GENE THERAPY FOR HEMOPHILIA B

B 型血友病的基因治疗

• 批准号: 2231783
• 负责人: KOTOKU KURACHI
• 金额: $ 30.12万
• 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/2231783
• 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.

（改编自申请人摘要）血友病B是常见的 X染色体连锁出血性疾病，这是由于缺乏 血浆中的生物活性凝血因子IX。 电流蛋白 血友病B的替代疗法仅对暂时性 缓解出血发作，但其重复应用需要 血友病B患者使他们暴露于几种严重的 并发症，特别是感染病原性病毒，如HIV-1， 肝炎病毒。 在此，我们建议建立一个坚实的实验基地 为血友病B提供安全、有效、实用的基因治疗， 原代成肌细胞介导的基因转移 构建了一套高度优化的新一代逆转录病毒载体 具有因子IX小基因的载体，以及可以在所述载体上表达因子IX的载体。 在培养的小鼠原代成肌细胞和肌管中， 鉴定 用逆转录病毒载体转导原代成肌细胞， 并植入同基因小鼠的骨骼肌中。 全身 分析重组因子IX的产生，并使其最大化以达到 治疗水平的重组因子IX（>5%的正常人血浆 通过单独优化和各种参数的组合， 以及涉及细胞分离和植入过程的条件。 任何 由于注射的成肌细胞对动物的有害作用，例如 致瘤性和对肌肉力量的有害影响， 经过长时间的观察和研究。 在广泛的 在小鼠中进行测试，然后简单地应用开发的优化方法 正常犬，然后进行血友病B的最终详尽试验 狗 在血友病狗的实验中， 构建了犬因子IX小基因类似于优化的 人因子IX逆转录病毒载体被用于转染骨骼肌成肌细胞 分离自血友病B犬。 然后将转导的细胞植入 回到同一动物的骨骼肌中，使我们能够评估 在完全同质的情况下， 系统至少几年。 最后，我们提出了优化的人 因子IX逆转录病毒进行了一系列广泛的安全性测试， 以及猴子，这些猴子需要证明它的最终用途， 在本研究的下一阶段进行人体应用。 获得的信息 从拟议的研究应该为我们提供了一个良好的实验基础 用于开发持久的离体基因治疗的临床方案， 血友病B。建立一个高效、安全、实用的基因 血友病B的治疗也应该为其开辟一条令人兴奋的途径 应用于许多其他疾病，不仅是各种血液和 需要有效的全身和局部递送的代谢紊乱 转基因产品，还有肌肉疾病。