RESUMOS QUE SERÃO APRESENTADOS NO CONGRESSO ANUAL DA SOCIEDADE AMERICANA DE TERAPIA GÊNICA, BOSTON, MAIO DE 2008

[531] Gait Disturbances in Animals with Muscular Dystrophy

Thomas G. Hampton, Ajit Kale, Ivo Amende, Hemmi N. Bhagavan, Case G. VanDongen R&D, The CuraVita Corporation, Boston, MA; Medicine, Saint Elizabeths Medical Center at Tufts, Boston, MA; R&D, BioBreeders, Inc., Watertown, MA

Gene therapy holds great promise as a treatment for muscular dystrophy. Gait analysis is increasingly becoming of interest to researchers in muscular dystrophy. Clinically, muscular dystrophy results in gait disturbances, and gait analysis is routinely used to aid in diagnosis and treatment. The dystrophin-deficient [mdx] mouse, and the delta-sarcoglycan-deficient [BIO TO2] hamster are excellent models to study muscular dystrophy and the efficacy of gene therapy. Yet, little is known about gait in these animal models. Here, we examine gait in mdx mice and BIO TO2 hamsters to identify physio-markers of muscular weakness in animals as they walk on a motorized treadmill belt. We demonstrate, for the first time, significant functional differences in forelimb gait vs. hind limb gait in dystrophin-deficient mice and BIO TO2 hamsters. The gait of 1 month old hamsters is similar to that of 12 week old mice, with comparable stride lengths [7 cm], stepping frequencies[4 Hz], and relative contributions of stance [65%] and swing [35%] to stride duration. Decreases in hind limb propulsion and increases in hind paw eversion are characteristic of muscle weakness in mice and dystrophic hamsters. We further demonstrate that hind limb propulsion decreases and hind paw eversion are characteristic of neonatal mouse pups that are just able to support their weight and walk on a treadmill [ 16 days of age]. Taken together, two distinct gait metrics are common to two different animal models of muscle weakness, indicating that gait analysis in rodent models may provide phenotypic information regarding muscle strength. Identification of muscular dysfunction in the mdx mouse and BIO TO2 hamster will be important in examining the effectiveness of gene therapy for muscular dystrophy.
 

[537] Effect of Gender on Phenotype in Golden Retriever Muscular Dystrophy

Joe N. Kornegay, Daniel J. Bogan, Janet R. Bogan Department of Pathology and Laboratory Medicine and the Gene Therapy Center, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC

Background. Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting approximately 1 of 3,500 newborn human males in whom absence of the protein dystrophin causes progressive degeneration of skeletal and cardiac muscle. Spontaneous forms of X-linked muscular dystrophy due to dystrophin deficiency have been identified in mice, multiple dog breeds, and cats. Unlike the dystrophin-deficient mdx mouse, which remains relatively normal clinically, affected dogs develop progressive, fatal disease similar to DMD. The most studied canine condition is golden retriever muscular dystrophy (GRMD). So as to better utilize the GRMD model in therapeutic trials, we have evaluated the diseases natural history using phenotypic tests. Affected dogs have joint contractures and weakness of individual and grouped muscles. Importantly, by comparing serial measurements of these tests, one can document improvement or delayed progression of disease. Previously published studies have suggested that gender affects phenotype in both the mdx mouse and GRMD dog. Homozygous females reportedly have less severe clinical signs, due presumably to various factors, including effects of estrogen on muscle regeneration and inflammation. Such a gender effect could influence interpretation of preclinical studies in which both male and female animals are used. Materials and Methods. To further clarify the effect of gender on the GRMD phenotype, we evaluated several phenotypic tests in untreated homozygous females (F; n = 24) and heterozygous males (M; n = 27) at 6 months of age. Newborn GRMD dogs were identified based on elevation of serum creatine kinase and subsequently developed characteristic clinical signs. Genotype was confirmed by PCR in most dogs. Methods for measuring tibiotarsal joint angle and torque force have been published. Cranial sartorius circumference measurements were made by encircling the muscle with suture at the time of biopsy. Paired t-tests (parametric) or Mann-Whitney rank sum test (non-parametric) were used. Results. Male and female values (mean SD) for tibiotarsal joint angles (M = 150.59 10.86^o, F = 145.25 14.47^o; p = 0.14); torque force generated by either isometric tibiotarsal tetanic flexion (M = 0.438 0.114 N/kg, F = 0.448 0.152 N/kg; p = 0.79) or extension (M = 2.109 1.071 N/kg, F = 2.171 0.722 N/kg; p = 0.604); tetanic extension/flexion ratio (M = 5.71 4.24, F = 5.64 3.14; p = 0.95); and cranial sartorius circumference (M = 3.03 0.69 mm/kg, F = 3.40 0.96 mm/kg; p = 0.134) did not differ. Conclusions. Results from these tests do not confirm an effect of gender on phenotype in GRMD dogs at 6 months of age. However, given that gender-based differences in the degree of muscle regeneration and inflammation have been documented in mdx mice and other murine models of muscle injury, care should be taken in designing and evaluating preclinical studies in which both male and female GRMD dogs are used.

[536] Functional Correlation in Golden Retriever Muscular Dystrophy

Joe N. Kornegay, Daniel J. Bogan, Janet R. Bogan Department of Pathology and Laboratory Medicine and the Gene Therapy Center, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC

Background. Most preclinical studies using the golden retriever muscular dystrophy (GRMD) model of Duchenne muscular dystrophy (DMD) have documented benefit based on pathologic or genetic features rather than functional tests. We have shown that GRMD dogs treated with prednisone have increased isometric tibiotarsal joint tetanic extensor force and a paradoxical decrease in flexor force. The decrease in flexor force may have occurred because prednisone reduced early flexor muscle necrosis and subsequent functional hypertrophy. In contrast to our finding of a paradoxical decrease in tibiotarsal joint flexion force in prednisone-treated dogs, another group showed that GRMD dogs given mesoangioblasts had increased values, perhaps reflecting differences in the timing or mechanism of the two treatments. These results emphasize the need to carefully consider functional endpoints used in the GRMD model. Materials and Methods. Newborn GRMD dogs were identified based on elevation of serum creatine kinase. Genotype was confirmed by PCR in most dogs. Functional tests were evaluated at 6 months of age. Tibiotarsal joint angle and torque force were measured as previously described and correlated in 51 dogs. Cranial sartorius circumference was measured by encircling the muscle with suture at the time of biopsy and correlated with tibiotarsal joint angle in 49 dogs. Correlation coefficients were done using a simple coefficient equation. Results. There was a high correlation between tibiotarsal joint isometric tetanic force and angle. Values (mean SD) for extension (2.138 0.915 N/kg) correlated directly (r = 0.54; p < 0.0001; power = 0.987), while those for flexion (0.443 0.132 N/kg) correlated inversely (r = - 0.70; p < 0.0001; power = 1.00) with joint angle (148.08 12.84^o). Tibiotarsal joint angle (148.00 12.97^o) and cranial sartorius circumference (3.204 0.839 mm/kg) correlated inversely (r = -0.70; p < 0.0001; power = 1.00). Dogs with weak extension and strong flexion force values and larger cranial sartorius muscles tended to have tibiotarsal joint flexor contractures. Conclusions. Contracture and muscle strength scores in DMD patients generally correlate and deteriorate synchronously. Joint contractures occur due to an imbalance in the strength of agonist and antagonist muscles. Weakness of the antagonist extensor muscle correlates highly with flexor contracture severity. Thus, as opposing extensor muscles weaken, flexor contractures worsen. A similar high correlation was seen between tibiotarsal joint angle and extensor force in GRMD dogs of this study. In addition, tibiotarsal joint flexor force and joint angle correlated inversely, suggesting that flexor muscle functional hypertrophy could contribute to contractures. The inverse correlation between cranial sartorius circumference and tibiotarsal joint angle is consistent with our previously published findings and suggests that hypertrophy of this muscle may play a role analogous to iliotibial band tightening in DMD.
 

[540] RNAi Targeting of FRG1: A Potential Therapy for Facioscapulohumeral Muscular Dystrophy (FSHD)

Sara E. Garwick, Jennifer L. Allen, Lindsay M. Wallace, Jorge A. Torres, Rossella Tupler, Scott Q. Harper Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH; Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH; Biomedical Sciences, Universita d Modena e Reggio Emilia, Modena, Italy; Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common muscular dystrophy, affecting 1 in 20,000 people. It is a dominantly inherited disorder characterized by progressive and asymmetric wasting of facial, shoulder, and limb muscles. In 20% of patients, pelvic and abdominal muscles are also affected, resulting in wheelchair dependence. Symptoms typically arise in late adolescence to adulthood, though more severe juvenile onset cases occur in 5-10% of cases. FSHD is caused by contraction of a specific haplotype of subtelomeric repetitive elements (D4Z4 repeats) on human chromosome 4q. Though this mutation was identified 15 years ago, efforts to develop FSHD therapies have been hindered by the inability to establish a clear underlying pathogenic mechanism for the disease. Currently, no treatment exists. One well-supported model suggests that D4Z4 contractions alter normal chromatin structure leading to aberrant up-regulation of chromosome 4 genes. To date, the best FSHD candidate gene is FRG1, which is localized to human 4q, elevated in some FSHD patient biopsies, and recent transgenic mouse studies support that FRG1 over-expression in muscle leads to FSHD-associated phenotypes. We hypothesize that reducing expression of FRG1 levels may offer a potential treatment for the FSHD. RNA interference (RNAi) has emerged as a powerful tool to reduce expression of any gene of interest in a sequence-specific manner. As such, RNAi is a leading candidate strategy for FSHD therapy. Here we use vector- and transgenic-based approaches to test treatment of dominant FSHD through FRG1 inhibition. First, we developed 14 different microRNA shuttle vectors targeting human FRG1 (miFRG1). Using adeno-associated viral vectors, we are testing the ability of our lead miFRG1 sequences to reduce over-expressed FRG1 and improve FSHD-associated symptoms in FRG1^-high mice. Second, we created a method to investigate the potential reversibility of FSHD, by generating 4 lines of doxycycline (dox)-responsive mice to inducibly over-express, or normalize, FRG1 in muscle (TRE.FRG1 x MCK.rTTA). These mice represent a genetic method to reduce FRG1 to normal levels after onset of dystrophy, thereby serving as a gold standard model for RNAi therapy. This work is an important first step toward establishing a potential treatment for FSHD targeting increased FRG1 expression. Moreover, we are establishing proof-of-principle for a method that can be broadly applied if additional FSHD-related genes are identified in the future.
 

[528] Development of Strong Muscle-Specific Promoters for Gene Therapy of Duchenne Muscular Dystrophy

Yue Zeng, Marilyne Blain, Mehdi Bendjelloul, Claire Guilbault, Kenneth E. Hastings, George Karpati, Bernard Massie, Renald Gilbert Genomics & Gene Therapy Vectors, Biotechnology Research Institut, NRC, Montreal, QC, Canada; Neuromuscular Research Group, Montreal Neurological Institute, Montreal, QC, Canada; Molecular Biology, University of Montreal, Montreal, QC, Canada

Duchenne muscular dystrophy (DMD) is fatal muscle disease caused by mutations in the dystrophin gene. Delivery of a functional dystrophin cDNA to muscle fibers is a promising approach for the treatment of DMD. The ideal vector that carries the dystrophin expression cassette should not only allow high short term dystrophin expression level in muscle, but it should also provide stable and long-term expression. To reach this goal, we have developed powerful muscle specific promoters derived from the genetic elements of the human slow isoform of troponin I gene (TnIS). We generated several constructs containing one to four copies of the TnIS upstream enhancer (USE) or truncated USE (USE) fused to the minimal promoter of the TnIS gene. Transient transfection studies in myotube cultures showed that constructs containing three or more USE or USE were stronger than CMV and comparable to the powerful hybrid CMV enhancer/-actin promoter (CB). Moreover, these constructs had only a very weak activity in non-muscle cells. The strength of the constructs containing three copies of USE or USE (USEx3 and USEx3) was similar to CMV and CB after in vivo electroporation of plasmid DNA into mouse muscle. We have also prepared gutless adenovirus expressing -galactosidase regulated by USEx3 andUSEx3 and we are currently testing their efficacy. In summary, multimerization of USE and USE generated powerful muscle-specific regulatory elements that could be useful for dystrophin gene replacement therapy of DMD.
 

[347] The Use of CpG Modifications Enhances Gene Correction Levels Mediated by Oligonucleotides in the Mdx Mouse Model for Duchenne Muscular Dystrophy

Carmen Bertoni, Arjun Rustagi, Thomas A. Rando Neurology, UCLA, Los Angeles, CA; Neurology and Neurological Sciences, Stanford, Palo Alto, CA

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder characterized by complete absence of dystrophin expression in skeletal muscles. Gene editing mediated by single stranded oligodeoxynucleotides (ssODNs) has the potential to treat both single point mutations as well as deletions that cause frame shift of the dystrophin mRNA. We have focused on the development of new vectors capable of activating specific repair mechanisms to direct the repair process specifically on the sequence of the genomic DNA targeted for correction. The Methyl Binding Protein 4 (MBD4) is a glycosylase capable of recognizing a T to G transversion at CpG sites and direct the conversion of the thymine into methylcytosine. CpG modifications were introduced on the mutating base of the targeting oligonucleotide in the attempt to mimic a deamination of methylcytosine and activate MBD4. The ability of modified ssODN to increase gene correction levels were assessed in muscle precursor cells in vitro using a reporter construct carrying a single point mutation in the gene encoding Green Fluorescent Protein (GFP). Muscle precursor cells were transfected with the reporter gene and stable clones were selected. CpG-mediated ssODNs showed a two to four fold increase in gene correction frequencies compared with unmodified ssODNs. Similarly, targeting oligonucleotides containing CpG modifications but unable to act through the base excision repair pathway failed to show significant increases in gene repair. The feasibility of using modified ssODNs for the treatment of DMD was tested in the mdx mouse. We have designed ssODNs complimentary to the coding or the non-coding strand of the donor site of exon 23 to induce skipping of the exon responsible for the lack of dystrophin in mdx and restore its expression. The ability of ssODNs containing CpG modifications to increase gene repair was studied in vitro and in vivo. The amount of dystrophin protein restored was significantly increased by the use of ssODNs designed to activate MBD4. Studies conducted on muscle cells in culture demonstrated up-regulation of MBD4 mRNA and the activation of the base excision repair mechanism through which MBD4 acts. Correction of the dystrophin gene was shown to occur at the genomic level and was stable over prolonged periods of time. In muscle cells in culture, restoration of dystrophin expression was analyzed at the protein level by western blot and immunohistochemistry and at the mRNA level by RT-PCR. Immunostaining analysis of mdx-injected muscles demonstrated the efficacy of ssODN containing CpG modifications of increasing the expression of functional dystrophin in vivo. The single base pair alteration was confirmed at the genomic level using restriction endonuclease analysis of total DNA isolated from muscles injected with targeting ssODN. Dystrophin expression was stable for at least four months after injection (the latest time point analyzed). Control oligonucleotides homologous to the region of the genomic DNA targeted for repair but unable to induce the single base pair alteration had no effects.

[427] AAV-Mediated Gene Therapy Strategies in a Canine Model of Duchenne Muscular Dystrophy

Zejing Wang, Stanly Riddell, Donghoon Lee, James Allen, Martin Kushmeric, Jeffrey Chamberlain, Stephen Tapscott, Rainer Storb Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle; Immunology, Fred Hutchinson Cancer Research Center, Seattle; Radiology, University of Washington, Seattle; Neurology, University of Washington, Seattle; Human Biology, Fred Hutchinson Cancer Research Center, Seattle

Duchenne Muscular Dystrophy (DMD) in both humans and dogs (cxmd) is a lethal, X-linked muscle disease due to lack of an anchor protein, dystrophin, cause by deletions or mutations in the dystrophin gene. Adeno-associated virus (AAV)-mediated micro-dystrophin (m-dys) delivery to skeletal muscle has been successful in restoring muscle function in mdx mice. Our previous studies in wild-type and cxmd dogs have demonstrated that direct intramuscular injection of AAV2 or AAV6 carrying different promoter-transgene cassettes resulted in robust cellular immune responses to AAV capsid proteins. Recent evidence of immune-mediated loss of AAV vector persistence in human trials also suggested that immune modulation might be necessary to achieve long-term transgene expression. We now demonstrated that the induced immune response to AAV vectors could be averted by a brief course of immunosuppression with a combination of anti-thymocyte globulin, cyclosporine, and mycophenolate mofetil, which permitted long-term and robust expression of a canine m-dys transgene in the skeletal muscle of cxmd dogs, restored localization of components of the dystrophin-associated protein complex at the muscle membrane, and decreased need for muscle regeneration. To further characterize the specificity of the observed T cell responses, we generated an overlapping peptide panel spanning the full- length capsid protein VP1 of AAV6 and used an ELISpot assay to measure antigen specific T cell secretion of interferon-g. Our preliminary results identified 7 peptides containing potential immunogenic epitopes from two different dogs, including one common sequence recognized by both dogs, and suggested that AAV capsid proteins played at least partial roles in induction of T cell responses following intra-muscular injection in dogs. We have initiated studies to determine the usefulness of non-invasive magnetic resonance imaging (MRI) compared to muscle biopsies, in assessing immune responses to AAV vector and the effect of restoring expression of dystrophin in dystrophic muscle. Our preliminary data suggested a trend for water relaxation properties (transverse relaxation time) T2 values to be longer in cxmd dogs than in normal controls. The same trend was also found in AAV-treated limbs with T2 tends to return towards control values. In conclusion, our studies suggested that AAV capsid proteins can induce immune responses in dog muscles, and transient immunosuppressive modulation can diminish this response for sustained transgene expression. Furthermore, MRI technology has the potential for non-invasive monitoring efficiency of AAV-mediated gene therapy. These methodologies can be directly translated to treat DMD patients.

[784] Systemic Delivery of AAV8 In Utero Results in High Level Gene Expression in Diaphragm: Treatment Implications for Duchenne Muscular Dystrophy

Bhanu M. Koppanati, Juan Li, Bing Wang, Molly Daood, Xiao Xiao, Jon F. Watchko, Paula R. Clemens Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA; Department of Pediatrics, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA; Neurology Service, Department of Veterans Affairs Medical Center, Pittsburgh, PA

One of the major challenges in the treatment of muscle disorders such as Duchenne muscular dystrophy (DMD) which affects many muscle groups is achieving efficient, widespread transgene expression in muscle. In utero gene transfer can potentially address this problem by accomplishing gene delivery when the tissue mass is small and the immune system is still immature. In our previous study we tested in utero AAV1 gene delivery using intraperitoneal administration to embryonic day 16 (E-16) pups. We observed high levels of transduction in diaphragm and intercostal muscle, but no detectable levels in limb muscle. Recently newer AAV serotypes such as AAV8 have demonstrated widespread and high transgene expression in skeletal muscles and diaphragm by systemic injections in adults and neonates. We have, therefore, tested AAV8 gene delivery by intraperitoneal administration in E-16 mice in utero. Using a AAV8 carrying a lacZ transgene, we observed high levels of transduction of diaphragm and more moderate levels of transduction of multiple limb muscles and heart. Furthermore we are extending these studies to the DMD disease model, the mdx mouse, using an AAV8 vector carrying an internally-deleted dystrophin cDNA. Preliminary data show high levels of recombinant dystrophin expression in the diaphragm with functional benefit measured by in vitro force studies. Our current studies demonstrate the potential of AAV8 to achieve widespread muscle transduction in utero and suggest the possibility of therapeutic potential for DMD.
 

[378] Body-Wide Restoration of Dystrophin Expression and Amelioration of Pathology in Dystrophic Dogs Using a Morpholino Cocktail

Toshifumi Yokota, Qi-long Lu, Terence A. Partridge, Masanori Kobayashi, Akinori Nakamura, Shin'ichi Takeda, Eric P. Hoffman Center for Genetic Medicine, Childrens National Medical Center, Washington, DC; Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC; Dpt of Molecular Therapy, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

Duchenne muscular dystrophy (DMD) is one of the most prevalent types of muscular dystrophy and is characterized by rapid progression of muscle degeneration that occurs early in life. Mutations in the dystrophin gene cause DMD and Becker muscular dystrophy (BMD), a milder allelic form of DMD. In general, DMD patients carry mutations which cause premature translation termination (nonsense or frame shift mutations), while in BMD patients dystrophin is reduced either in molecular weight (derived from in-frame deletions) or in expression level. It is noteworthy that some BMD patients with quite large deletions, nonetheless show only very mild or asymptomatic clinical or laboratory evidence of muscle disease. This raises the possibility of using anti-sense-mediated removal of one or more exons around the site of the original mutation so as to induce loss of additional exons from DMD mRNA and thus restore the translational reading frame to convert DMD to a milder BMD phenotype. Restoration of reading frame of dystrophin by antisense-mediated exon skipping of mRNA has been demonstrated in the mdx mouse model of DMD. Here, we describe the development, testing and systemic application of a cocktail of antisense phosphorodiamidate morpholino oligomers (PMOs; morpholinos) designed to promote the skipping of exons 6 and 8 to restore dystrophin expression in canine X-linked muscular dystrophy (CXMD), a clinically severe dog (beagle) model of DMD. In muscle cultures, each anti exon-6 antisense oligo (AO) alone induced efficient 6-9 skipping whereas, for intramuscular injection a three morpholino cocktail was required to restore dystrophin expression. Systemic infusions of 120-200 mg/Kg of this cocktail, weekly or bi-weekly for 5-22 weeks into three 2-7 months old dystrophic dogs induced recovery of dystrophin expression in skeletal muscle throughout the body accompanied by decreased muscle inflammation and improved exercise ability with no evidence of toxicity. Such multi-exon skipping could be potentially applicable for more than 90% of DMD patients with dystrophin deletion mutations and offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein.
 

[535] Electrocardiographic Improvement of Mdx Heart by Transduction with rAAV9-Microdystrophin

Jin-Hong Shin, Sachiko Ohshima, Kasahara Yuko, Takashi Okada, Shin'ichi Takeda Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan; Neurology, Graduate School of Medicine, Pusan National University, Busan, Republic of Korea; Neurology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Background: Duchenne muscular dystrophy is a lethal X-linked disorder due to deficiency of the dystrophin and characterized by progressive deterioration of skeletal and cardiac muscles. Cardiomyopathy, although not evident in early cases, comprises major cause of mortality in advanced patients. Common electrocardiographic abnormalities in human patients include tachycardia, shortened PR interval, and reversed R/S ratio. We have previously reported improvement of skeletal muscle pathology and specific force by the transduction of microdystrophin into mdx mice, a most well-known model of Duchenne muscular dystrophy. To investigate the therapeutic effect of the microdystrophin on the mdx mice heart, we systemically transduced mdx mice by the AAV9 vector expressing the microdystrophin (rAAV9-microdystrophin). Methods: Four weeks old mdx mice (n=5) were transduced with the rAAV9-microdystrophin driven by the CMV promoter (3.0 x 10¹² v.g./body) via the tail vein. Four weeks as well as 20 weeks after transduction, the electrocardiogram was taken under anesthesia, and compared with that of age-matched C57BL10 mice (n=5) and untransduced mdx mice (n=5). Autonomic blockade was performed with intraperitoneal injection of either atropine (0.5 mg/kg body weight) or propranolol (1.0 mg/kg), or both. Baroreflex sensitivity was tested by intraperitoneal injection of phenylephrine (3.0 mg/kg) after adrenergic blockade with propranolol. Results: Immunofluorescence staining revealed that more than 90% of the cardiac myofibers were transduced with the microdystrophin. HR was elevated (p<0.01) in the mdx mice group compared to C57BL10 mice, while there was not significant difference between transduced and untransduced group. PR interval was significantly shorter (p<0.05) in the untransduced mdx mice than C57BL10 mice at the age of 8 weeks, while the microdystrophin-transduced mdx mice showed prolonged PR interval toward that of the C57BL10. R/S ratio from right precordial lead markedly increased in the untransduced mdx mice compared to C57BL10 at the age of 6 months, which became significantly decreased in the transduced mdx mice (p<0.05). The differences persisted more than 20 weeks after transduction. PR interval, heart rate, and heart rate variability were all responsive to autonomic challenges in any group, although sympathetic blockade could not completely revert the PR shortening of mdx mice. Conclusion: Although cardiac pathology in mdx mice is not evident before 3 months of age, we could detect several abnormalities in the electrocardiographic profiles, as early as 8 weeks old. The changes were improved by systemic transduction of the microdystrophin, supporting therapeutic effect on cardiac dysfunction. The response to autonomic challenge suggests that these electrocardiographic abnormalities are partly due to upregulated sympathetic tone to compensate the cardiac function in the presymptomatic mdx heart, leaving the possibility of abnormalies in the conduction system.
 

[539] Long Term Benefit of AAV/Antisense-Mediated Exon Skipping in Dystrophic Mice

Fernanda G. De Angelis, Michela A. Denti, Tania Incitti, Valentina Cazzella, Irene Bozzoni Genetics and Molecular Biology, University of Rome La Sapienza, Rome, Italy

Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne Muscular Dystrophy (DMD) or the milder Becker Muscular Dystrophy, depending on whether the translational reading frame is lost or maintained. Since internal in-frame deletions in the protein produce only mild myopathic symptoms it is possible, by skipping specific mutated exons, to restore a partially corrected phenotype. Exon skipping in the dystrophin mRNA can be achieved in the mdx mouse model by the use of chimeric antisense U1snRNA. We designed and produced Adeno-Associated Viral (AAV) vectors carrying antisense sequences against the splice junctions of dystrophin exon 23 of the mdx mouse and we tested their activity in vivo. Local intramuscular delivery of AAV-antisense RNAs restores dystrophin and muscle strength, while systemic delivery results in body-wide rescue of dystrophin synthesis and functional recovery. Finally we have analyzed the long-term antisense efficacy 18 months after a single systemic injection. We showed that the U1-antisense construct is still present and able to rescue dystrophin synthesis at levels sufficient to mantain an effective regeneration capacity and an almost normal muscle phenotype. The absence of immune response against the transgene, together with data coming from non human primates showing the persistence of expression from AAV vectors in muscle for more than 6 years, suggest that a long-term treatment for human DMD patients might be feasible.
 

[529] Preservation of Muscle Force in Mdx3cv Mice Correlates with the Low-Level Expression of a near Full-Length Dystrophin Protein

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

Complete absence of dystrophin causes Duchenne muscular dystrophy (DMD). Dystrophin restoration at 20% level reduces muscle pathology and improves muscle force. Levels lower than this are considered therapeutically irrelevant. Interestingly, less than 20% dystrophin expression is seen in some Becker muscular dystrophy (BMD) patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mice. But mdx3cv mice expressed a near full-length dystrophin protein at 5% of the normal level. Consistent with previous reports, we found dystrophic skeletal muscle pathology in both strains. Surprisingly, mdx3cv extensor digitorium longus (EDL) muscle showed significantly higher tetanic force and it was also more resistant to eccentric contraction-induced injury. Furthermore, mdx3cv forelimb grip force was stronger. Immunostaining revealed utrophin up-regulation and detectable dystrophin-associated glycoprotein complex assembly on the sarcolemma in both strains. Our results suggest that a sub-therapeutic level expression of a near full-length membrane-bound dystrophin may have contributed to muscle force preservation in mdx3cv mice. This finding may help to explain the benign clinical phenotype in some BMD patients.
 

[432] Full Rescue of Dystrophin Expression in Cardiac, Smooth and Skeletal Muscles by Antisense Oligonucelotide-Induced Exon Skipping

Qi Long Lu, Peijuan Lu, Bo Wu McColl-Lockwood Laboratory for Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC

Frameshift and nonsense mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD). Skipping the mutated exon(s) with antisense oligonucleotides (AON) can restore reading-frame and production of dystrophin proteins. We demonstrated early that functional levels of dystrophin can be induced by intramuscular delivery of 2-O-methyl phosphorothioate AONs (2O MePS) in the mdx mouse, a model of DMD. Effective exon skipping and dystrophin induction can also be achieved by systemic delivery of the AONs. To improve the efficiency of exon skipping, we examined AONs with various chemistries and found that phosphorodiamidate morpholino oligomer (PMO) offers significantly higher efficiency than 2O MePS in exon skipping and dystrophin induction. PMO delivered systemically is able to restore functional levels of dystrophin in skeletal muscles. However, considerable variation exists within and between skeletal muscles. More disappointing, AON of all chemistries fail to induce meaningful levels of exon skipping and dystrophy production in the cardiac muscle. However, restoration of functional levels of dystrophin in heart and respiratory muscles is critical for treating DMD. To overcome the delivery difficiency with bare AONs, we examined several delivery-enabling modifications to PMO. We are now able to induce effective exon skipping and restore near normal levels of dystrophin expression in all body muscles, including smooth muscles and cardiac muscle by systemic delivery of delivery-enabled PMOs. This is associated with significantly improved muscle functions. Antisense therapy offers realistic hope for the treatment of majority of DMD patients.
 

[1000] Methodologies To Enhance Systemic Gene Transfer to Musculature in Large Animal Models Using AAV6 Vectors

Paul Gregorevic, Brian R. Schultz, James M. Allen, Eric Finn, Caitlin Doremus, Jeffrey B. Halldorson, Jeffrey S. Chamberlain Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, Department of Neurology, University of Washington, Seattle, WA; Molecular and Cellular Biology, Medical Scientist Training Program, University of Washington, Seattle, WA

Severe neuromuscular disorders such as Duchenne muscular dystrophy are associated with profound structural and functional disruption of muscles throughout the body. Therefore, it is necessary to develop genetic interventions of therapeutic potential that can access affected muscle fibers body-wide. We have previously reported therapeutically successful delivery of rAAV6 vectors throughout the musculature of dystrophic mice. Towards a systemic intervention with clinical potential, we are currently seeking to enhance the efficiency of rAAV6 transduction in small and larger mammals. In mice, we have compared the tissue-specific expression levels of reporter genes driven by either the CMV or CAG promoter, finding that CAG produces stronger transgene expression levels overall, but also increases expression in non-muscle tissue, especially liver. Simultaneous injection of proteasome inhibitors along with rAAV6 has little effect in striated muscle; however, transgene expression in other tissues is dramatically increased. This suggests that different mechanisms of transduction may occur in different tissues. For larger animal studies, we are utilizing a wild-type dog model for systemic rAAV6 dissemination experiments. Although a single rAAV6 injection in a mouse can lead to whole-body transduction, including heart, diaphragm, and limb muscles, a larger animal may benefit from multiple routes of vector administration. In initial experiments, we performed either a jugular vein infusion or a localized systemic injection into the femoral artery. Jugular vein delivery of 10^13 vector genomes produces substantial reporter gene expression in both the heart and the diaphragm. Localized infusion of the same vector genome quantity into the femoral artery produces robust expression in muscles immediately distal to the infusion site. In these studies, using human reporter genes, animals receiving immune suppression exhibit greater transgene expression than animals not immune suppressed. Additional studies in progress are comparing different infusion protocols for the relative ability to transduce limb muscles versus internal muscles, such as heart and diaphragm, and the use of single versus multiple injections for systemic gene transfer.
 

[999] Effective Transduction of Dystrophic Dogs with rAAV Serotype 8

Sachiko Ohshima, Jin-Hong Shin, Akiyo Nishiyama, Katsutoshi Yuasa, Yuko Kasahara, Takashi Okada, Shin'ichi Takeda Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan

Background: Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder of the striated muscle caused by mutations in the dystrophin gene, which encodes a large sub-sarcolemmal cytoskeletal protein dystrophin. The absence of dystrophin associated with the loss of dystrophin-glycoprotein complex from the sarcolemma results in progressive muscle weakness, cardiomyopathy, and early mortality. Several treatment modalities have been attempted to correct the dystrophic phenotypes, but more effective therapy still needs to be developed. A recombinant adeno-associated virus (rAAV) has been utilized in the various preclinical and clinical studies. However, many questions associated with the host immune reaction have been raised and innate immune response against the rAAV has not been studied. Here we investigated the transduction efficiency and immune response by using the rAAV8 and rAAV2 in the muscles of normal Beages and canine X-linked muscular dystrophy in Japan (CXMD_J). Methods: The rAAV8 or rAAV2 encoding the lacZ gene driven by the CMV promoter in the range of 1 x 10¹¹ to 1 x 10¹³ g.c./muscle was directly injected into the anterior tibial muscle. The rAAV8 at a dose of 1 x 10¹⁴ g.c./kg was also injected into the unilateral hind limb via the lateral saphenous vein of the normal Beagles at 5-12 weeks old by using the limb-perfusion method. The CXMD_J at 5-12 weeks old were also transduced with the rAAV8 encoding the microdystrophin gene by the same method. The transduced muscles were sampled 4 weeks after the injection to analyze histological findings. To investigate innate immunity against the rAAV, bone marrow-derived dendritic cells were differentiated using GM-CSF as well as IL-4 and infected by the rAAV2 or 8. Levels of the cytokine and costimulating factor mRNA of the transduced dendritic cells of Beages were evaluated by qRT-PCR. Results: Efficient -galactosidase transduction was confirmed in the canine skeletal muscles with either intramuscular or intravenous injection of the rAAV8. Microdystrophin expression in the CXMD_J muscle with limb-perfusion method was more extensive than that with intramuscular injection. rAAV-mediated transduction with either injection protocol was associated with the lymphocyte infiltration. qRT-PCR analysis of the rAAV-transduced dendritic cells suggested that mRNA levels of the costimulating factors as well as interferon were higher in the cells transduced with the rAAV2 than that with the rAAV8. Discussion: The rAAV8 is the efficient tool for the therapeutic gene delivery into the dystrophic canine skeletal muscle. rAAV8-mediated gene transfer showed effective transgene expression, but roles of dendritic cells in the innate immune response must be further investigated to improve transduction protocol. We are currently conducting MR imaging and torque measurement of the microdystrophin-transduced CXMD_J to investigate the therapeutic efficiency.
 

[349] The Functional Capacity of R4-R23 Microdystrophin Is Improved by Switching Hinge 2 with Hinge 3

Glen B. Banks, Ariana C. Combs, Caitlin Doremus, Leonard Meuse, Eric E. Finn, James M. Allen, Jeffrey S. Chamberlain Senator Paul D Wellstone Muscular Dystrophy Cooperative Research Center, Department of Neurology, University of Washington, Seattle, WA

In skeletal muscle, dystrophin provides a flexible connection between actin and the dystrophin glycoprotein complex at the myotendinous junction (MTJ), sarcolemma and neuromuscular synapse. Dystrophin-deficient muscles are highly susceptible to contraction-induced injury and they undergo repeated cycles of necrosis and regeneration. Dystrophin contains an N-terminal actin binding domain, a large central rod domain, a cysteine rich region and a C-terminal domain (Fig. 1A). The central rod domain contains 24 spectrin repeats, 4 hinge regions and a second actin-binding domain (Fig. 1A). Large deletions in the rod domain can minimally affect the functional capacity of dystrophin and usually lead to a more mild form of DMD, called Becker muscular dystrophy. Our laboratory previously developed a highly functional truncated dystrophin called R4-R23/CT microdystrophin that prevents muscle degeneration in dystrophin-deficient mdx mice when intravenously delivered using recombinant adeno-associated virus pseudotyped with serotype 6 capsids (rAAV6). We found here that expression of R4-R23/CT microdystrophin led to chronic myotendinous strain injury. This injury led to an increase in utrophin and 7-integrin expression in addition to ringed fibers, where the peripheral myofibrils form rings around the central myofibrils. Interestingly, the sarcolemma of these muscles was protected from contraction-induced injury in an isometric stretch assay, better than wild-type mice. We could circumvent these abnormalities by replacing hinge 2 from the central rod domain of R4-R23/CT microdystrophin with hinge 3 (H2-R24/CT+H3 microdystrophin). To compare the efficacy of these two microdystrophins we delivered a non-saturating dose (2e12 vg) of rAAV6-microdystrophin intravenously into 3 week old mdx mice. We examined the gastrocnemius and tibialis anterior muscles 5 months after injection. Greater than 60% of the mdx muscle fibers expressed both of the microdystrophins. We found that R4-R23/CT microdystrophin significantly reduced the central nuclei from 80% in mdx mice to 16% in dystrophin positive fibers (P < 0.001). However, H2-R24/CT+H3 microdystrophin was more effective, reducing the central nuclei to 2% in dystrophin positive fibers (P < 0.001). The muscle fiber area was significantly increased in H2-R24/CT+H3 microdystrophin positive fibers compared to R4-R23/CT microdystrophin positive fibers (P < 0.001). Both of these microdystrophins were equally effective at restoring peak force production and protecting the muscles from contraction-induced injury. Thus, we have developed a highly functional microdystrophin with greater potential for gene therapy of DMD.
 

[233] Pre-Clinical Biodistribution and Toxicity Analysis Following Intravenous Delivery of AAV6 Vectors

Daniel Stone, Ying Liu, Zong-Yi Li, Robert Strauss, Eric E. Finn, James M. Allen, Jeff S. Chamberlain, Andre Lieber Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA; Department of Pathology, University of Washington, Seattle, WA; Department of Neurology, University of Washington, Seattle, WA; California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, CA

In recent years a common strategy to increase the gene delivery efficiency of a parental viral vector has been to use different serotypes of the same virus. Specifically, vectors based entirely on novel serotypes, or containing regions of other serotypes, have been utilized. Using this approach adeno-associated virus (AAV) vectors based on serotype 6 (AAV6) were found to efficiently transduce muscle in vivo after intravenous delivery, when compared to parental AAV2 vectors. A number of studies have demonstrated widespread transduction of skeletal muscle after AAV6 delivery, and due to these encouraging pre-clinical results it is being considered for use in the treatment of muscular dystrophy. As a prelude to future clinical studies, we investigated the biodistribution and safety profile of AAV6 following intravenous delivery to mice. Mice were injected with a dose of 2 10¹² viral genomes and analyzed for CBC, serum markers, vector biodistribution, cytokine/chemokine induction and tissue inflammation at 30 minutes, 6 or 72 hours post injection. AAV6 had minimal effect on circulating blood cell levels, with only a moderate increase in leukocyte levels at 72 hours, while serum levels of ALT and sCD62p were unaffected, indicating no hepatotoxicity or coagulation activation. Vector biodistribution showed more vector in serum than blood cells after 30 minutes and 6 hours, while the major sites of tissue sequestration were the liver and spleen. Vector was found in all tissues tested and for all tissues the level of vector genomes dropped significantly between 6 and 72 hours. Analysis of gene transcription for IL-1, IL-6, TNF-, MCP-1 and IP-10 revealed that AAV6 induced significant transcription in liver, while in spleen only transcription of MCP-1 was seen. Analysis of serum showed significant rises in levels of TNF- at 30 minutes post injection, MCP-1 at 6 hours post injection, and IL-6 at 30 minutes and 6 hours post injection. Immunohistochemistry using liver sections and antibodies against markers for leukocytes, monocytes/macrophages, granulocytes/neutrophils, lymphocytes or NK cells demonstrated a lack of inflammatory infiltrate at any time point tested. When taken together our data suggest that AAV6 vectors show non-specific biodistribution, and are relatively non-toxic following intravenous delivery. Although there is some indication of AAV6 induced toxicity, it is mostly transient, and has almost completely subsided by 72 hours post injection. AAV6 remains a promising tool for future use in the treatment of muscular dystrophy.
 

[1001] Experimental Targeted Gene Therapy for Quadriceps Muscle Weakness for Sporadic Inclusion Body Myositis (sIBM) with Implications for Other Neuromuscular Disorders

Janaiah Kota, Chrystal L. Montgomery, Amy Eagle, Danielle Tucker, Chalonda Handy, Amanda M. Haidet, Louise R. Rodino-Klapac, Kim M. Shontz, Christopher J. Shilling, K. Reed Clark, Zarife Sahenk, Jerry R. Mendell, Brian K. Kaspar The Research Institue at Nationwide Childrens Hospital, Columbus, OH

Sporadic inclusion body myositis (sIBM) is the most common acquired muscle disease occurring over age 50 with a prevalence of 5 per 100,000. Clinical features include: male preponderance (males 2:1 females), selective muscle atrophy (particularly quadriceps muscle), and weakness in knee extension with frequent falls and loss of ambulation. The cause remains enigmatic. Muscle pathology demonstrates combined muscle degeneration and inflammation. Immunosuppressive (IS) therapy can reduce mononuclear cell infiltration in muscle without sustained clinical benefit. A translational strategy targeting sIBM would improve quadriceps muscle strength and diminish inflammation. Therapeutic strategies for neuromuscular disorders have focused on enhancement of muscle mass and strength. Follistatin has been demonstrated to bind to myostatin, a negative regulator of muscle mass, and functions as a potent myostatin antagonist. Several studies, including work from our laboratory have demonstrated the potential of follistatin based on rodent models of muscular dystrophy. In our studies, delivery of follistatin led to increased muscle mass and size along with decreased pathological markers of the disease, demonstrating significant therapeutic promise for advancing to clinical studies. To this end we combined gene transfer of rAAV1.follistatin to the quadriceps of Cynamologous macaque with immunosuppressive therapy employing tacrolimus and myocophenylate mofetil (MMF). This IS regimen serves two roles in sIBM: promoting safe passage for gene transfer and reduction of the inflammatory milieu. Four months post injection, the effect of rAAV.follistatin was assessed on the injected muscle in comparison to nave control. Gross observation of the muscle demonstrated unequivocal increase in muscle size. By histological exam, fiber size was significantly increased predominantly affecting type 2 muscle fibers (nave, 68.4 um10.0, follistatin, 87.015 um). The effects were greatest at the injection site but spread throughout the muscle. This may be related to the secretion of the peptide reaching sites beyond the immediate injection area in light of detection of follistatin in the serum of treated animals exceeding the baseline levels. In support of secreted follistatin reaching sites beyond the transduced muscle, we found a shift in fiber size in remote muscles such as tibialis anterior and gastrocnemius muscles. The immunosuppressive drugs had no adverse effects on the general health of animals, assessed by observation and blood chemistries and there were no organ abnormalities at necropsy. In summary, the ability of follistatin to cause hypertrophy in higher animals warrants its consideration for clinical development to treat human muscle disorders. sIBM is one example but other conditions would also benefit, including several forms of muscular dystrophy. The necessity for IS therapy in non-inflammatory neuromuscular disorders will require further study.
 

[266] Transplantation of Muscle-Derived Stem Cells Genetically Engineered To Express Vascular Endothelial Growth Factor (VEGF) Decreases Fibrosis in Dystrophic Muscle

Bridget M. Deasy, Joseph M. Feduska, Thomas R. Payne, Johnny Huard Stem Cell Research Center, Childrens Hospital of Pittsburgh, Pittsburgh, PA; Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA; Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Department of Molecular Genetics & Biochemistry, University of Pittsburgh, Pittsburgh, PA; Cook Myosite Inc., Pittsburgh, PA

Transplantation of allogeneic or genetically-engineered autologous muscle-derived stem cells (MDSCs) into the skeletal muscle of dystrophin-deficient mdx mice, a model for Duchenne Muscular Dystrophy, is able to regenerate dystrophin-positive skeletal muscle fibers. Here, we examined the role of VEGF signaling in MDSC-mediated cell therapy for muscular dystrophy. We used MDSCs which were genetically engineered to express human vascular endothelial growth factor (VEGF₁₆₅, MDSC-VEGF) or the VEGF-specific antagonist, soluble Flt1 (sFlt1, MDSC-Flt). After transducing the cells, there was no change in the marker profile of the cells (CD34, Sca-1, or desmin) or in the ability of the cells to differentiate into myotubes in vitro. In vivo, our studies show a significant decrease in fibrosis at the site of transplantation of cells engineered with VEGF₁₆₅ (VEGF secretion levels 10⁵ 10⁶ ng/mL/5E⁵ cells) when compared to non-engineered cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the injected muscle with cells engineered to express sFlt1 as compared to the transplantation of control MDSCs. We detected a significant positive correlation between vascularization and skeletal muscle regeneration. At the same time, we did not observe any significant increase of skeletal muscle regeneration (as measured by the number of new dystrophin-positive fibers) in the engraftments using MDSC-VEGF or MDSC-Flt cells as compared to control MDSCs. These findings suggest that an increase in vascularization and a decrease in skeletal muscle fibrosis in dystrophic tissue are induced by the secretion of VEGF by donor MDSCs.
 

[998] Long-Term Mini-Dystrophin Expression without Immunosuppression in GRMD Dogs after AAV8-Mediated Gene Delivery by Hydrodynamic Limb Vein Injection

Joseph Kornegay, Chunlian Chen, Juan Li, Janet Bogan, Dan Bogan, Jiwei Chen, Bing Wang, Tong Zhou, Richard J. Samulski, Xiao Xiao Department of Pathology School of Medicine, School of Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC; Division of Molecular Pharmaceutics, School of Pharmacy, Univerity of North Carolina at Chapel Hill, Chape Hill, NC; Gene Therapy Center,, School of Medicine, Univerity of North Carolina at Chapel Hill, Chapel Hill, NC; Dept. of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA

Golden retriever muscular dystrophy (GRMD) dogs are employed as a large animal model of Duchenne muscular dystrophy (DMD) for AAV vector mediated gene therapy studies. Specifically, AAV serotype 8 (AAV8) is selected as the vector of choice for the delivery of a canine mini-dystrophin gene, which is under the transcriptional control of CMV promoter. The AAV8-CMV-cMinidys vector was injected into the hind legs of multiple young adult GRMD dogs (5 kg to 10 kg in weight) by the hydrodynamic limb vein injection method (isolated retrograde limb vein perfusion). The vector dose used in each dog was at 1 x 10e13 v.g/kg body weight. The injection volumes ranged from 10 ml/kg to 50ml/kg with a consistent injection rate of 1 ml/second. During and immediate after injection, blood circulation in the hindlimbs was block by a tourniquet for a total of 10 minutes. No overt vector-related adverse effect was observed during and after the procedure. At various time points, muscle biopsy samples were taken and analyzed for mini-dystrophin expression and immune responses. Canine minidystrophin gene expression was detected at both short term and long term (6 months) time points post vector injection. There was no discernable CTL responses against the canine minidystrophin gene. Immunofluorescent staining of CD4+ and CD8+ cells on muscles from vector-injected legs and the contralateral saline-injected control legs revealed no statistic differences. Furthermore, a normal dog similarly perfused with an AAV8-CMV-GFP vector also showed strong GFP expression at 2 week and 10 week time points without CTL immune responses. By contrast, a normal dog similarly perfused with an AAV2-CMV-GFP vector triggered robust CTL responses. These results suggest that the AAV8 vectors triggers minimal or no CTL responses against the canine minidystrophin in GRMD dogs and GFP in normal dogs when delivered by the limb perfusion method.
 

[527] Gene Delivery to Dystrophic Diaphragm by the Helper-Dependent Adenovirus Vector (HDAdv) Mediated Full-Length Dystrophin Expression

Masatoshi Ishizaki, Ryoko Kawano, Tomohiro Suga, Yuji Uchida, En KImura, Yasushi Maeda, Makoto Uchino Neurology, Kumamoto University, Kumamoto City, Japan; Sojo Univetsity, Kumamoto City, Japan

Backgrouud Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory or cardiac failure and results in death at about 20 years of age. Ventilatory insufficiency is a central problem in the management of DMD patients. One of the most important problem against the DMD gene therapy is the huge size of dystrophin cDNA. We have generated a helper-dependent adenovirus vector, which has a cloning capacity of up to 37kb, that carried myc-tagged murine full-length dystrophin cDNA. In this study we evaluated the therapeutic effect of the HDAd-mediated full-length dystrophin gene transfer into severe dystrophic diaphragm of utrophin/dystrophin double knockout mice (dko mice) by the intraperitoneal injection. Method and Results ?We have constructed HDAd vector contained the murine full-length dystrophin expression cassette and myc-tag,integral protein (HDAdv-mFLmyc-dys). Each 7-day-old dko mice were injected with the HDAdv-mFLmyc-dys (1.41012 particles/ml, 100?l) by the intraperitoneal injection. In the diaphragm, the transgene was widely expressed (mean 38%, max 61%) and prevented the dystrophic changes pathologically in injected dko mice. The contractile property of the dko diaphragm was measured to assess the correction of muscle function as well as pathological improvements. Furthermore, we showed that respiratory function of injected dko mice recovered by using whole-body plethysmography and their lifespan became longer. Conclusion These results offer a hopeful prospect for DMD therapy. Therapeutic gene transfer with HDAd may ameliorate DMD patients.
 

[538] Transgenic Mini-Dystrophin Expression in Skeletal Muscles of Mdx:utrn-/- Double KO Mice Ameliorates Dystrophic Phenotypes and Remarkably Extends Life Span

Peiqi Hu, Bing Wang, Juan Li, Jiwei Chen, Chunping Qiao, Chunlian Chen, Xiao Xiao Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, NC

Mice deficient in both dystrophin and utrophin (mdx:utrn-/-) manifest phenotypes similar to that seen in Duchenne muscular dystrophy (DMD) patients, including severe muscle wasting, skeletal deformities, joint contractures and premature death, and have been considered as a better animal model than the mdx mice. We previously created mini-dystrophin genes and demonstrated that AAV-mediated local intramuscular delivery of the minigenes into the muscle of mdx mice ameliorated mdx dystrophic histopathology and improved contractile functions. In this study, we have generated transgenic mdx:utrn-/- mice expressing mini-dystrophin 3849 (containing 5 central rods) under the control of a shortened muscle-specific creatine kinase promoter (dMCK). Immunofluorescent staining revealed widespread expression in multiple skeletal muscles, mostly in the fast twitch myofibers, but no detectable mini-dystrophin in the cardiac and diaphragm muscles. Compared with phenotypic mdx:utrn-/- double knockout mice, the expression of mini-dystrophin 3849 transgene in the transgenic mdx:utrn-/- mice resulted in 1) dramatically restoring life span. All observed mice (n=9) lived longer than 40 weeks and 70% of them (6 in 9 mice) lived longer than 80 weeks, whereas 50% survival rate for dKO mice (n=17) were 9 weeks. 2) restoring fertility of both males and females. 3) preventing growth retardation, kyphosis formation and joint contractures.

4) significantly improving muscle strength and contractile function. 5) ameliorating dystrophic pathology. We conclude that mini-dystrophin 3849 gene is effective to genetically rescue muscle deficiencies, improve the overall health and prolong the life span of the severe DMD mice, and interestingly, the these data indicate that restoration of dystrophin in heart and diaphragm is not critically required for the prolongation of life span.
 

[532] Promoter, Vector Serotype and Route of Delivery Influence -Sarcoglycan Persitent Expression after Gene Transfer in Mouse

Marc Bartoli, Jérôme Poupiot, Isabelle Richard LGMD Department, Genethon, Evry, France

We previously demonstrated a wide and sustained transgene expression in gene transfer experiments using recombinant pseudotype-1 adeno-associated virus (rAAV1) vectors expressing the human -sarcoglycan cDNA, constituting a promising approach for the treatment of limb girdle muscular dystrophy type 2D (LGMD2D). However, we observed that injected mice developed serologic-mediated immune response towards the transgene. We evaluated this immune response against -sarcoglycan by testing different promoters (CMV, C5-12, desmin), AAV serotypes (1, 6 and 9) and administration-routes (intramuscular, intraarterial and intravenous). We observed that i) use of a weak and muscle-specific promoter (desmin) induced the lowest cellular infiltration, ii) AAV6 serotype is more immunogenic than AAV1 and 9 and iii) systemic administration by intravenous injection of the vector prevented all cellular infiltrations and allowed -sarcoglycan expression persistence. In parallel, to further examine the observed immune response, we carried-out detection of serotype specific antibodies and demonstrated that conditions leading to the loss of expression of -sarcolgycan correlated with the presence of IgG2a isotypes. These findings showed that the gene transfer design influences -sarcolgycan persitent expression and that IgG2a anti--sarcoglycan antibodies detection could be useful to monitor deleterious immune response.
 

[534] A Gene Therapy Strategy Utilizing Follistatin Combined with Micro-Dystrophin Gene Replacement Dramatically Improves Force Generation in Mdx Mice

Louise R. Rodino-Klapac, Paul M. L. Janssen, Anil Birdi, Amy Eagle, Kim M. Shontz, Chrystal L. Montgomery, K. Reed Clark, Jerry R. Mendell, Brian K. Kaspar Center for Gene Therapy, Research Institute at Nationwide Childrens Hospital, Columbus, OH; Pediatrics, The Ohio State University, Columbus, OH; Physiology and Cell Biology, The Ohio State University, Columbus, OH

Duchenne muscular dystrophy (DMD) is a devastating disease of childhood characterized by severe muscle weakness leading to loss of ambulation and premature death. Gene replacement strategies using mini- or micro-dystrophin cassettes small enough for AAV packaging incompletely restore muscle function. Clinically this mandates a complementary approach for functional restoration. Prior studies demonstrated a role for muscle specific expression of IGF-1 (Barton et al 2002; Abmayr et al 2005) to enhance muscle function. Our goal is to expand the repertoire of potential agents augmenting function for translational treatment for DMD patients. Follistatin 344 is an alternatively spliced circulating myostatin inhibitor with predominate effects limited to skeletal muscle, resulting in increased muscle mass. To test its capacity in a pre-clinical setting, we treated aged 6 month old mdx mice with a combinatorial therapy of rAAV.follistatin 344 and rAAV.micro-dystrophin by direct muscle injection into the tibialis anterior/extensor digitorum (EDL) complex. The physiologic affects of combinatorial therapy were established six months after gene transfer including studies addressing the order of transgene delivery with comparisons to the effects of either transgene alone. We found significant improvements in both maximum force generation and resistance to damage by repeated eccentric contractions in all treatment groups with the most significant improvement in muscles treated with both follistatin and micro-dystrophin (p<0.05). These findings correlated with improved hindlimb grip strength and reversal of dystrophic features by histopathology. Increased force by follistatin expression exceeded mere increase in muscle size in mdx muscle, addressing concerns that size alone might account for improved muscle function. Complete necropsies and reproductive capacity of mice undergoing gene therapy demonstrated no evidence of off target affects. In summary, although AAV gene therapy treatment for DMD is limited by the inability to deliver full-length dystrophin, combinational therapy with follistatin provides a means to compensate for the limitations of small dystrophin gene replacement expanding our translational options.
 

[533] MMP-1 Gene Therapy Enhances Myoblast Migration after Implanting into MDX Mice

Yong Li, William Wang, Haiying Pan, Mia Jefferson Dept. of Orthopaedic Surgery, University of Pittsburgh, School of Medicine, Pittsburgh, PA; Stem Cell Research Center, Childrens Hospital of Pittsburgh, Pittsburgh, PA; Dept. of Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, PA

The challenge in myogenic cell transplantation for Duchenne Muscular Dystrophy is quick death and poor migration of donor cells that significantly limits this technique application. Current treatment involves numerous injections which result in not only pain and discomfort, but also the development of additional scar tissue after the injections. Therefore, the development of a novel therapeutic approach by which to enhance the migratory properties of the transplanted cells that will reduce the number of injection sites, promote widespread cell fusion, and improve muscle healing would be very significant. Matrix metalloproteinase type 1 (MMP1), a naturally occurring collagen-digesting enzyme, can eliminate the existing fibrous scar in different tissues including skeletal muscle tissues. Additionally, MMP1 also is able to enhance cell migration. Therefore, the current experiment use of retrovirus vector transfer MMP1 gene into C2C12 myoblasts prior transplanted into skeletal muscle of MDX/SCID, a dystrophic/immunodeficient mouse model. Results show that MMP1 gene transfer increases C2C12 myoblast migration, differentiation and significantly enlarges the dystrophin-positive muscle graft within the dystrophic skeletal muscle after implanting into MDX/SCID mice. We also detected some LacZ, a transplanted myoblasts tracker marker co-localized with dystrophin positive myofibers within diaphragm muscles at two weeks following systemic injection of the MMP1 gene-transferred C2C12 myoblast. These results support the notion that MMP1 is able to increase myoblast migration and may help spread donor cells to all types of muscles after systemic delivery. Our overall goal is to identify a novel technique via local and/or systemic delivery that upon transplantation will stimulate cell migration in vivo, improving dystrophin gene transfer into the dystrophic muscle fibers.
 

[997] Systemic AAV-9 Delivery in Normal Dog Leads to High-Level Persistent Transduction in Whole Body Skeletal Muscle

Yongping Yue, Arkasubhra Ghosh, Chun Long, Brian Bostick, Bruce F. Smith, Joe N. Kornegay, Dongsheng Duan Dept of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO; Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO; Scott-Ritchey Research Center, Auburn University, Auburn, AL; Dept of Pathology and Laboratory Medicine, University of North Carolina - Chapel Hill, Chapel Hill, NC

Adeno-associated virus (AAV) is a promising vector for muscle disease gene therapy. The dog model represents an ideal intermediate system prior to human trials. Previous attempts to deliver AAV directly to canine muscle have largely failed to achieve efficient transduction because of a strong immune response. In this study, we evaluated systemic AAV-9 gene delivery in newborn dogs by local and systemic delivery. Transgene expression was examined at different time points after AAV infection by biopsy or whole body necropsy. In contrast to the previous reports of low expression and strong immune reaction in dog muscle, we observed efficient transduction at 4 weeks following intramuscular gene delivery. Importantly, systemic gene delivery resulted in impressive whole body skeletal muscles transduction for up to 6 months. In more than 20 different muscle groups (including head, neck, chest, abdominal, thoracic and pelvic limbs), we observed 80% transduction in the majority of muscles throughout the entire muscle length. Taken together, our results provide the first evidence that systemic AAV delivery can reach multiple muscles in a large animal and that body size is not a barrier to intravascular AAV gene transfer. Our results raise the hope of whole body correction for many muscle diseases such as Duchenne muscular dystrophy. (Supported by NIH and MDA).
 

[1004] Oligodeoxynucleotide-Mediated Gene Correction of the Dystrophin Gene In Utero

Lingzhi Cai, Carmen Bertoni, Thomas A. Rando, Paula R. Clemens Neurology, University of Pittsburgh, Pittsburgh, PA; Neurology, UCLA, Los Angeles, CA; Neurology, Stanford University, Stanford, CA; Neurology Service and GRECC, VAPAHCS, Stanford, CA; Neurology Service, Veterans Affairs Medical Center, Pittsburgh, PA

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that is caused by mutations in the dystrophin gene. There is pathologic evidence that the disease process in DMD is ongoing at birth, supporting efforts to develop treatments that could be instituted at the earliest point during fetal life. Gene correction strategies are attractive because of their ongoing benefit to all progeny of the corrected cell. Previous studies demonstrated the feasibility of making single base changes in the dystrophin gene using single-stranded oligodeoxynucleotides (ssODNs) in skeletal muscle. In this study, ssODN gene correction technology was applied to fetal skeletal muscle in utero, with a goal of gene correction in muscle progenitor cells. MDX92b and MDX93b are ssODNs that are complementary to the transcribed and nontranscribed strands, respectively, of the intron 22/exon 23 splice site of the mdx mouse dystrophin gene. Specific gene correction was expected to result in skipping of exon 23, creating an in-frame deletion of the exon that contains the nonsense mutation that results in dystrophin deficiency in the mdx mouse. An ssODN, homologous to the coding strand of the targeted sequence but lacking the mismatch and therefore unable to induce the specific single base alteration, was used for negative control experiments. Doses of 25g or 50g of ssODN were injected intramuscularly into the hind limb of embryonic day 16 (E-16) mdx mice in utero. Muscles from mice treated with control or targeting ssODNs were harvested and tested for dystrophin protein expression by immunostaining at 4 weeks, 9 weeks, 4 months and 6 months of age. There was an average increase from 8 (4 weeks) to 53 (6 months) dystrophin-positive fibers with the 25g dose of ssODN and an average increase from 47 (9 weeks) to 130 (6 months) dystrophin-positive fibers with the 50g dose. No dystrophin-positive fibers were observed in untreated mdx mice nor those treated with the control ssODN at any time-point. In ssODN treated muscles, dystrophin-positive fibers harbored fewer centrally-placed nuclei, as compared to uncorrected muscle fibers, indicating that gene correction protected against muscle fiber degeneration. In summary, these data demonstrate that gene correction of muscle cells in utero is feasible. The temporal increase in the number of dystrophin-positive fibers following a single injection of a targeting ssODN suggests that muscle precursor cells underwent gene correction in utero.
 

[433] Restoration of Dystrophin Expression in Skeletal and Cardiac Muscle by Systemic Delivery of MorpholinoE23-Vivo Porter Oligonucleotide

Bo Wu, Yongfu Li, Paul A. Morcos, Timothy J. Doran, Peijuan Lu, Qi Long Lu McColl-Lockwood Laboratory for Muscular Dystrophy Laboratory, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC; Gene Tools, LLC, Philomath, OR

Antisense oligonucleotide (AONs) is able to restore dystrophin expression by targeted skipping of exon(s) disrupting reading frame of the gene in the dystrophic mdx mouse. However, low delivery efficiency leads to high variability and failure of dystrophin induction in skeletal and cardiac muscle respectively. In this study, we examined a Morpholino oligomer conjugated with an guanidine significantly improved dystrophin production in both skeletal and cardiac muscles in the mdx mice in vivo. Intramuscular injection of morpholinoE23-vivoporter induced dystrophin expression in almost 100% fibers of TA muscle. Single intravenous injection of morpholinoE23-vivoporter restored dystrophin expression in skeletal muscles at the levels equivalent to the injection of 50 times unmodified morpholinoE23. Repeated injection at biweekly interval achieved almost normal levels of dystrophin expression in all skeletal muscles body-wide and partially in cardiac muscle without detectable immune response. Intraperitoneal injections of the morpholinoE23-vivoporter achieved normalization of dystrophin expression only in the diaphragm and abdomen muscles, indicating a local rather than systemic delivery effect. Morpholino with delivery-enabling modifications offers realistic prospects for the treatment of a majority of DMD patients.
 

[431] Targeted Restoration of Dystrophin Expression in DMD by Peptide-Conjugated Antisense Oligonucleotides

HaiFang Yin, YiQi Seow, Hong M. Moulton, Patrick L. Iversen, Jordan K. Boutilier, Matthew Wood Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom; AVI BioPharma Inc., Corvallis, OR

Duchene Muscular Dystrophy (DMD) is a severe muscle disorder caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) alone or in combination with tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs alone or in combination with homing peptides and evaluated in mdx mice following weekly systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles at extremely low AO doses of 3mg/kg. This is the first time that targeted AO delivery to muscle and successful body-wide restoration of dystrophin expression have been achieved at such low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.
 

[920] Over-Expression of Follistatin in Myoblasts Increases Their Proliferation and Differentiation, and Improves the Graft Success

Basma F. Benabdallah, Manaf Bouchentouf, Joel Rousseau, Jacques P. Tremblay Pharmacologie, CRH Sainte-Justine, Montreal, QC, Canada; Medecine Experimentale, Hopital General Juif - Universite Mc Gill, Montreal, QC, Canada; Genetique Humaine, CHUL, Quebec, QC, Canada; Genetique Humaine, CHUL, Quebec, QC, Canada

Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy for the disease, as it permits the long term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair which follows myoblast transplantation. Myostatin, known as a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by blocking myoblast proliferation and delaying myoblast differentiation. Follistatin forms a complex with myostatin preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to over-express the follistatin protein in normal myoblasts to block the myostatin signaling. Our results first confirmed the over-expression of the human follistatin into lentivirus transduced myoblasts, and second showed that the over-expression of the follistatin protein in normal human myoblasts improved in vitro their proliferation rate by about 1,5 folds after 96 h and also their differentiation rate by about 1,6 and 1,8 folds respectively in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engrafment of those transduced human normal myoblasts with the follistatin lentivirus into SCID mouse muscles was enhanced by 2 folds.

[1003] Myostatin Propeptide Gene Delivery in Normal Dogs Via AAV Vector Increased Muscle Fiber Sizes

Chunping Qiao, Janet Bogan, Juan Li, Hui Zheng, Zhenhua Yuan, Jianbin Li, Dan Bogan, Joe Kornegay, Xiao Xiao Molecular Pharmaceutics, UNC School of Pharmacy, Chapel Hill; Department of Pathology & Laboratory Medicine, UNC, Chapel Hill

The ultimate purpose of this study is to evaluate the therapeutic efficacy of myostatin inhibition by means of AAV-mediated gene transfer in the DMD large animal model golden retriever muscular dystrophy (GRMD) dogs, hopefully paving the way for future clinical studies. The immediate purpose of this study is to see whether delivery AAV vector encoding myostatin propeptide (AAV-MPRO) gene in normal dogs can increase their muscle fiber sizes, as it does in mouse. We have delivered AAV8 vector containing the MRPO gene by the hydrodynamic limb vein injection technique in the hind leg of 3-month-old normal dogs. The body weight ranged from 6.3 kg to 9.7 kg. For each dog, the vector was delivered into one-leg, and the contralateral leg served as controls. A tourniquet was used to block the blood circulation of the entire injected hind leg (lower and upper legs and buttocks) for 10 minutes during vector injection. The vector dose was 10¹³ v.g/kg body weight. During and after vector injection in 3 months, no vector-related adverse event was observed. ELISA assay of sera has detected the presence of MRPO protein in the vector-injected dogs over the background levels of uninjected dogs. MRI imaging of the hind legs revealed increased sizes in some dogs but not in other. Muscle biopsy on a number of muscles of both legs was performed. Samples were examined for vector distribution by Real-time PCR and muscle histology by HE staining. Real-time PCR detected AAV-MPRO-Fc vector DNA in a majority of biopsied muscles from the vector injected legs, The copy numbers ranged between 0.01 v.g. to 1.5 v.g./nucleus. In two of the vector-perfused dogs, we observed muscle myofiber size increases on the injected legs when compared to the same muscle of the uninjected control legs. Particularly for dog Ramone, the gastrocnemius muscle from the injected leg displayed at least 50% myofiber size increase comparing with the muscle from control leg. Our preliminary studies demonstrated that delivering AAV vector encoding myostatin propeptide gene into normal dog could induce muscle hypertrophy and increase their myofiber sizes. Currently we are still analyzing the data and plan to do more experiments.

[351] A Novel Mini-Dystrophin Gene Restores Neuronal Nitric Oxide Synthase (nNOS) to the Sarcolemma

Yi Lai, Yongping Yue, Dejia Li, Chun Long, Luke Judge, Brian Bostick, Jeffrey S. Chamberlain, Dongsheng Duan Dept of Mole Microbio and Immun, Univ of Missouri-Columbia, Columbia, MO; Dept of Neurology, Univ of Washington, Seattle, WA

Duchenne Muscular Dystrophy (DMD) is the inherent disease due to the loss of dystrophin protein. Gene therapy is one of the most promising means to cure this disease. Dystrophin is composed of the N-terminal, rod, cysteine-rich and C-terminal domains. The rod domain consists of 24 spectrin-like repeats. Dystrophin is located on the sarcolemma of myofibers and it connects the extracelluar matrix with cytoskeletal F-actin filaments. Dystrophin assembles dystroglycans, sarcoglycans, dystrobrevins, syntrophins and nNOS into the dystrophin-associated protein complex (DGC) on the sarcolemma. The loss of dystrophin leads to the secondary loss of DGC members, including nNOS from the sarcolemma. The absence of sarcolemmal nNOS is a critical factor in DMD pathogenesis. Restoring sarcolemmal nNOS is thus considered an important goal in DMD therapy. The prevailing hypothesis is that dystrophin C-terminal domain recruits syntrophin to DGC and syntrophin then recruits nNOS to DGC through a PDZ-PDZ domain interaction. However, recent studies suggest that syntrophin can also be restored to the sarcolemma through a C-terminal domain independent pathway. The presence of syntrophin is insufficient by itself to restore nNOS. We hypothesized that a region in dystrophin rod domain is also required to recruit nNOS to DGC. To test this hypothesis, we generated a series of mini-dystorphin genes carrying different lengths of rod domain. We used the H2-R19 minigene as starting template and added back the missing repeats one by one. The H2-R19 minigene is the best characterized mini-dystrophin gene and it is also the most potent candidate gene besides full-length gene. To identify regions responsible for nNOS restoration, we transfected our newly synthesized minigenes into mdx muscles and performed single and double immunostaining as well as in situ nNOS activity assays. We also included a full-length human dystrophin plasmid as positive control. The transfected plasmids were distinguished from revertant fibers by a human dystrophin specific antibody and a sereis of epitope-specific antibodies. Minigene/nNOS double positive, minigene positive/nNOS negative and minigene negative/nNOS positive fibers were quantified from a total of 58 transfected muscles. Our results demonstrated that adding an additional 1, 2, or 3 repeats was not sufficient to restore nNOS. However, in the presence of 4 additional repeats, 97.8 % of transfected myofibers restored nNOS. This is very close to what we observed with full-length gene plasmid (98.9 %). Taken together, we have shown that dystrophin rod domain is critical for restoring sarcolemmal nNOS. This finding challenges the prevailying hypothesis and sheds new light on the mechanism of nNOS restoration in muscle. Importantly, the new minigene we described here represents an ideal candidate gene for adeno-associated virus (AAV) mediated gene therapy and lentiviral mediated stem cell therapy. Therapies based on this novel minigene are predicted to lead to a better recovery of muscle function than current mini-/micro-genes. (Supported by NIH

[1002] Delivery of NEMO Binding Domain-Protien Transduction Domain Fusion Peptide to Young Mdx Mice Yields Increased Regeneration and Decreased Necrosis in Hindlimb and Diaphragm Muscles

Daniel P. Reay, Michele Yang, Khaleel K. Rheman, Denis C. Guttridge, Paul D. Robbins, Paula R. Clemens Department of Neurology, University of Pittsburgh, Pittsburgh, PA; Childrens Hospital of Philadelphia, Philadelphia, PA; Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; Human Cancer Genetics Program, The Ohio State University, Columbus, OH; Neurology Service, Veterans Affairs Medical Center, Pittsburgh, PA

Peptide-mediated therapies are a potential addition to the treatment modalities for Duchenne muscular dystrophy (DMD), providing the opportunity to complement viral and non-viral gene transfer and cellular transfer approaches. The activation of NF-B is a critical factor that results in muscle degeneration and wasting and is observed in muscle of human DMD and in the mdx mouse, a genetic and biochemical model for DMD. The NF-B intracellular pathway marks a critical point at which muscle degeneration may be averted, as NF-B plays a key role in increasing transcription of pro-inflammatory cytokines and decreasing transcription of muscle regulatory factors. In the absence of dystrophin, signaling pathways activate NF-B, leading to nuclear translocation and increased transcription of many genes that are ultimately detrimental to muscle. In order to circumvent this process, specific peptides containing the NEMO binding domain (NBD), have been generated that interrupt the IKK subunit (NEMO) of the IB kinase complex, such that IKK can no longer phosphorylate IB, effectively blocking activation of NF-B. To increase the delivery of the peptide to widespread muscle cells throughout the body, a fusion peptide was prepared by linking the NBD peptide to different protein transduction domains (PTDs). In a prior study, treatment of mdx mice with an NBD peptide fused to the antennapedia gene homeobox domain (ANTP) resulted in decreased muscle inflammation and increased muscle fiber regeneration. In this study, we treated mdx mice at 4-5 weeks of age with an intraperitoneal injection of different PTD-NBD peptides (HIV Tat homeodomain (TAT), poly-lysine (8K), and ANTP fused to either wild type or mutant NBD) on a time course of three injections per week for a total of 4 and 7 weeks of treatment. Upon completion of the study, the tibialis anterior (TA) muscle and diaphragm were excised and analyzed for morphologic features of muscle regeneration and necrosis and for NF-B activity. Increased regeneration and decreased necrosis was observed after 4 weeks of treatment in the TA muscle and after both 4 and 7 weeks of treatment in the diaphragm muscles. Additionally, decreased NF-B activity was revealed by electrophoretic mobility shift assay (EMSA). There was some variation in both morphologic results and in the degree of effect on NF-B activity depending on the specific PTD. These studies provide evidence that PTD-NBD peptide therapy has the potential for therapeutic effect in DMD by modulating downstream pathways of dystrophin-deficiency.
 

[930] Gastrocnemius Targeted Vascular Delivery of AAV8 in Non-Human Primates

Louis G. Chicoine, Louise R. Rodino-Klapac, Chrystal L. Montgomery, Nathaniel A. Walton, Janaiah Kota, Brian D. Coley, Reed K. Clark, Jerry R. Mendell Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH; Radiology, Nationwide Childrens Hospital, Columbus, OH

DMD is the most common severe form of childhood muscular dystrophy. Ambulation is lost in early teenage years and premature death is encountered in the early twenties from loss of pulmonary function and cardiomyopathy. Effective gene replacement therapy using adeno-associated virus (AAV) requires vascular delivery to reach multiple muscle groups. In the mdx mouse we demonstrated that rAAV8 can successfully cross the vascular barrier and deliver the micro-dystrophin gene when perfused through the femoral artery. Before such a major undertaking can be proposed clinically, it is critical to optimize delivery conditions and volumes in the non-human primate, a model more closely simulating the clinical paradigm. The premise for our current study is that efficiency of AAV gene delivery through a vascular barrier is enhanced in a larger fluid volume by exposing vector to a greater endothelial surface area and or greater number of capillary junctions, permitting greater muscle transduction. We tested this hypothesis by targeting the left gastrocnemius muscle of 3 adult rhesus macaques with AAV8.CMF.eGFP (1 x 10¹² vg/kg) using a fluoroscopically guided catheter, which was positioned beyond the popliteal artery entering the sural artery of the left gastrocnemius. Two tourniquets were then placed on the leg, one just proximal to the catheter tip and one below the gastrocnemius to create a tight compartment for gene delivery. The region was pre-flushed in each of the macaques with one of the following volumes 0.5, 1.0, or 2.5 ml/kg of PBS with tourniquets snug but not tight enough to completely occlude the vessels. After this pre-flush, tourniquets were tightened and the eGFP containing vector was delivered in volumes equal to 0.5, 1.0, and 2.5 ml/kg over 1 minute. The region remained isolated for 10 minutes (dwell time) and was then flushed with an equal volume of PBS. The tourniquets were released and the arterial catheter removed. The gastrocnemius muscles and other organs were harvested 3 weeks later. qPCR analysis showed that the 2.5 ml/kg treated gastrocnemius contained the greatest abundance of eGFP transduced gene as well as the widest distribution of eGFP expression within the muscle. The levels of transduction were confirmed by western blot analysis. Biodistribution analysis by qPCR also showed limited delivery to systemic organs and contralateral muscle. Only the spleen and liver contained measurable amounts of eGFP by qPCR. We conclude that AAV8.CMV.eGFP delivered to the non-human primate through a fluoroscopically-guided catheter efficiently transduces the gastrocnemius muscle, increasing in proportion to volume of fluid used for vector delivery. We anticipate using this approach for clinical delivery of rAAV carrying micro-dystrophin or in other appropriate gene restoration strategies.
 

[430] A Morpholino-Cell-Penetrating Peptide Conjugate Caused Effective Exon-Skipping in Heart and Skeletal Muscles of MDX Mice

Natee Jearawiriyapaisarn, Hong M. Moulton, Brian Buckley, Jennifer Roberts, Peter Sazani, Suthat Fucharoen, Patrick L. Iversen, Ryszard Kole Thalassemia Research Center and Institute of Molecular Biology and Genetics, Mahidol University, Nakhonpathom, Thailand; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC; AVI BioPharma, Inc., Corvallis, OR; Ercole Biotech, Research Triangle Park, Durham, NC

Conjugation of arginine-rich cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligomers (PMO) has been shown to enhance the intracellular delivery of PMO to modulate gene expression, in particular to alter pre-mRNA splicing. In searching for more active, stable and less toxic peptide-PMO conjugates (PPMOs), we designed a series of PPMOs (termed A through I) carrying CPPs that consisted of eight arginine (R) and a variable number of 6-aminohexanoic acid and/or -alanine residues. Their functional biodistribution and toxicity were initially evaluated in EGFP-654 transgenic mice, a positive readout mouse model for splice switching oligonucleotides. After four once daily intraperitoneal injections at 12.5 mg/kg/day, all conjugates exhibited broad tissue distribution with varied degree of splicing correction in different tissues. The B conjugate showed high splicing correction in the heart (60%), diaphragm (100%) and quadriceps (100%) of the mice. The muscles of heart, diaphragm and quadriceps are important targets for the treatment of Duchenne muscular dystrophy (DMD) with splice switching oligonucleotides. To determine whether the B peptide-conjugated PMO would be applicable for DMD treatment, the M23D-B PPMO, targeted to a donor splice site of dystrophin intron 23, was assessed for exon 23 skipping efficiency in mdx mice, a model for DMD. In these mice, a nonsense mutation in exon 23 of the DMD gene prevents dystrophin production. Four once daily intravenous injections of M23D-B at 12.5 mg/kg/day resulted in persistent exon skipping in dystrophin mRNA and produced high and sustained dystrophin protein expression in the heart, diaphragm and multiple skeletal muscles of treated mice without causing any detectable toxicity. The results suggest that the X/B containing PPMO can be applicable to therapeutic modulation of gene expression.
 

[844] An Improved Protocol for Intra-Arterial Injection in Mouse

Qiang Liu, Zhong Yang, Yaming Wang Anesthesia, Brigham & Womens Hospital, Harvard Medical School, Boston, MA

An improved protocol for intra-arterial injection in mice Qiang Liu, Zhong Yang, Yaming Wang Department of Anesthesia, Brigham Womens Hospital, Harvard Medical School, Boston MA 02115 Duchenne muscular dystrophy (DMD) is an X-linked lethal degenerative disease characterized by widespread muscle damage throughout the body. It is caused by a mutation in the coding region of a large membrane protein, dystrophin. Dystrophin deficiency reduces the stability of skeletal muscle cell membranes. Given the syncitial nature of skeletal myofibers, fusion of diseased muscle cells with therapeutic cells expressing wild type dystrophin may lead to a cell based therapy for this devastating disease. However, systemic delivery of therapeutic cells to muscles remains a major challenge. Recent studies shown that some cells with stem cell like properties can transmigrate from blood into damaged muscles and consequentially contribute to muscle regeneration upon intra-arterial injection. Thus, the use of intra-arterial injection techniques has increased rapidly. The small size of the mouse femoral artery coupled with its high blood pressure presents technical challenges for such delivery methods. Here we describe a new protocol that has improved our success rate substantially while avoiding permanent occlusion of the femoral artery and the usage of heparin. In Brief: 1. Separation of the femoral artery: In order to expose the artery and avoid collateral damage to adjacent structures, the artery will be separated from associated nerves and veins carefully with fine-tip forceps. 2. Creation of a small incision on the wall of the femoral artery: After occlusion of the proximal end of the femoral artery and vein with a microvessel clip, a small incision will be made on the wall of the artery using a 30G1/2 needle tip. 3. Insertion of a blunt-tipped MicroFilTm 36 gauge needle through the incision into the lumen of the artery: The titanium alloy MicroFilTm 36 gauge needle, is 10cm long, 20 m ID and 90 m OD and is equipped with a syringe adapter at one end (World Precision Instruments, USA). It is flexible, yet rigid enough for this use. 4. Cell injection: Cells diluted in 100l HBSS will be injected through the MicroFilTm needle slowly into the artery. The MicroFilTm 36 gauge needle will be carefully removed 5 minutes after injection. Pressure will be applied to the incision site by a wet cotton tip for 2 minutes to minimize bleeding before releasing the microvessel clip. This protocol has allowed us to achieve a greater than 90% success rate. We encountered no post-operative complications related to the procedure. This is a simple, reliable and practical approach that resolves a technical difficulty faced by many labs.
 

[93] High-Level Muscle Specific Transgene Expression in Cultures of Myotubes Using Lentiviral Vectors

Marc-Andre Robert, Marilyne Blain, Sofien Dessolin, Antoine Caron, Sophie Broussau, Lucie Bourget, Bernard Massie, Renald Gilbert Genomics and Gene Therapy Vectors, Biotechnology Reasearch Institute, Montreal, QC, Canada; Molecular Biology, University of Montreal, Montreal, QC, Canada; Neuromuscular Research Group, Montreal Neurological Institute (McGill University), Montreal, QC, Canada

Lentiviral vectors (LV) are promising vehicles for gene transfer applications, because they can integrate their genome into the cells chromosomes and thus provide stable transgene expression. LV can accommodate relatively large transgene (at least 6 kb) and they can transduce dividing and non-dividing cells. LV carrying minidystrophin could thus be used to treat patients suffering from Duchenne muscular dystrophy by restoring the dystrophin function with a stem cells or a gene therapy approach. Because unrestricted and widespread dystrophin expression could be immunogenic or even toxic to dividing cells (myoblasts or stem cells), we have constructed a small and efficient muscle specific promoter (USEx3, 600pb) derived from the slow troponin I gene. In the present study, we compared the strength, the stability and the muscle specificity of LV carrying GFP regulated by USEx3 (LV-USEx3) or by the cytomegalovirus (CMV) promoter (LV-CMV). Mouse C2C12 myoblasts were transduced with both LVs and GFP expression was monitored by flow cytometry in actively dividing myoblasts, or by fluorescent microscopy and by western blot in differentiated myotube cultures. In the myoblasts, the fluorescence index of the cell population ([% of fluorescent cells] X [mean fluorescence intensity]) was stable for at least three weeks and was 5 to 20-folds higher in the cells transduced with LV-CMV. However, after differentiation into myotubes, the GFP expression level of the cells transduced with LV-USEx3 was greater than for LV-CMV. These data show that USEx3, in the context of LV, can provide stable, specific and high-level transgene expression in differentiated muscle cultures.
 

[346] Heart Specific Min-Dystrophin Expression Rescues Systolic but Not Diastolic Function in Mdx Mice

Brian P. Bostick, Yongping Yue, Chun Long, Nathaniel Marschalk, Jing Chen, Dongsheng Duan Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO

Heart failure is a leading cause of death in Duchenne muscular dystrophy (DMD). Significant progress has been made in developing gene and cell therapies for treating DMD skeletal muscle disease. Unfortunately, there is little knowledge of the suitability of these therapies for the heart. Notable among the skeletal muscle therapies is the 6 kb H2-R19 mini-dystrophin gene. The H2-R19 minigene has been shown to completely rescue skeletal muscle pathology and restore muscle force. In this study, we set out to test the suitability of the H2-R19 minigene for the treatment of DMD cardiomyopathy. We developed a series of transgenic mice with heart specific expression of the H2-R19 dystrophin. After backcrossing these mice to the congenic mdx background, we performed a comprehensive evaluation of heart structure and function. Morphological examination revealed a rescue from dystrophic pathology. Most notable was the strengthened cardiomyocyte sarcolemmal integrity in the Evans blue uptake assay. Additionally, Masson trichrome staining demonstrated a complete absence of fibrosis. Heart restricted expression of the H2-R19 minigene improved uphill treadmill endurance compared to mdx mice. ECG analysis showed normalization of the PR interval and cardiomyopathy index. However, the heart rate, QRS duration and QT interval did not reach normal levels. Left ventricular catheterization proved the most informative. The H2-R19 transgenic mdx mice had normalization of baseline systolic parameters. Notably, end-systolic pressure, end-systolic volume and maximal rate of pressure development (dP/dt max) returned to normal levels. Analysis of baseline diastolic parameters showed an incomplete rescue. The end-diastolic volume, time constant of isovolumetric relaxation (tau) and maximal rate of left ventricular relaxation (dP/dt min) remained uncorrected. This systolic-diastolic discrepancy resulted in a normal ejection fraction, while stroke volume and cardiac output were only partially improved. Challenging the heart with dobutamine revealed that the H2-R19 minigene was capable of restoring normal dobutamine response. Further, H2-R19 transgenic mdx mice undergoing dobutamine stress had a normal survival. To explore the mechanisms underlying the incomplete rescue, we expressed the H2-R19 minigene on the normal C57BL/10 (BL10) background. The H2-R19 dystrophin profoundly displaced wild-type dystrophin. However, the cardiovascular profile of these mice was not affected. Taken together, our results provide the first comprehensive analysis of a therapeutic mini-dystrophin gene in the heart. The incomplete rescue of DMD heart disease by the H2-R19 minigene suggests that the heart may have different requirements for gene therapy than skeletal muscle. Additionally, these findings highlight the potential importance of the skeletal muscle in modulating heart function. (Supported by grants from the NIH and the MDA).
 

[348] A Human Artificial Chromosome (HAC) Vector with about 2.4 Mb-Human Dystrophin Genome Including Native Expression Control Elements

Hidetoshi Hoshiya, Yasuhiro Kazuki, Satoshi Abe, Masato Takiguchi, Yuichi Iida, Yoshinori Watanabe, Mitsuhiko Osaki, Naoyo Kajitani, Toko Yoshino, Yasuaki Shirayoshi, Mitsuo Oshimura Department of Biomedical Science, Regenerative Medicine and Biofunction,, Graduate School of Medical Science,Tottori University, Yonago, Tottori, Japan; Research Center for Bioscience and Technology, Tottori University, Yonago, Tottori, Japan; Division of Regenerative Medicine and Therapeutics, Tottori University, Yonago, Tottori, Japan

Duchenne muscular dystrophy (DMD) is caused by mutation or deletion in the 2.4 Mb dystrophin gene. This gene has highly complex expression patterns, and various isoforms are expressed by at least seven promoters and alternative splicing. For gene therapy of DMD, there are a number of points that should be considered, i.e., the tissue-specific expression, period, localization, expression level, and isoforms. Moreover, it is necessary to avoid damaging hosts chromosome, and to be expressed semipermanently. We have previously produced a Human Artificial Chromosome (HAC) vector by deleting all genes on human chromosome 21. The purpose of this study is to develop a dystrophin expression vector that has the potential for physiological expression of dystrophin. Because the dystrophin is the biggest gene, no vector with whole dystrophin genomic region has been developed. Thus, we constructed a HAC vector with only 2.4Mb human dystrophin genomic region including its own transcriptional regulatory elements. A loxP site was inserted to a proximal locus of the dystrophin genome of human chromosome X (h.Chr.X) in homologous recombination proficient chicken DT40 cells. Genes on telomere-side from the dystrophin genome were deleted by the telomere truncation in the DT40 cells. This modified h.Chr.X fragment including the dystrophin genome was transferred to CHO cells containing the HAC vector by microcell-mediated chromosome transfer (MMCT). The dystrophin genome was cloned into the HAC vector by the Cre-mediated site-specific translocation. This vector was designated as Dys-HAC. The Dys-HAC was transferred to mouse embryonic stem (ES) cells from the CHO cells. To test the human dystrophin expression in vivo, the chimeric mice were produced from the ES cells with the Dys-HAC. We confirmed the human dystrophin expression of various tissue-specific isoforms in the chimeric mice with the Dys-HAC by RT-PCR analysis. In addition, the GFP on the Dys-HAC was expressed in all chimeric tissues, suggesting that the Dys-HAC was stably maintained in vivo. Mesenchymal stem cells (MSCs) have great potential to differentiate into functional skeletal muscle and are considered as candidates for transplantation therapy. Thus, the Dys-HAC was transferred to human MSCs and to test the mitotic stability of the Dys-HAC. The Dys-HAC was stably maintained up to 75 PDLs without a selection in human MSCs. In conclusion, the Dys-HAC may be a useful tool for an ex vivo therapy for DMD. Furthermore, using stem cells derived from multiple potential sources combined with the HAC-mediated gene delivery may comprise this useful treatment for genetic defects such as DMD in a near future.
 

[864] Polyplex Nanomicelle Promotes Intravenous Gene Transfer to Skeletal Muscle

Keiji Itaka, Katsue Morii, Kensuke Osada, Kazunori Kataoka Division of Clinical Biotechnology,Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

Skeletal muscle is an attractive target for clinical gene therapy, especially for muscular dystrophy and peripheral ischemic diseases. Long-term secretion of functional protein such as erythropoietin from the transgene into the muscle is also beneficial for the treatment of various chronic diseases. For this purpose, a variety of delivery methods using naked DNA has been investigated. In this study, a novel gene carrier for intravenous transfer to muscle tissue using increased pressure was proposed. This carrier is a polyplex nanomicelle composed of plasmid DNA and poly(ethylene glycol)-poly(lysine) (PEG-PLys) block copolymer. The nanomicelle has the core-shell structure with dense PEG palisades surrounding the core to compartmentalize the condensed pDNA. The nanomicelles containing luciferase-encoding pDNA were injected from great saphenous vein with a tourniquet placed on the upper hind limb to restrict blood flow into and out of the hind limb. A substantial transgene expression was observed on the skeletal muscles of the injected limb by IVIS observation and the histological analysis. Compared to naked DNA, the nanomicelle achieved more than one-order higher luciferase expression as well as the more sustained expression profile up to two months. Interestingly, the transgene expressions by nanomicelles on the skeletal muscles were strongly correlated to the expressions using a cell-free transcription and translation assay system, where the ideal composition of nanomicelles showed much higher expressions than naked DNA. These results suggest that, by the intravenous injection with isolation of the limb by tourniquet, the nanomicelles transferred the pDNA under the optimal condition for the gene expression into the muscle fibers. Thus, the intravenous injection of nanomicelles is a promising technique to achieve sufficient and long-term transgene expressions on the skeletal muscles.