DEVELOPMENT OF GENE THERAPIES FOR HEMOPHILIA

血友病B是一种X染色体连锁的出血性疾病，原因是 凝血因子IX缺乏。当前的蛋白质替代物 治疗对暂时缓解出血发作是有效的，但 血友病B患者需要反复使用的药物使他们暴露在 几种严重并发症的风险，特别是病原体感染 HIV-1和肝炎病毒等病毒。在这项提案中，我们试图 开发一种有效且安全的治疗方法 基于利用骨骼的体细胞基因治疗的血友病B 成肌细胞作为基因输送载体的高效系统输送 重组人第IX因子。复制缺陷型重组逆转录病毒 高效表达重组人因子的载体 将构建小鼠骨骼肌成肌细胞的Ix。小鼠初级 然后用优化的逆转录病毒载体(S)转导成肌细胞 注射到同基因裸鼠骨骼肌中。生产和销售 具有生物活性的重组第IX因子在小鼠体内的系统递送 动物将通过确定最优组合 可变条件包括成肌细胞的纯度、细胞总数 注射对象、注射部位、每只动物的注射次数、 刺激肌纤维再生。转导的成肌细胞 注射到小鼠骨骼肌中的能力将被分析 以产生高水平的重组因子IX进入系统 发行量。注射的成肌细胞对 动物，如致瘤性和对肌肉的有害影响 力量，如果它发生了，也将在很长一段时间内被广泛检查 一段时间。在小鼠身上进行了广泛的测试和优化后，EX 活体方法将在正常狗身上进行测试，为最后一次测试做准备 在血友病B犬上的应用。正常骨骼肌成肌细胞的分离 用在小鼠身上优化的逆转录病毒载体转导狗，并 测试其表达重组第IX因子的全部能力 体外培养。然后，转导的犬成肌细胞将被注射回 相同的狗，用于进一步优化细胞所涉及的条件 调职。体外方法，在小鼠和 正常的狗，最终将应用于无价的血友病B犬。通过 使用含有犬类的优化逆转录病毒载体，而不是 人凝血因子IX的cDNA，其长期有效性和安全性将是 在一个完全相同的系统中展示。所获得的信息 这些研究将为我们提供坚实的实验基础 制定有效、安全、持久的体细胞移植临床方案 血友病B的细胞基因治疗。

Hemophilia B is an X chromosome-linked bleeding disorder due to a deficiency of blood coagulation factor IX. Current protein replacement therapy is effective to temporarily relieve bleeding episodes, but the 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. In this proposal, we attempt to develop an alternative approach of an effective and safe treatment for hemophilia B based upon somatic cell gene therapy which utilizes skeletal myoblasts as a gene delivery vehicle for an efficient systemic delivery of recombinant factor IX. Replication-deficient recombinant retroviral vectors which can give high-level expressions of recombinant human factor IX in mouse skeletal myoblasts will be constructed. Mouse primary myoblasts transduced with an optimized retroviral vector(s) will then be injected into skeletal muscles of syngenic nude mice. Production and systemic delivery of biologically-active recombinant factor IX in the animals will be maximized by determining the optimal combination of variable conditions including purity of myoblasts, the total cell number to be injected, sites for injection, number of injections for each animal, and stimulation of myofiber regeneration. The transduced myoblasts injected into mouse skeletal muscles will be analyzed for their capability to produce a high-level recombinant factor IX into the systemic circulation. Any deleterious effect of the injected myoblasts to the animals, such as tumorigenicity and detrimental effects on the muscle strength, if it occurs, will also be extensively examined for a long period of time. After extensive testing and optimization in mice, the ex vivo approach will be tested on normal dogs preparing for its final application on hemophilia B dogs. Skeletal myoblasts isolated from normal dogs are transduced with the retroviral vector optimized in mice and tested for their full capability to express recombinant factor IX in vitro. The transduced dog myoblasts will then be injected back into the same dogs for further optimization of conditions involved in the cell transfer. The ex vivo approach, well tested and optimized in mice and normal dogs, will finally be applied to invaluable hemophilia B dogs. By employing an optimized retroviral vector containing a canine, instead of human, factor IX cDNA, its long-term effectiveness and safety will be demonstrated in a totally homogeneous system. The information obtained from these studies will provide us with a solid experimental base for developing an effective, safe and durable clinical protocol of somatic cell gene therapy for hemophilia B.