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卓有成效的模型，似乎有可能在不久的将来开发出这样的方法来广泛应用。相当大比例的严重血友病患者会产生抑制抗体来输注凝血因子，他们认为凝血因子是一种“外来”蛋白质。这些人对凝血因子浓缩物没有反应，直到最近出现了血友病中最困难的管理问题之一，过去十年的经验表明，给予足以达到循环水平2-4mug/ml水平的重组F.VIIa可以导致使用抑制剂的个人有效止血。在这项应用中，我们建议开发一种基于基因的方法来在血友病动物中给药，在那里已经诱导了抑制物的形成。在我们成功将AAV载体应用于肝脏的基础上，我们将开发表达经工程处理的F.VII结构的AAV载体，该结构被切割到F.VIIa细胞内，并以激活形式分泌。在目标1中，我们将进行短期实验，以确定是否可以通过门静脉注射AAV-F.VIIa载体在血友病抑制剂小鼠模型中实现止血。在第二个目标中，我们将对在一系列确定水平上持续表达F.VIIa的小鼠的凝血参数进行长期研究。这些实验的目的是确定是否有任何基线水平的F.VIIa表达将导致凝血参数的改善而不会产生严重的不良反应。在这个目标的第二部分，我们将开发由药物多西环素激活的“开关”控制的AAV-VIIa载体，并确定这样的系统是否可以用于减少与长期表达VIIa相关的不良副作用，同时仍然用于预防止血挑战。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.VIIa作为分泌产品的修改的F.VII结构的免疫原性。这些临床前研究的成功完成应该有助于确定基于基因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.