Prolongation of Factor VIII Lifetime in Circulation

描述(申请人提供)：凝血因子VIII(FVIII)的遗传缺陷是凝血的内在途径的重要组成部分，会导致危及生命的出血性疾病血友病A，治疗方法是反复输注昂贵的重组和血浆衍生的FVIII产品。目前的应用目标是基于对FVIII分解代谢机制的突破性知识，开发具有更长循环寿命的重组FVIII，以更有效地治疗血友病A。我们已经证实，低密度脂蛋白受体相关蛋白(LRP)是由低密度脂蛋白受体超家族成员之一的低密度脂蛋白受体相关蛋白(LRP)介导的，并受细胞表面硫酸乙酰肝素蛋白多糖(HSPGs)的促进。在小鼠模型中同时阻断LRP和HSPG，导致FVIII半衰期显著延长5.5倍。我们已经将主要的LRP和HSPG结合位点分别定位在FVIII A2亚基的484-509和558-565残基内。这些部位暴露在FVIII与von Willebrand因子(VWF)的复合体中，其中FVIII在循环中存在。我们建议在LRP和HSPGs结合位点及其附近找到一个最佳的突变组合，这将大大减少FVIII清除的相应成分，并且不会影响FVIII的功能特性。我们将使用分离的A2亚基作为FVIII的模型，基于A2与VWF的复合体的分解代谢与FVIII的同一性，进行全面的定点突变。一种A2突变的最佳组合(S)被发现可以最大限度地减少LRP和HSPGs介导的细胞模型中分解代谢的成分，而不会影响重组激活的FVIII的功能活性和稳定性，接下来将被引入FVIII结构。我们计划使用编码B结构域缺失的FVIII的构建体，它提供高水平的FVIII表达，以及携带B结构域区域741-956的FVIII构建体，这是细胞高效分泌FVIII所必需的。我们将应用各种方法来评估产生的突变体FVIII维持对其正常功能至关重要的相互作用的能力，包括与VWF形成复合体的能力，与Xase复合体成分的相互作用，以及正常的激活/失活动力学。我们还将研究在FVIII缺陷的血友病A小鼠模型中重复使用突变FVIII是否与增强免疫反应无关。为了获得在血友病A患者中使用突变FVIII的预后，我们将比较突变和野生型FVIII刺激严重血友病A患者外周血T淋巴细胞体外增殖的能力。完成目前的项目将导致产生在循环中寿命更长的突变FVIII，这将满足主要的功能、生化和免疫学标准。

DESCRIPTION (provided by applicant): Genetic deficiency in factor VIII (fVIII), an essential component of the intrinsic pathway of blood coagulation, results in a life-threatening bleeding disorder Haemophilia A, which is treated by repeated infusions of expensive recombinant and plasma-derived fVIII products. The goal of the current application is to develop recombinant fVIII with a prolonged lifetime in circulation for more efficient therapy of Haemophilia A based on the breakthrough knowledge on the mechanisms of fVIII catabolism. We have established that fVIII clearance from circulation is mediated by low-density lipoprotein receptor-related protein (LRP), a member of LDL receptor superfamily, and facilitated by cell surface heparan sulfate proteoglycans (HSPGs). Simultaneous blocking of LRP and HSPGs in a mouse model, led to a significant, 5.5-fold prolongation of fVIII half-life. We have localized the major LRP- and HSPGs- binding sites within residues 484-509 and 558-565, respectively, of the A2 subunit of fVIII. These sites are exposed within fVIII complex with von Willebrand factor (vWf), in which fVIII is present in circulation. We propose to find an optimal combination of mutations within LRP- and HSPGs-binding sites and in their proximity, which would substantially reduce the corresponding components of fVIII clearance and will not affect the functional properties of fVIII. We will perform comprehensive site-specific mutagenesis using the isolated A2 subunit as a model of fVIII based on the identity of catabolism of A2 to that of fVIII from it complex with vWf. An optimal combination(s) of A2 mutations found to maximally reduce LRP- and HSPGs-mediated components of catabolism in a cell model without affecting the functional activity and stability of reconstituted activated fVIII, will be next introduced into fVIII constructs. We plan to use a construct encoding B domain-deleted fVIII, which provides high fVIII expression levels, and a fVIII construct carrying the B domain region 741-956, which is required for efficient fVIII secretion from the cell. We will apply a variety of methods to assess the ability of generated mutant fVIII to maintain interactions critical for its normal functioning, including ability for complex formation with vWf, interaction with components of the Xase complex, and normal activation/inactivation kinetics. We will also examine whether repeated use of mutant fVIII is not associated with increased immune response in fVIII-deficient mouse model of Haemophilia A. To obtain prognosis of the use of mutant fVIII in Haemophilia A patients, we will compare the ability of mutant and wild-type fVIII to stimulate in vitro proliferation of peripheral blood T lymphocytes from patients with severe Haemophilia A. Accomplishment of the current project will result in generation of mutant fVIII with prolonged lifetime in circulation, which will meet major functional, biochemical and immunological criteria.