Gene based approach to treating hemophilic inhibitors

血友病已被证明是研究基于基因的疾病治疗方法的一个富有成效的模型，而且这种方法似乎很可能在不久的将来得到广泛应用。相当大比例的严重血友病患者对输注的凝血因子产生抑制抗体，他们认为这是一种“外来”蛋白质。这些个体对凝血因子浓缩物没有反应，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，重组F.VIIa的剂量足以达到循环水平2-4马克杯/毫升，可导致具有抑制剂的个体有效止血。在这个应用中，我们建议开发一种基于基因的方法来在已经诱导抑制剂形成的血友病动物中给药VIIa。在AAV载体成功应用于肝脏的基础上，我们将开发AAV载体，表达工程化的F.VII结构，该结构与F.VII在细胞内结合，并以活化形式分泌。在aim 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来实现血友病抑制剂小鼠模型的止血。VIIa向量。在第二个目标中，我们将在连续表达一系列确定水平的f.v ia的小鼠中进行凝血参数的长期研究。这些实验的目的是确定是否存在f.v ia表达的基线水平，从而改善凝血参数而不会产生严重的不良反应。在该目标的第二部分，我们将开发由药物多西环素激活的“开关”控制的AAV-VIIa载体，并确定这种系统是否可以用于减少与VIIa长期表达相关的不良副作用，同时仍然有助于防止出血。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狗，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狗的免疫原性，在第四个目标中，我们将评估用于产生完全加工的F.VII构建物的免疫原性。这些临床前研究的成功完成应该有助于确定以基因为基础的方法治疗血友病抑制剂是否可行。

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.