Gene transfer into hematopoietic stem cells and gene therapy for chronic granulomatous disease

为了提高造血干细胞基因治疗的疗效，我们开发了一种具有高转导/表达效率的逆转录病毒载体和转导后的造血细胞体内扩增系统。1)将MSCV的长末端重复序列和引物tRNA结合位点与gag-kill MFG的包装和剪接信号组装在一起，构建新的逆转录病毒载体（MGK）。2)为了在体内扩增转导的造血细胞，开发了一种名为“选择性放大基因”的新系统。用含有他莫昔芬反应性选择性放大基因和EGFP标记基因的双链逆转录病毒载体转导小鼠骨髓细胞，并将其移植到致命辐射的宿主中。造血重建后，一部分受体接受他莫昔芬治疗。激毒动物的egfp表达频率明显高于对照动物。结果表明，经选择性扩增基因/他莫昔芬系统扩增的造血干细胞/祖细胞在体内扩增是可行的。这些结果在美国基因治疗学会和美国血液学学会的年会上发表。我们邀请了美国印第安纳大学的m.c. Dinauer博士来讨论他们对x连锁慢性肉芽肿病的临床基因转移试验。我们还与东京、长崎和熊本的合作者进行了讨论，建立了慢性肉芽肿病基因治疗的临床方案。

In order to improve the efficacy of hematopoietic stem cell gene therapy, a retroviral vector with high transduction/expression efficiency and an in vivo expansion system of transduced hematopoietic cells were developed. 1) A new retroviral vector (MGK) was constructed, by assembling the long terminal repeats and the primer tRNA binding site from MSCV and the packaging and splicing signals of gag-killed MFG. 2) For in vivo expansion of transduced hematopoietic cells, a novel system named 'selective amplifier genes' was developed. Murine bone marrow cells were transduced with a bicistronic retroviral vector containing a tamoxifen-responsive selective amplifier gene and the EGFP marker gene, and transplanted to lethally irradiated hosts. After hematopoietic reconstitution, a subset of the recipients were challenged with tamoxifen. The challenged animals showed significantly higher frequency of EGFP-expressing cells than the control animals. The results indicated that the transduced hematopoietic stem/progenitor cells were expanded by the selective amplifier gene/tamoxifen system, and in vivo expansion of the gene-modified cells is feasible.These results were presented at the annual meetings of American Society of Gene Therapy and the American Society of Hematology. We invited Dr. M. C. Dinauer from Indiana University, U.S.A., to discuss about their clinical gene transfer trial for X-linked chronic granulomatous disease. We also discussed with collaborators in Tokyo, Nagasaki and Kumamoto to set up a clinical protocol of gene therapy for chronic granulomatous disease.