Prolongation of Factor VIII Lifetime in Circulation

延长因子 VIII 在循环中的寿命

• 批准号: 6900877
• 负责人: EVGUENI L. SAENKO
• 金额: $ 34.93万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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/6900877
• 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) 是凝血内在途径的重要组成部分，其遗传缺陷会导致危及生命的出血性疾病 A 型血友病，可通过重复输注昂贵的重组和血浆衍生的 fVIII 产品来治疗。当前申请的目标是基于对 fVIII 分解代谢机制的突破性认识，开发具有延长循环寿命的重组 fVIII，以更有效地治疗 A 型血友病。我们已经确定，fVIII 从循环中的清除是由低密度脂蛋白受体相关蛋白 (LRP)（LDL 受体超家族的成员）介导的，并由细胞表面硫酸乙酰肝素蛋白聚糖 (HSPG) 促进。在小鼠模型中同时阻断 LRP 和 HSPG 导致 fVIII 半衰期显着延长 5.5 倍。我们已分别将主要的 LRP 和 HSPG 结合位点定位在 fVIII A2 亚基的残基 484-509 和 558-565 内。这些位点暴露在具有冯维勒布兰德因子 (vWf) 的 fVIII 复合物中，其中 fVIII 存在于循环中。我们建议在 LRP 和 HSPG 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 复合物的 fVIII 分解代谢的特性，进行全面的位点特异性诱变。发现 A2 突变的最佳组合可以最大程度地减少细胞模型中 LRP 和 HSPG 介导的分解代谢成分，而不影响重构的活化 fVIII 的功能活性和稳定性，接下来将被引入 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，其提供高 fVIII 表达水平，以及携带 B 结构域区域 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。我们还将研究在血友病 A 的 fVIII 缺陷小鼠模型中，重复使用突变型 fVIII 是否与免疫反应增强无关。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 在体外刺激重症患者外周血 T 淋巴细胞增殖的能力。 血友病A。当前项目的完成将导致产生具有延长的循环寿命的突变体fVIII，其将满足主要的功能、生化和免疫学标准。