TARGETED GENE THERAPY FOR HEMOPHILIA A

血友病A是第二常见的先天性出血性疾病 在全球范围内，约占血友病的90%。 足够 止血需要最低血浆因子VIII（FVIII）浓度 (10- 50 ng/ml; < 0.25 nM），表明即使是低效的全身性 输送系统可能会成功。 在这个过程中， 腺相关病毒（AAV）是一种腺相关病毒，它在缺乏腺相关病毒的情况下， 辅助病毒作为稳定的前病毒整合到人类基因组中， 独立于细胞的增殖状态。 除了终点站 重复，AAV整合到基因组中不需要转录 调节元件，降低肿瘤发生的风险， 外源启动子插入这些病毒中以保持正常功能。 这笔赠款将集中在产生适当的FVIII删除 用于所提出的rAAV/FVIII递送系统的构建体。 基于先前 在这些和其他实验室工作，这些结构的大小应该 不干扰rAAV包装和复制限制，也 功能性FVIII表达。 一种最小长度的细胞特异性启动子 将被表征并用于驱动细胞特异性表达。 小说 设计用于克服rAAV大小限制和适度病毒感染的方法 将探索滴度。 在最初的体外研究之后，动物模型 将用于进一步研究和开发体内表达系统。 尽管该提案具体涉及FVIII递送的方法， 该结果可普遍适用于多种基因， 瞄准战略。

Hemophilia A is the second most common congenital bleeding disorder worldwide, accounting for approximately 90% of hemophilia. Adequate hemostasis requires a minimal plasma factor VIII (FVIII) concentration (10 - 50 ng/ml; < 0.25 nM), suggesting that even inefficient systemic delivery systems may be successful. The vector of choice in this proposal is adeno-associated virus (AAV), which in the absence of its helper virus integrates as a stable provirus into the human genome, independent of the cell's proliferative state. Except for the terminal repeats, integration of AAV into the genome requires no transcriptional regulatory elements, reducing the risk of oncogenesis and allowing foreign promoters inserted into these viruses to retain normal function. This grant will focus on the generation of appropriate FVIII deletional constructs for proposed rAAV/FVIII delivery systems. Based on previous work in these and other laboratories, the size of these constructs should not interfere with rAAV packaging and replication constraints, nor functional FVIII expression. A minimum-length cell-specific promoter will be characterized and used to drive cell-specific expression. Novel methods designed to overcome rAAV size constraints and modest viral titers will be explored. After initial in-vitro studies, animal models will be used to further study and develop in-vivo expression systems. Although this proposal specifically addresses methods for FVIII delivery, the results may be generally applicable to a wide variety of gene- targeting strategies.