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

在本研究中，我们确定了在构建FVIII载体之前利用AAV载体进行交易的最佳条件。为此，我们通过比较不同血清型和启动子来研究小鼠体循环中促红细胞生成素（Epo）的含量。对于肌肉转导，我们比较了血清型与巨细胞病毒衍生（CMV）启动子的差异。每只小鼠共接受6 × 10^<10>个血清型载体基因组拷贝到双侧腓肠肌中。对于肌肉介导的表达，血清1型Epo浓度最高，其次是5、4、3和2。利用AAV血清2型平台比较CMV、CAG、EF-1α和PGK启动子在肝脏中的转导作用。载体经静脉注射，剂量分别为1 × 10^<10>vg和1 × 10^<11>vg/体。在肝脏启动子分析中，CAG启动子的Epo浓度最高，其次是CMV、PGK和EF-1α。对于血清型的比较，使用CAG启动子，血清5型在肝脏中效果最好。根据这些结果，我们设计了用于系统递送因子VIII的AAV载体结构。在肝脏和肌肉中，利用CAG启动子+ AAV5衣壳和CMV启动子+ AAV1结构的组合表达因子VIII的重链和轻链。这种策略的能力以及这些质粒的效力在转染实验后得到证实。利用这些质粒，我们制备了必要数量的AAV载体用于体内转导。同时，获得了因子VIII敲除小鼠。我们现在正在准备注射到小鼠模型中，并追求这种策略的治疗效果。如果效果良好，我们将转向更大的动物模型，以验证其对人类基因治疗的效用。

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.