TARGETED GENE THERAPY FOR HEMOPHILIA A

血友病A（先天性凝血因子VIII缺乏症）是世界范围内最常见（严重）的先天性出血性疾病。A型血友病是基因治疗的典型系统，原因如下：(1)它是由单基因缺陷引起的，(2)其生物加工和合成已被广泛研究，(3)血浆凝血因子的治疗水平可改善出血事件，(4)人体内的生理水平较低（100 - 200 ng/mL），临床表现与循环fVIII水平密切相关，因此5% fVIII活性（5 - 10 ng/mL）即可达到治疗效果。作为一种明确的基因治疗手段，本实验室一直致力于研究腺相关病毒（AAV）的fVIII基因治疗策略，因为AAV感染与人类疾病无关，并且携带前病毒的细胞不能表达新的细胞表面抗原，因此不能作为免疫靶标。如果在AAV感染期间不存在辅助病毒，则AAV基因组在人类染色体19q上的位点特异性区域（AAVS1）整合，这一特性依赖于AAV rep68/78和反向末端重复序列（TR's）。由于肝细胞主要在肝脏中产生，因此肝细胞是血友病A基因替代策略的逻辑位点，尽管血管内皮细胞仍然是一个有吸引力的靶标，可以通过与血管性血友病因子的共表达来调节递送。在本提案中，我们将继续开发用于血友病A基因治疗的新型病毒载体，这些病毒共同具有AAV末端重复序列的独特整合特性，作为最终治疗血友病A的手段。迄今为止，我们是唯一一个成功生成fVIII/rAAV病毒的实验室，为该方向的进一步研究提供了原理证明。三种病毒的特点是它们能够传递b结构域缺失因子VIII重组AAV，腺/腺相关杂交（Ad/AAV）病毒和含有AAV TR的迷你腺病毒（mAD），这是作为亲本Ad/AAV杂交病毒的独特转录副产物产生的。另外的目标将是建立血管内皮细胞作为fVIII递送靶点的效用，使用体外和体内模型靶向递送到这些细胞类型中。最佳病毒载体将在小鼠和犬血友病a模型中进行研究。这项工作旨在为预期的人类试验奠定基础。

Hemophilia A (congenital deficiency of coagulation factor VIII) is the most common (serious) congenital bleeding disorder worldwide. Hemophilia A is a model system for definitive genetic therapy for the following reasons: (I) it is caused by a single- gene defect, (ii) its biological processing and synthesis have been extensively studied, (iii) therapeutic levels of plasmatic clotting factors ameliorate hemorrhagic episodes, and (iv) physiologic levels in human are low (100 - 200 ng/mL), with clinical manifestations closely paralleling circulating fVIII levels such that therapeutic benefit can be reached with 5 percent fVIII activity (5 - 10 ng/mL). As a definitive means of genetic treatment, this laboratory has focused on adeno- associated virus (AAV) for fVIII gene therapy strategies, as AAV infection is not associated with human diseases, and cells carrying proviruses fail to express novel cell-surface antigens, thereby failing to serve as immunological targets. If a helper virus is not present during an AAV infection, the AAV genome integrates in a site-specific region (AAVS1) on human chromosome 19q, a property that is dependent on AAV rep68/78 and inverted terminal repeats (TR's). By virtue of being produced primarily in the liver, the hepatocyte is the logical site for gene replacement strategies for hemophilia A, although vascular endothelial cells remain an attractive target as a means of regulated delivery by co-expression with Von Willebrand factor. In this proposal, we will continue to develop novel viral vectors for hemophilia A gene therapy, viruses which collectively share the unique integrating properties of the AAV terminal repeats, as a means of definitive treatment for hemophilia A. To date, we have been the only laboratory that has successfully generated a fVIII/rAAV virus, serving as proof-of-principle for further research in this direction. Three viruses will be characterized for their ability to deliver B-domain-deleted factor VIII recombinant AAV, adeno/adeno-associated hybrid (Ad/AAV) virus, and mini-adenovirus (mAD) containing the AAV TR's, generated as a unique transcriptional byproduct of parental Ad/AAV hybrid viruses. Additional aims will be directed at establishing the utility of vascular endothelial cells as targets for fVIII delivery, using both in vitro and in vivo models for targeted delivery into these cellular types. Optimal viral vectors will be studied in murine and canine models of hemophilia A. The proposed work is designed to lay the foundation for anticipated trials in humans.