GENE THERAPY FOR HEMOPHILIA B

(摘自申请人的摘要)血友病B是一种常见的 X染色体连锁出血性疾病是由于缺乏 血浆中具有生物活性的凝血因子IX。当前蛋白质 血友病B的替代疗法仅对暂时性有效 止血，但它的重复使用需要 血友病B患者使他们面临几个严重的风险 并发症，特别是感染致病病毒，如艾滋病毒-1和 肝炎病毒。在这里，我们建议建立一个坚实的实验基地 为安全、有效、实用的血友病B基因治疗奠定基础 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 有一个因子IX微型基因，和一个可以表达因子IX在 小鼠原代成肌细胞和肌管的最高水平是 已确认身份。原代成肌细胞被逆转录病毒载体转导， 并植入同基因小鼠的骨骼肌中。系统性的 对重组因子IX的生产进行了分析，并将其最大化以达到 重组人凝血因子IX(&gt；5%)的治疗水平 水平)通过单独和各种参数的组合进行优化 以及细胞分离和植入过程中所涉及的条件。任何 注射成肌细胞对动物的有害影响，如 致瘤性和对肌肉力量的有害影响，是 经过长时间的监测和检查。在经历了广泛的 在小鼠身上进行了测试，然后简单地应用了所开发的优化方法。 给正常的狗，然后对血友病B进行最后的、详尽的测试 狗。在血友病狗的实验中，一种逆转录病毒载体 是用犬类因子IX迷你基因构建的，类似于优化的 人凝血因子IX逆转录病毒载体转导骨骼肌成肌细胞 从血友病B犬身上分离出来的。然后将被转导的细胞植入 回到相同动物的骨骼肌中，使我们能够评估 在完全同质的情况下，程序的有效性和安全性 系统至少运行了几年。最后，我们提交了优化后的人 因子IX逆转录病毒在培养中进行了一系列广泛的安全性测试 在猴子身上也是如此，猴子需要证明它的最终用途 人类在这项研究的下一阶段的应用。获取的信息 应该为我们提供一个可靠的实验基础 开发一种持久的体外基因治疗的临床方案 血友病B.建立有效、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道路 应用于许多其他疾病，不仅是各种血液学和 需要有效的全身性和局部性分娩的代谢紊乱 转基因产品，但也有肌肉疾病。

(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.