 71.0 ± 6.5 vs. Proximal 26.0 ± 3.2; Central 35.7 ± 1.8; Distal 29.3 ± 0.3, respectively; P ≤ 0.003). From these data we conclude that the presence of pre-existing binding antibodies attenuate transgene expression. We speculate that immune modulation of patients with plasmapheresis may enhance transgene expression in cases where pre-existing vector antibodies are present.

49) Regulatory Cassettes Derived from the Slow Troponin I Gene Confer High-Level Expression in Skeletal Muscle after Gene Transfer Mediated by Helper-Dependent Adenovirus and Recombinant AAV

Rénald Gilbert, Mehdi Bendjelloul, Yué Zeng, Claude Guérin, Nancy Larochelle, George Karpati, Bernard Massie, Josephine Nalbantoglu.  Genomics & Gene Therapy Vectors, Biotechnology Research Institute, Montreal, QC, Canada; Neuromuscular Research Group, Montreal Neurological Institute, Montreal, QC, Canada; Département de Microbiologie et Immunologie, Université de Montréal, Montreal, QC, Canada.

Viral vectors used for gene replacement therapy of muscle diseases, such as Duchenne muscular dystrophy, require regulatory elements that can confer strong and muscle-specific expression of the therapeutic gene product. To achieve this goal, we generated regulatory cassettes by linking three (ΔUSEx3) or four (ΔUSEx4) copies of the truncated 60-pb upstream enhancer (ΔUSE) of human slow troponin I gene. ΔUSE has been reported to drive pan-muscle specific expression in transgenic mice. In addition, we have previously shown, using naked DNA, that ΔUSEx3 confers strong and skeletal muscle-specific expression in cell culture and in vivo. To evaluate the activity of ΔUSEx3 and ΔUSEx4 in the context of viral vectors, we constructed helper-dependent adenovirus (HD) expressing β-galactosidase (β-gal) regulated by ΔUSEx3, ΔUSEx4, or by the hybrid CMV enhancer/β-actin (CB) promoter. We also constructed recombinant AAV vectors expressing the green fluorescent protein (GFP) regulated by ΔUSEx4 or CMV. The β-gal activity of HD-ΔUSEx3(β) and HD-ΔUSEx4(β) was 1% of HD-CB(β) in cultured non-muscle cells and 20 to 90% in differentiated myotubes. After intramuscular injection of normal and mdx mouse muscle, the number of transduced fibers and the β-gal activity of HD-ΔUSEx3(β) and HD-ΔUSEx4(β) corresponded to 20 to 80% of the value obtained with HD-CB(β). Notably, the number of transduced fibres after intramuscular injection of AAV-ΔUSEx4(GFP) in adult mdx muscle was similar to the value obtained under the same conditions using AAV-CMV(GFP). In summary, the strength, muscle specificity and small size of ΔUSEx3 and ΔUSEx4 render them very attractive for gene transfer applications in skeletal muscle.

50) Blocking the Myostatin Signal Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Raouia Fakhfakh, Annick Michaud, Jacques P. Tremblay.  Human Genetics Unit, Chul-Chuq, Quebec, QC, Canada.

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin is a negative regulator of skeletal-muscle development and responsible for limiting regeneration. It binds with high affinity to the ActRIIB, which initiates signaling through a smad2/3-dependent pathway. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with expression of dominant negatif mutants of activin type IIB receptor, (dnActRIIB). In vitro, human myoblasts is infected with a lentivirus carrying or not dnActRIIB. Increase proliferation and fusion of dnActRIIB myoblastes is the result of blocking myostatin activity. This inhibition regulates the expression of myogenic regulatory factors (MRLFs). In vivo, dnActRIIB myoblastes was transplanted in immunodifiscient dystrophic mice (Rag/mdx). Dystrophin immunostaining in the tissue cross-sections of tibialis anterior muscles of RAG/mdx mice 3 weeks post-transplantation revealed more human dystrophin-positive myofibers with dnActRIIB myoblasts than controls. Blocking the myostatine signal allowed the success of myoblast transplantation to improve, the fusion and the proliferation of myoblastes to increase and regulat expression of myogenic regulator factors.