Résumé :
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Communication n° 293 Introduction : Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, making it amenable to gene- or cell-based therapies. Another possible treatment entails the combination of both principles by transplantation of autologous myogenic cells after their genetic complementation. This approach requires efficient and stable transduction of these cells with recombinant dystrophin genes. Recently, we generated a dual high-capacity adenovirus (Ad)/adeno-associated virus (AAV) hybrid vector (hcAd/AAV) for the delivery of two full-length dystrophin (FL-Dys)-encoding modules into target cells. Furthermore, we showed that in the presence AAV Rep78/68 proteins hcAd/AAV DNA is integrated in a specific region of human chromosome 19. Here, we focused on improving hybrid vector entry into myoblasts as these cells barely express CAR, which constitutes the primary receptor for the human Ad serotype 5 (Ad5) fibers, displayed at the surface of the hcAd/AAVs capsids. To this end, hybrid vector particles were equipped with human Ad serotype 50 (Ad50) fiber domains to achieve CAR-independent uptake. Objectives : To increase the efficiency of FL-Dys delivery to DMD myoblasts by a high-capacity vector system with site-specific integration ability. Methods : Dual hcAd/AAV DNA encoding FL-Dys was rescued into Ad5 capsids displaying Ad50 fiber domains in producer cells infected with a first-generation helper Ad vector encoding Ad50 fiber domains (hAd5F50). The resulting hybrid vector particles were amplified by serial propagation. Contamination of vector preparations with hAd5F50 particles was prevented to a great extent by Cre/loxP-mediated excision of the hAd5F50 packaging elements. Results : Tropism-modified vectors could be produced at similar titers as unmodified vectors (1-3û109 HeLa cell-transducing units [HTU]/ml). Importantly, at 1, 3, and 10 HTU/cell, the percentage of FL-Dys-positive DMD myoblasts was 112-, 54-, and 18-fold higher for the tropism-modified hcAd/AAVs than for the control vectors. Conclusions : Dual hcAd/AAV hybrid vectors carrying Ad50 fiber domains complement DMD myoblasts much more efficiently than their counterparts with Ad5 fibers.
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