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

为提高造血干细胞基因治疗的效率，研制了高效转导/表达逆转录病毒载体和转导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灭活的MFG的包装和剪接信号进行拼接，构建了新的逆转录病毒载体MGK。2)针对转导的造血细胞的体内扩增，开发了一种新的选择性扩增基因系统。用含有他莫昔芬反应选择性扩增基因和EGFP标记基因的双顺反子逆转录病毒载体转导小鼠骨髓细胞，并将其移植到致死照射的宿主体内。在造血重建后，一部分受者接受三苯氧胺治疗。实验动物的EGFP表达细胞频率显著高于对照动物。结果表明，利用选择性扩增基因/三苯氧胺系统扩增了转导的造血干/祖细胞，转基因细胞体内扩增是可行的。我们邀请了美国印第安纳大学的M.C.Dinauer博士讨论他们关于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讨论了建立慢性肉芽肿疾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.