Establishment of the protocol of gene therapy for inborn errors of metabolism using purified peripheral blood stem cells

基因治疗技术的最新进展导致了基因治疗在遗传性代谢性疾病中的应用。在针对先天代谢疾病的基因治疗方案中，大多以骨髓细胞和纯化的骨髓干细胞为靶点。对先天代谢异常的患者在确诊后应尽快开始治疗。因此，与骨髓干细胞相比，外周血干细胞从婴儿体内更安全、更可重复地获得，是遗传性代谢性疾病患者基因治疗的良好靶细胞。在本项目中，我们首先建立了从外周血和脐带血中分离纯化CD34~+细胞的有效方法。然后，我们对逆转录病毒介导的NeoR基因在从儿童G-CSF动员的外周血和脐带血中纯化的CD34+细胞的转导和长期表达进行了评估，结果表明CD34+细胞在脐血或动员的外周血中是适合和现实的逆转录病毒基因转移方案中治疗先天性代谢疾病的靶细胞。此外，我们还构建了含CAG启动子和葡萄糖脑苷酶基因的逆转录病毒载体，为高谢病的有效基因治疗奠定了基础。

Recent progress in gene therapy techniques has led to an application of gene therapy to inherited metabolic diseases. In most protocols of gene therapy to inborn errors of metabolism, bone marrow cells and purified bone marrow stem cells are used as the targets. Therapy to patients with inborn errors of metabolism should be started as soon as possible after diagnosis. Therefore, peripheral blood stem cells, more safely and repeatably obtainable from infants than bone marrow stem cells, are a good candidate for the target cells for gene therapy in patients with inherited metabolic diseases. In this projectat, first we developed the effective protocol of purificetion of CD34^+ cells from peripheral and cord blood. Then, we evaluated retro-viral mediated transduction and long-term expression of NeoR gene in CD34_+ cells purified from G-CSF-mobilized peripheral blood of small children and cord blood, and showed that CD34^+ cells in CB or mobilized PB are suitable and realistic targets for retroviral gene transfer protocols for inborn errors of metabolism. Furthermore, we constructed the retro-viral vector containing CAG promoterand glucocerebrosidase cDNA for the effective gene therapy for Gaucher disease.