Résumé :
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Communication n° 635 Introduction : Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder affecting 1:3500 male individuals, caused by recessive mutations in the dystrophin gene. The size of the gene (2.4Mb) and mRNA (14kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of gene therapy. rAAV vectors overcome many of the problems associated with other vector systems but accommodate limited transgene capacity (<5kb), currently incorporating only 'micro' versions of dystrophin cDNA (mdys). More than 8 AAV vector serotypes have been identified to date with certain serotypes (1, 5, 6, 7) displaying more favourable tropism in transducing muscle fibers compared to the traditionally used AAV2. Objectives : The project is designed to contribute a second generation of clinically-valuable microdystrophin vectors which have undergone rigorous preclinical testing in relevant DMD models and could then selectively used in phase I clinical programmes. Methods : Overlapping PCR using genotype-phenotype patient data and bio-informatic relationships, molecular subcloning, rAAV pseudotyping, intramuscular, intraperitoneal, tail vein and femoral artery injection studies, Q-PCR/RT-PCR, immuno-histochemical and western blotting analyses, centro-nucleation morphometric and Evans Blue exclusion assays, muscle creatine kinase (mCK) activity and physiological assays. Results : Production of various human, canine and murine mdys versions and reporter cDNAs, incorporating either constitutive or muscle-specific promoter elements and optimal Kozak sequences for transcriptional improvement and packaging of the cassettes into recombinant AAV vectors of alternative serotypes (1, 2, 5 and 6) by pseudotyping has been achieved. These vectors are currently fully screened and their functionality/immunogenicity is determined by extensive in vitro and in vivo studies utilising local and systemic routes of administration (i.m., i.p., i.v. and intra-arterial) in dystrophic preclinical models (mdx, GRMD). Conclusions : In this work we demonstrate a selective comparison between suitable species-specific micro-dystrophin gene configurations, muscle-specific/constitutive expression systems, rAAV vector serotypic variations and route-administration protocols for DMD.
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