TARGETED GENE THERAPY FOR HEMOPHILIA A

血友病A(先天性凝血因子缺乏)是世界范围内最常见(严重)的先天性出血性疾病。血友病A是一个明确的遗传治疗的模式系统，原因如下：(I)它是由单基因缺陷引起的，(Ii)它的生物处理和合成已经被广泛研究，(Iii)血浆凝血因子的治疗水平可以改善出血发作，(Iv)人类的生理水平很低(100-200 ng/mL)，临床表现与循环FVIII水平密切相关，因此FVIII活性(5-10 ng/mL)可以达到5%的治疗效益。作为一种明确的基因治疗手段，本实验室将重点放在腺相关病毒(AAV)的FVIII基因治疗策略上，因为AAV感染与人类疾病无关，并且携带前病毒的细胞无法表达新的细胞表面抗原，因此无法作为免疫学靶点。如果在AAV感染期间不存在辅助病毒，AAV基因组整合在人类染色体19q上的一个位点特定区(AAVS1)，这一特性取决于AAVrep68/78和反向末端重复(Tr)。由于肝细胞主要在肝脏产生，因此肝细胞是血友病A基因替换策略的合理场所，尽管血管内皮细胞通过与Von Willebrand因子共表达而仍然是一种有吸引力的靶点，作为一种受调控的递送方式。在这个方案中，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，作为血友病A的最终治疗手段。到目前为止，我们是唯一成功产生FVIII/rAAV病毒的实验室，为这一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运送B结构域缺失因子重组腺病毒、腺/腺相关杂交病毒(Ad/AAV)和含有AAVtR的迷你腺病毒(MAD)，后者是作为亲本Ad/AAV杂交病毒的独特转录副产物产生的。其他目标将针对建立血管内皮细胞作为FVIII传递的目标，使用体外和体内模型定向传递到这些细胞类型。最优的病毒载体将在血友病A的小鼠和犬模型中进行研究。这项拟议的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.