Gene based approach to treating hemophilic inhibitors

血友病已被证明是一个富有成效的模式，为研究基于基因的方法来治疗疾病，它似乎有可能，这种方法将在不久的将来被开发的广泛应用。很大一部分重度血友病患者会对输注的凝血因子产生抑制性抗体，他们认为凝血因子是一种“外来”蛋白。这些个体对凝血因子浓缩物没有反应，并且直到最近才出现血友病中最困难的管理问题之一。过去十年的经验表明，以足以达到2- 4 μ g/ml水平的循环水平的剂量施用重组F.VIIa可以在具有抑制剂的个体中导致有效止血。在本申请中，我们建议开发一种基于基因的方法，在血友病动物中给予VIIa，其中抑制剂的形成已被诱导。在我们成功将AAV载体施用于肝脏的基础上，我们将开发表达工程化F.VII构建体的AAV载体，所述工程化F.VII构建体在细胞内裂解为F.VIIa并以活化形式分泌。在目标1中，我们将进行短期实验以确定我们是否可以通过门静脉注射AAV-F.VIIa载体在血友病抑制剂的小鼠模型中实现止血。在第二个目标中，我们将在小鼠中进行凝血参数的长期研究，这些小鼠在一系列确定的水平上连续表达F.VIIa。这些实验的目的是确定是否存在任何基线水平的F.VIIa表达，其将导致凝血参数的改善而没有严重的副作用。在该目标的第二部分中，我们将开发由可被药物强力霉素激活的“开关”控制的AAV-VIIa载体，并确定这样的系统是否可用于减少与VIIa的长期表达相关的不希望的副作用，但仍然用于防止响应于止血挑战的出血。在第三个目标中，我们将寻求将这些发现扩展到患有血友病和抑制剂的犬，在第四个目标中，我们将评估这些发现对患有血友病和抑制剂的犬的免疫原性，在第四个目标中，我们将评估用于产生作为分泌产物的完全加工的F.VIIa的修饰的F.VII构建体的免疫原性。这些临床前研究的成功完成将有助于确定基于基因的血友病抑制剂治疗方法是否可行。

Hemophilia has proven a fruitful model for the study of gene-based approaches to the treatment of disease, and it seems likely that such an approach will be developed for widespread application in the near future. A substantial proportion of patients with severe hemophilia, develop inhibitory antibodies to infused clotting factor, which they perceive as a "foreign" protein. These individuals fail to respond to clotting factor concentrates and until recently presented one of the most difficult management problems in hemophilia Experience over the past decade has shown that administration of recombinant F.VIIa in doses sufficient to achieve circulating levels of 2-4mug/ml levels can result in effective hemostasis in individuals with inhibitors. In this application we propose to develop a gene-based approach to administration of VIIa in hemophilic animals where inhibitor formation has been induced. Building on our success with AAV vectors administered to liver, we will develop AAV vectors that express an engineered F.VII construct that is cleaved to F.VIIa intracellular and secreted as the activated form. In aim 1, we will carry out short-term experiments to determine whether we can achieve hemostasis in a mouse model of hemophilic inhibitors by portal vein injection of an AAV-F.VIIa vector. In the second aim we will carry out long-term studies of clotting parameters in mice that continuously express F.VIIa at a series of defined levels. The purpose of these experiments is to determine whether there is any baseline level of F.VIIa expression that will result in improvement in clotting parameters without serious adverse effects. In the second part of this aim we will develop AAV-VIIa vectors controlled by a "switch" that can be activated by the drug doxycycline, and determine whether such a system can be used to reduce unwanted side effects associated with long-term expression of VIIa, yet still serve to prevent bleeding in response to a hemostatic challenge. In the third aim we will seek to extend these findings to dogs with hemophilia and inhibitors, and in the fourth aim we will assess the immunogenicity of these findings to dogs with hemophilia and inhibitors, and in the fourth aim we will assess the immunogenicity of the modified F.VII constructs used to generate fully processed F.VIIa as a secreted product. Successful completion of these pre-clinical studies should help to establish whether a gene-based approach to treatment of hemophilic inhibitors is feasible.