Prolongation of Factor VIII Lifetime in Circulation

延长因子 VIII 在循环中的寿命

• 批准号: 6742426
• 负责人: EVGUENI L. SAENKO
• 金额: $ 4.23万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742426
• 关键词: binding sites; biotechnology; cell proliferation; chemical stability; clinical research; coagulation factor VIII; genetically modified animals; hemophilia As; heparan sulfate; human subject; laboratory mouse; low density lipoprotein receptor; patient oriented research; protein engineering; protein metabolism; protein protein interaction; protein structure function; recombinant proteins; site directed mutagenesis; tissue /cell culture; von Willebrand factor

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.

描述（由申请方提供）：凝血因子VIII（fVIII）（凝血的内源性途径的重要组分）的遗传缺陷导致危及生命的出血性疾病血友病A，通过重复输注昂贵的重组和血浆衍生的fVIII产品进行治疗。本申请的目的是基于对fVIII催化剂机制的突破性认识，开发在循环中具有延长寿命的重组fVIII，以更有效地治疗血友病A。我们已经确定，凝血因子VIII从循环中的清除是介导的低密度脂蛋白受体相关蛋白（LRP），LDL受体超家族的成员，并促进细胞表面硫酸乙酰肝素蛋白聚糖（HSPGs）。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HSPGs-结合位点分别定位在FVIII的A2亚基的残基484-509和558-565内。这些位点暴露于具有血管性血友病因子（vWf）的FVIII复合物内，其中FVIII存在于循环中。我们建议在LRP和HSPGs结合位点及其附近找到突变的最佳组合，这将大大减少FVIII清除的相应组分，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的催化剂与来自其与vWf复合物的fVIII的催化剂的同一性，进行全面的位点特异性诱变。A2突变的最佳组合被发现最大限度地减少细胞模型中LRP和HSPG介导的catalysts组分，而不影响重构的活化fVIII的功能活性和稳定性，接下来将其引入fVIII构建体中。我们计划使用编码B结构域缺失的fVIII的构建体和携带B结构域区域741-956的fVIII构建体，所述结构域缺失的fVIII提供高的fVIII表达水平，所述结构域区域741-956是从细胞有效分泌fVIII所需的。我们将采用多种方法评估生成的突变体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形成复合物的能力、与Xase复合物组分的相互作用以及正常的活化/失活动力学。我们还将研究在血友病A的fVIII缺陷小鼠模型中重复使用突变体fVIII是否与免疫应答增加无关。为了获得在血友病A患者中使用突变型fVIII的预后，我们将比较突变型和野生型fVIII刺激重度血友病A患者外周血T淋巴细胞体外增殖的能力。本项目的完成将导致产生在循环中具有延长寿命的突变体FVIII，其将满足主要功能、生化和免疫学标准。