NEW THERAPIES FOR HEMOPHILIA

本项目提案的主题是为血友病开发新的治疗方法。特别是，重点将放在该疾病最显著的并发症上，即对通常用于治疗出血发作的凝血因子的抑制性抗体的发生。抑制的发展是一个毁灭性的事件，它不可避免地导致发病率的增加和治疗费用的增加（对于没有这种并发症的患者来说，这已经非常高了）。此外，抑制剂的开发仍然是血友病基因治疗的主要关注点之一。我们将采用多学科的方法来解决这个问题，集中研究生物工程凝血因子（具有增强促凝活性的VIIa因子突变体）和免疫原性和抗原性降低的人-猪因子VIII杂交分子。虽然不是这个提议的一部分，但涉及这些蛋白质的临床试验可以作为一个长期目标来现实地预期。在项目1(“涉及这些蛋白质的组织试验的作用可以现实地预期为一个长期目标。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，并开发测量组织因子加密和促凝形式在体内表达的方法。在项目（“血友病中增强的维生素K依赖性蛋白”）中，重组因子VII分子在膜接触区域的特定残基上发生突变，将对其促凝活性进行表征。选择的突变体的抗出血功效（和潜在的不良血栓形成性）将在血友病动物模型中进行测试。在项目3（“CD4+ T细胞对猪因子VIII的反应”）中，CD4+ T细胞对人-猪杂交FVIII分子的反应将被检测，主要是在具有FVIII抑制剂的血友病A小鼠模型中。我们的假设是，这些杂交分子的免疫原性可能低于人类的FVIII。最后，在项目4（“因子IX和因子VII基因表达的嵌合成型术”）中，我们将使用嵌合RNA/DNA构建体“修复”犬血友病因子IX基因的点突变，并故意诱导因子VII基因的选定点突变，以促进体内高活性FVII(a)的表达，这将“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.