NEW THERAPIES FOR HEMOPHILIA

本项目提案的主题是开发血友病的新疗法。特别是，重点将放在疾病的最重要的并发症，即通常用于治疗出血事件的凝血因子的抑制性抗体的发生。抑制性的发展是一个毁灭性的事件，这不可避免地导致发病率和治疗成本的增加（对于没有这种并发症的患者来说已经非常高了）。此外，抑制剂的开发仍然是围绕血友病基因治疗的主要问题之一。我们将采用多学科方法来解决这个问题，集中于生物工程凝血因子（具有增强的促凝血活性的因子VIIa的突变体，以及具有降低的免疫原性和抗原性的人-猪因子VIII杂合分子）。虽然不是该提案的一部分，但涉及这些蛋白质的临床试验可以作为一个长期目标进行实际预期。在项目1中（“涉及这些蛋白质的组织试验的作用可以作为一个长期目标来实际预期。在项目1（“组织因子在血友病中的作用”）中，我们将研究体外组织因子加密的生化基础，并开发测量体内组织因子加密和促凝形式表达的方法。在项目（“血友病中的增强型维生素K依赖性蛋白”）中，将对在膜接触区特定残基处突变的重组因子VII分子的促凝血活性进行表征。将在血友病动物模型中检测所选突变体的抗出血疗效（和潜在的不良血栓形成性）。在项目3（“对猪因子VIII的CD 4 + T细胞应答”）中，将主要在使用FVIII抑制剂的血友病A小鼠模型中检查对人-猪杂合FVIII分子的CD 4 + T细胞应答。我们假设这些杂合分子的免疫原性可能低于人FVIII。最后，在项目4（“因子IX和因子VII基因表达的嵌合体成形术”）中，我们将使用嵌合RNA/DNA构建体“修复”犬血友病因子IX基因中的点突变，并故意诱导因子VII基因中的选定点突变，以促进体内高活性FVII（a）的表达，从而“绕过”具有抑制剂的患者对因子VIII（或IX）的需求。

The theme of this Program Project proposal is to develop novel therapies for hemophilia. In particular, the focus will be on the most significant complication of the disease, namely the occurrence of inhibitory antibodies to the clotting factors that are normally used to treat bleeding episodes. The development of an inhibitory is a devastating event, which leads inevitably to increased morbidity, and cost of therapy (which is already exceptionally high for patients without this complication). Furthermore, development of inhibitors remains one of the principal concerns surrounding gene therapy for hemophilia. We will adopt a multi- disciplinary approach to this problem, concentrating on bio-engineered clotting factors (mutants of factor VIIa with enhanced pro-coagulant activity, and human-porcine factor VIII hybrid molecules with reduced immunogenicity and antigenicity. Although not part of this proposal, clinical trials involving these proteins can be realistically anticipated as a longer term goal. In Project 1 ("The Role of Tissue trials involving these proteins can be realistically anticipated as a longer term goal. In Project 1 ("The Role of Tissue Factor in Hemophilia"), we will examine the biochemical basis for tissue factor encryption in vitro, and develop methods to measure expression of both the encrypted and pro-coagulant forms of tissue factor in vivo. In Project ("Enhanced Vitamin K- dependent Proteins in Hemophilia"), recombinant factor VII molecules that have been mutated at specific residues in the membrane contact region will be characterized with respect to their pro-coagulant activity. The anti-hemorrhagic efficacy (and potential undesirable thrombogenicity) of selected mutants will be tested in animal models of hemophilia. In Project 3 ("CD4+ T Cell Response to Porcine Factor VIII"), CD4+ T cell responses to human-porcine hybrid FVIII molecules will be examined, primarily in the murine model of hemophilia A with a FVIII inhibitor. It is our hypotheses that these hybrid molecules may be less immunogenic than human FVIII. Finally, in Project 4 ("Chimeraplasty for Factor IX and Factor VII Gene Expression"), we will use chimeric RNA/DNA constructs to "repair" the point mutation in the factor IX gene in canine hemophilia, and deliberately induce a selected point mutation in the factor VII gene to promote expression of high activity FVII(a) in vivo that will "by-pass" the need for factor VIII (or IX) in patients with inhibitors.