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37. Long-Term Expression of Mini-Human Dystrophin in Transgenic mdx Mice Improves Dystrophic Muscle Functions
MUSCLE AND CONNECTIVE TISSUE: MUSCLE 37. Long-Term Expression of Mini-Human Dystrophin in Transgenic mdx Mice Improves Dystrophic Muscle Functions Bing Wang,1 Juan Li,1 Chunlian Chen,1 Xiancheng Jiang,3 Terry O’ Day,2 Jon Watchko,2 Xiao Xiao.1 1 Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Pediatrics, Magee-Women’s Research Institute, University of Pittsburgh, Pittsburgh, PA; 3Anatomy and Molecular Cell Biology, The State University of New York, New York, NY. BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene and affects one in every 3500 males. The clinical and pathological characteristics of DMD patients show progressive myopathy of skeletal and cardiac muscles and premature death of patients. The human “mini- or micro -dystrophines” with different truncated central rod domains have been proven to ameliorate dystrophic pathology. In this study, our purpose is to investigate the long-term expression of human mini-dystrophin and therapeutic benefits in transgenic mdx mice. METHODS: Four lines of transgenic mdx mouse (Tg-Dys3849mdx) were generated. The 3.8 kb human mini-dystrophin containing five rods (R1-2, R22-24) of the central region and two hinges (H1 and H4) was driven by a truncated creatine-kinase promoter with two modified enhancers (dMCK). The gene expression cassette was cloned in an AAV vector plasmid for creation of transgenic mice. After five generations of back-crossing with the mdx mice, one of the lines containing a single copy of the minigene was used to test the gene expressions and muscle functions. RESULTS: 1) the human mini-dystrophin expression was found a majority of the of skeletal muscles (Quad, GAS, TA, forelimb, abdominal, intercostals, thoracic and lumbar spinal muscles), but only very limited gene expressions in DIA and no expression in cardiac muscle; 2) Preference of gene expression was found in fast twitch myofibers over the slow fibers; 3) the DAGs such as sarcoglycans and nNOS were restored at sarcolemma and coincided with human mini dystrophin expression; 4) the morphology of dystrophic muscle expressing the human mini-dystorphin was improved and central nuclei were reduced to < 2%; 5) myofiber membrane integrity was improved by EBD test; 6) Improvement in treadmill running and grip force was observed in transgenic mice at 6 and 10 months when compared with the littermates (p<0.05); Also, tetanic force and specific force of TA muscle were significantly increased at 6, 10 and 20 months (p<0.05); 7) pseudohypertrophy was not found in GAS and TA mass (p<0.05). Taken together, this study demonstrates that the human minidystrophin with 5 central rods can effectively ameliorate the pathology and improve the functions of the dystrophic muscles in mdx mice.
38. Morpholino Antisense Oligonucleotide Induced Exon Skipping Efficiently Restores Dystrophin Expression in the mdx Mouse Dominic J. Wells,1 Caroline McCulley,1 Anna Graham,1 Ke Liu,1 Kim E. Wells.1 1 Cellular and Molecular Neuroscience, Imperial College London, London, United Kingdom. Although antisense oligonucleotides (AO) have been used most commonly to inhibit gene expression, they can also be used to modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3’/5’ splice sites. Recent studies have shown that the morpholino chemistry is particularly useful as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due to a premature stop codon in exon 23. Using an AO that targets exon 23, we have examined the longevity of exon skipping by RT-PCR and S16
dystrophin immunocytochemistry and western blots. Skipped dystrophin transcript can still be detected 14 weeks after a single intramuscular injection of morpholino AO and dystrophin is clearly present at least 4 months post treatment. This is substantially longer than the expression seen in similar studies using a 2-O-methyl phosphorothioate AO. We have used similar techniques to examine the dose/volume relationship in preparation for clinical trials of this approach in Duchenne muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that most closely resembles the pathology in DMD, exhibiting substantial fibrosis and fibre loss, we have used the diaphragm to test the effect of increased fibrosis in reducing AO uptake by the muscle fibres by treating mice at different ages. Delivery of AO in mice younger than 2 weeks can largely prevent the development of dystrophic pathology and treatment at later ages reduces the membrane fragility as determined by uptake of Evans Blue dye. These pre-clinical studies demonstrate the effectiveness of the morpholino based AO in inducing long-lived expression of dystrophin and this approach is now being taken forward to a UK clinical trial in DMD by the MDEX Consortium.
39. Evaluation of rAAV-1 vs rAAV-8 Vectors and Their Mode of Delivery in Nonhuman Primate Skeletal Muscle Alice Toromanoff,1 Yan Cherel,2 Jack-Yves Deschamps,3 Valder R. Arruda,4 Katherine A. High,5 Hansell H. Stedman,6 Mark E. Haskins,7 Fabienne Rolling,1 Ignacio Anegon,8 Philippe Moullier,1 Caroline Le Guiner.1 1 INSERM, UMR649, Nantes, France; 2INRA UMR703, ENV, Nantes, France; 3Service d’Urgences, ENV, Nantes, France; 4 Department of Pediatrics, University of Pennsylvania, Philadephia; 5Children’s Hospital, Philadephia; 6Department of Surgery and Pennsylvania Muscle Institute, University of Pennsylvania, Philadephia; 7School of Veterinary Medecine, University of Pennsylvania, Philadephia; 8INSERM, UMR643ITERT, Nantes, France. AAV serotype and mode of delivery impact on gene transfer efficiency, immunological outcome, and clinical efficacy. Several groups recently described a drug-free limb perfusion technique in the dog, which allows the transduction of the entire limb using rAAV vectors. The aim of our study is to evaluate in the nonhuman primate model two distinct modes of delivery, and selected AAV serotypes with high skeletal muscle tropism, on several parameters including: (i) clinical tolerance, (ii) innate immune response, (iii) transgene expression pattern, (iv) vector genome copy per cell, (v) transgene distribution in muscle and (vi) vector shedding. For this, we compared rAAV-1 vs rAAV-8 encoding for LEA29Y, a secreted reporter gene, administered in skeletal muscle of a cohort of eight nonhuman primates, either intramuscularly (IM) or by limb perfusion. We demonstrated that the limb perfusion delivery method is remarkably simple and well tolerated with no adverse effect in 3 to 5 kg animals. Clinical and biological parameters remain unchanged after the procedure. Using the same amount of rAAV-1 (3,5x1012 vector genome/kg), the serum concentration of the reporter gene after limb perfusion is at least 6 to 10 times the levels achieved after IM delivery. rAAV-8 is also at least 6 to 10 times more potent than rAAV-1 when both are delivered IM. Long-term analysis of both serotypes and delivery modes with respect to expression levels of the transgene are under way. Transgene expression and vector DNA analyses on surgical biopsies, by immunohistochemistry and Q-PCR respectively, confirm that little diffusion occurs after IM rAAV-1 delivery, resulting in high local vector genome copy number. In contrast, limb Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
38. Morpholino Antisense Oligonucleotide Induced Exon Skipping Efficiently Restores Dystrophin Expression in the mdx Mouse Dominic J. Wells,1 Caroline McCulley,1 Anna Graham,1 Ke Liu,1 Kim E. Wells.1 1 Cellular and Molecular Neuroscience, Imperial College London, London, United Kingdom. Although antisense oligonucleotides (AO) have been used most commonly to inhibit gene expression, they can also be used to modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3’/5’ splice sites. Recent studies have shown that the morpholino chemistry is particularly useful as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due to a premature stop codon in exon 23. Using an AO that targets exon 23, we have examined the longevity of exon skipping by RT-PCR and S16
dystrophin immunocytochemistry and western blots. Skipped dystrophin transcript can still be detected 14 weeks after a single intramuscular injection of morpholino AO and dystrophin is clearly present at least 4 months post treatment. This is substantially longer than the expression seen in similar studies using a 2-O-methyl phosphorothioate AO. We have used similar techniques to examine the dose/volume relationship in preparation for clinical trials of this approach in Duchenne muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that most closely resembles the pathology in DMD, exhibiting substantial fibrosis and fibre loss, we have used the diaphragm to test the effect of increased fibrosis in reducing AO uptake by the muscle fibres by treating mice at different ages. Delivery of AO in mice younger than 2 weeks can largely prevent the development of dystrophic pathology and treatment at later ages reduces the membrane fragility as determined by uptake of Evans Blue dye. These pre-clinical studies demonstrate the effectiveness of the morpholino based AO in inducing long-lived expression of dystrophin and this approach is now being taken forward to a UK clinical trial in DMD by the MDEX Consortium.
39. Evaluation of rAAV-1 vs rAAV-8 Vectors and Their Mode of Delivery in Nonhuman Primate Skeletal Muscle Alice Toromanoff,1 Yan Cherel,2 Jack-Yves Deschamps,3 Valder R. Arruda,4 Katherine A. High,5 Hansell H. Stedman,6 Mark E. Haskins,7 Fabienne Rolling,1 Ignacio Anegon,8 Philippe Moullier,1 Caroline Le Guiner.1 1 INSERM, UMR649, Nantes, France; 2INRA UMR703, ENV, Nantes, France; 3Service d’Urgences, ENV, Nantes, France; 4 Department of Pediatrics, University of Pennsylvania, Philadephia; 5Children’s Hospital, Philadephia; 6Department of Surgery and Pennsylvania Muscle Institute, University of Pennsylvania, Philadephia; 7School of Veterinary Medecine, University of Pennsylvania, Philadephia; 8INSERM, UMR643ITERT, Nantes, France. AAV serotype and mode of delivery impact on gene transfer efficiency, immunological outcome, and clinical efficacy. Several groups recently described a drug-free limb perfusion technique in the dog, which allows the transduction of the entire limb using rAAV vectors. The aim of our study is to evaluate in the nonhuman primate model two distinct modes of delivery, and selected AAV serotypes with high skeletal muscle tropism, on several parameters including: (i) clinical tolerance, (ii) innate immune response, (iii) transgene expression pattern, (iv) vector genome copy per cell, (v) transgene distribution in muscle and (vi) vector shedding. For this, we compared rAAV-1 vs rAAV-8 encoding for LEA29Y, a secreted reporter gene, administered in skeletal muscle of a cohort of eight nonhuman primates, either intramuscularly (IM) or by limb perfusion. We demonstrated that the limb perfusion delivery method is remarkably simple and well tolerated with no adverse effect in 3 to 5 kg animals. Clinical and biological parameters remain unchanged after the procedure. Using the same amount of rAAV-1 (3,5x1012 vector genome/kg), the serum concentration of the reporter gene after limb perfusion is at least 6 to 10 times the levels achieved after IM delivery. rAAV-8 is also at least 6 to 10 times more potent than rAAV-1 when both are delivered IM. Long-term analysis of both serotypes and delivery modes with respect to expression levels of the transgene are under way. Transgene expression and vector DNA analyses on surgical biopsies, by immunohistochemistry and Q-PCR respectively, confirm that little diffusion occurs after IM rAAV-1 delivery, resulting in high local vector genome copy number. In contrast, limb Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy