Prolongation of Factor VIII Lifetime in Circulation

延长因子 VIII 在循环中的寿命

• 批准号: 6597896
• 负责人: EVGUENI L. SAENKO
• 金额: $ 34.7万
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6597896
• 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（由申请人提供）：因子VIII （fVIII）的遗传缺乏是血液凝固内在途径的重要组成部分，导致危及生命的出血性疾病血友病a，通过反复输注昂贵的重组和血浆来源的fVIII产品来治疗。当前应用的目标是基于fVIII分解代谢机制的突破性知识，开发具有延长循环寿命的重组fVIII，以更有效地治疗血友病a。我们已经确定fVIII从循环中清除是由低密度脂蛋白受体相关蛋白（LRP）介导的，LRP是低密度脂蛋白受体超家族的一员，并由细胞表面硫酸肝素蛋白聚糖（HSPGs）促进。在小鼠模型中，LRP和HSPGs同时阻断，导致fVIII半衰期显著延长5.5倍。我们在fVIII A2亚基的484-509和558-565残基中分别定位了LRP-和HSPGs-的主要结合位点。这些部位暴露于与血管性血友病因子（vWf）的fVIII复合体内，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。我们将利用分离的A2亚基作为fVIII的模型，基于A2与vWf复合物分解代谢的一致性，进行全面的位点特异性突变。A2突变的最佳组合被发现可以最大限度地减少细胞模型中LRP和hspgs介导的分解代谢成分，而不会影响重组激活fVIII的功能活性和稳定性，接下来将引入fVIII构建。我们计划使用编码B结构域缺失的fVIII的构建体，它提供高fVIII表达水平，以及携带B结构域741-956的fVIII构建体，这是细胞有效分泌fVIII所必需的。我们将应用多种方法来评估产生的突变体fVIII维持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，以及正常的激活/失活动力学。我们还将研究在A型血友病小鼠模型中反复使用突变型fVIII是否与免疫反应增加无关。我们将比较突变型和野生型fVIII对重度a型血友病患者外周血T淋巴细胞体外增殖的刺激能力。本项目的完成将产生循环寿命延长的突变型fVIII，满足主要的功能、生化和免疫学标准。