Fundamental researches toward gene therapy of Hemophilia A utilizing adeno-associated virus vectors

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In this study we determined the optimal conditions for the transaction utilizing AAV vectors prior to the construction of vectors for FVIII. For this purpose, we investigated the amount of murine erythropoietin (Epo) within systemic circulation by comparing different serotypes as well as promoters. For the muscle transduction, we compared the difference by serotypes with cytomegalovirus-derived (CMV) promoter. Each mouse received a total of 6 x 10^<10> genome copies of each serotype vector into bilateral gastrocunemius muscle. For the muscle-mediated expression, serotype 1 showed the highest Epo concentration, followed by 5, 4, 3 and 2 at 4 weeks. For the liver-mediated transduction, CMV, CAG, EF-1α and PGK promoters were compared utilizing AAV serotype 2 platform. Vectors were intraportally administered, with the dose of both 1 x 10^<10>vg and 1 x 10^<11>vg/body As for the promoter analysis in the liver, CAG promoter achieved the highest concentration of Epo, followed by CMV, PGK and EF-1α. For the comparison of serotypes, CAG promoter was used and serotype 5 worked best in the liver. According to these results, we designed the AAV vector structure for the systemic delivery of Factor VIII. For the liver and muscle, the combination of CAG promoter plus AAV5 capsid and CMV promoter with AAV1 structure were utilized to express both heavy and light chains of Factor VIII. The competence of this strategy along with the potency of these plasmids was confirmed following experiments with transfection. Utilizing these plasmids, we prepared the necessary amounts of AAV vectors for in vivo transduction. Also, knockout mice for Factor VIII were obtained. We are now preparing for the injection into the mouse model and pursuing the therapeutic outcome of this strategy. If it works well, we will move to the models of larger animals to validate its utility toward human gene therapy.