GENE THERAPY FOR HEMOPHILIA B

B 型血友病的基因治疗

• 批准号: 2231784
• 负责人: KOTOKU KURACHI
• 金额: $ 35.92万
• 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/2231784
• 关键词: Macaca mulatta; Retroviridae; SCID mouse; coagulation factor IX; dogs; gene therapy; genetic transduction; hemophilia As; muscle cells; recombinant proteins; technology /technique; tissue /cell culture; transfection /expression vector

(Adapted from applicant's abstract) Hemophilia B is a common X-chromosome-linked bleeding disorder which is due to a deficiency of biologically active blood coagulation factor IX in plasma. Current protein replacement therapy for hemophilia B is only effective to temporarily relieve bleeding episodes, but its repeated applications required by hemophilia B patients expose them to the risk of several serious complications, notably infection of pathogenic viruses such as HIV-1 and hepatitis virus. Here, we propose to establish a solid experimental base for a safe, effective, and practical gene therapy for hemophilia B based on primary myoblast-mediated gene transfer. A set of highly optimized new-generation retroviral vectors are constructed with a factor IX minigene, and a vector which can express factor IX at the highest level in mouse primary myoblasts and myotubes in culture is identified. Primary myoblast are transduced with the retroviral vector, and implanted into skeletal muscles of syngeneic mice. The systemic production of recombinant factor IX is analyzed, and maximized to reach a therapeutic level of recombinant factor IX (>5% the normal human plasma level) by optimizing individually and in combinations of various parameters and conditions involved in cell isolation and implantation procedures. Any deleterious effects to animals due to the injected myoblasts, such as tumorigenicity and detrimental effects on the muscle strength, are monitored and examined over a long period of time. After the extensive testing in mice, the optimized approach developed is then applied briefly to normal dogs, followed by the final, exhaustive testing with hemophilia B dogs. In the experiments with hemophilia dogs, a retroviral vector which is constructed with a canine factor IX minigene similar to the optimized human factor IX retroviral vector is used to transduce skeletal myoblasts isolated from hemophilia B dogs. The transduced cells are then implanted back into skeletal muscles of the same animals, permitting us to evaluate the effectiveness and safety to the procedure in a totally homogeneous system for at least several years. Finally, we submit the optimized human factor IX retrovirus to a series of extensive safety testings in culture as well as in monkeys which are required to certify it for its ultimate use in human applications in the next phase of this study. Information obtained from the proposed studies should provide us with a sound experimental base for developing a clinical protocol of durable ex vivo gene therapy for hemophilia B. Establishment of this effective, safe and practical gene therapy for hemophilia B should also open up an exciting avenue for its applications to many other diseases, not only various hematological and metabolic disorders which require an efficient systemic and local delivery of transgene products, but also muscular disorders.

（改编自申请人的摘要）血友病B是一种常见 X染色体相关的出血障碍，这是由于缺乏症 血浆中生物活性血液凝血因子IX。 当前蛋白质 血友病B的替代疗法仅对暂时有效 缓解出血情节，但其重复申请要求 血友病B患者暴露于几个严重的风险 并发症，特别是感染了HIV-1和HIV-1和 肝炎病毒。 在这里，我们建议建立一个可靠的实验基础 为基于血友病B的安全，有效和实用的基因治疗 原代肌细胞介导的基因转移。 构建了一组高度优化的新代逆转录病毒载体 使用一个因子IX Minigene和一个可以在 鼠标原代肌细胞和文化中的肌管中的最高水平是 确定。 原代肌细胞被逆转录病毒载体转导， 并植入合元小鼠的骨骼肌。 系统性 分析重组因子IX的产生，并最大化以达到 重组因子IX的治疗水平（> 5％正常人血浆 级别）通过单独优化各种参数的组合 以及涉及细胞分离和植入程序的条件。 任何 由于注射的肌细胞，对动物的有害影响，例如 肿瘤性和对肌肉强度的有害影响是 经过长时间的监视和检查。 大量之后 然后在小鼠中进行测试，然后简要采用优化的方法 对于普通狗，然后对血友病B进行最终的详尽测试 狗。 在使用血友病犬的实验中，逆转录病毒载体 用犬IX IX MINIGENE构建，类似于优化 人类因子IX逆转录病毒载体用于传递骨骼成肌细胞 从血友病B狗中分离出来。 然后将转导的细胞植入 回到同一动物的骨骼肌，允许我们评估 完全均匀的程序的有效性和安全性 系统至少几年。 最后，我们提交了优化的人 第IX因子逆转录病毒进行了一系列文化中广泛的安全测试 以及在猴子中证明其最终使用所需的猴子 在本研究的下一阶段的人类应用。 获得的信息 从拟议的研究中应为我们提供合理的实验基础 用于开发耐用的离体基因治疗的临床方案 血友病B.建立这种有效，安全和实用的基因 血友病B的治疗还应为其途径开辟一条令人兴奋的途径 对许多其他疾病的应用，不仅是各种血液学和 需要有效的全身和本地交付的代谢疾病 转基因产品，也是肌肉疾病。