ADENOVIRUS--MEDIATED DELIVERY OF NOVEL FACTOR VIII

腺病毒——介导的新型因子 VIII 的传递

• 批准号: 2231879
• 负责人: RANDAL J. KAUFMAN
• 金额: $ 25.43万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2231879
• 关键词: Adenoviridae; coagulation factor VIII; disease /disorder model; dogs; endoplasmic reticulum; gene expression; gene therapy; genetic regulation; genetically modified animals; hemostasis; laboratory mouse; liver cells; protein engineering; tissue /cell culture; von Willebrand factor

Factor VIII is the plasma protein that is functionally deficient in the clinical bleeding disorder hemophilia A. Patients require frequent infusions of factor VIII in order to maintain hemostasis. Factor VIII is synthesized in the hepatocyte and is secreted into the circulation where it is stabilized by non-covalent interactions with von Willebrand factor (vWF). Isolation of the factor VIII gene identified its domain structure of Al-A2-B-A3-Cl-C2 and enabled production of recombinant-derived factor VIII. Recombinant-derived factor VIII has minimized potential therapeutic complications associated with plasma-derived factor VIII, although significant limitations remain. Thus, the ultimate treatment for Hemophilia A would be a genetic cure for the disease. However, important questions need to be answered before gene therapy for hemophilia A becomes a reality. The proposed studies focus on three fundamental aspects that regulate factor VIII expression and activity. First, factor VIII expression in cultured mammalian cells is limited due to both inefficient rnRNA expression and protein secretion. The newly synthesized factor VIII is retained in the lumen of the endoplasmic reticulum (ER) in a complex with the immunoglobulin binding protein BiP and is inefficiently secreted. Recent results indicate that the limited factor VIII mRNA accumulation is coupled with factor VIII protein synthesis. The proposed studies will develop a hepatocyte-targeted adenoviral-based factor VIII gene delivery system to elucidate if similar limitations in factor VIll expression occur in vivo. Second, in the circulation, the ratio of vWF to factor VIII is tightly maintained at 50:1, although the mechanism responsible is not understood. We will identify the molecular mechanisms by which vWF regulates factor VIII levels using vWF-'knock-out' and transgenic mice. Finally, factor VIII activity is regulated by proteolytic activation and inactivation. The inactivation of factor VIII in vitro results from dissociation of the A2-domain peptide. Porcine factor VIll has increased specific activity and reduced A2-domain dissociation compared to human factor VIII. We have engineered a novel porcine/human hybrid factor VIII that has increased in vitro procoagulant activity. We propose to evaluate the hemostatic efficacy of this molecule in a hemophiliac dog model. The proposed studies will provide important insight into mechanisms that regulate fact6r VIII expression and activity in vitro and in vivo and the information obtained will be essential in considering avenues for ene therapy for hemophilia A.

因子VIII是血浆蛋白，在功能上缺乏 临床出血障碍血友病A.患者需要频繁 因子VIII的输注以维持止血。因子VIII是 在肝细胞中合成，分泌到循环中 通过与von Willebrand因子的非共价相互作用稳定 （vwf）。 VIII因子基因的隔离确定了其域结构 Al-A2-B-A3-CL-C2，并能够产生重组衍生的因子VIII。 重组衍生的因子VIII最大程度地减少了潜在的治疗性 与血浆衍生因子VIII相关的并发症，尽管 仍然存在重大限制。因此，血友病的最终治疗 A将是该疾病的遗传治疗方法。但是，重要的问题 在基因治疗的血友病A成为现实之前，需要回答。 拟议的研究集中于调节的三个基本方面 因子VIII表达和活性。首先，因子VIII表达 培养的哺乳动物细胞由于两个效率低下而受到限制 表达和蛋白质分泌。新合成的因子VIII是 保留在内质网（ER）的管腔中 免疫球蛋白结合蛋白BIP且效率低下。 最近的结果表明，有限因子VIII mRNA积累是 结合VIII蛋白合成因子。拟议的研究将 开发基于肝细胞的腺病毒因子VIII基因递送 阐明因子Vill表达中是否存在类似限制的系统 体内。其次，在循环中，VWF与因子VIII的比率为 尽管不是负责机制，但紧密保持在50：1 理解。我们将确定VWF的分子机制 使用vwf-'诺克输出和转基因小鼠来调节因子VIII水平。 最后，因子VIII活性受蛋白水解激活和 失活。 VIII因子在体外的失活是由 A2核苷肽的解离。猪因素Vill已增加 与人相比 因子VIII。我们设计了一种新型的猪/人类杂种VIII 这增加了体外突出凝胶活性。我们建议评估 该分子在血友病模型中的止血疗效。这 拟议的研究将提供对机制的重要见解 调节FACT6R VIII表达和体外和体内活性 获得的信息对于考虑ENE的途径至关重要 血友病A的治疗。