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

基因转移至造血干细胞和慢性肉芽肿病的基因治疗

基本信息

批准号：
11694309
负责人：
KUME Akihiro
金额：
$ 4.35万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

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.

为了提高造血干细胞基因治疗的效果，研制了一种高转导/表达效率的逆转录病毒载体和一种转导造血细胞的体内扩增系统。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的包装和剪接信号组装在一起，构建了一种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。用含有他莫昔芬响应性选择性放大基因和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，并移植到致死性照射的宿主中。造血重建后，接受者的一个子集与他莫昔芬挑战。与对照组动物相比，受攻击的动物显示出显著更高的EGFP表达细胞频率。结果表明，选择性扩增基因/他莫昔芬系统可扩增转导的造血干/祖细胞，基因修饰细胞的体内扩增是可行的，这一结果在美国基因治疗学会和美国血液学会年会上发表。我们邀请了M博士。C.来自美国印第安纳州大学的Dinauer，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讨论建立慢性肉芽肿病基因治疗的临床方案。