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疾病中的应用。在遗传性代谢缺陷的基因治疗中，大多数方案都是以骨髓细胞或纯化的骨髓干细胞为靶点。对先天性代谢缺陷患者应在确诊后尽快开始治疗。因此，外周血干细胞比骨髓干细胞更安全和可重复地从婴儿获得，是遗传代谢疾病患者基因治疗靶细胞的良好候选者。在本课题中，我们首先建立了从外周血和脐带血中分离纯化CD 34 ^+细胞的有效方法。然后，我们评估了逆转录病毒介导的NeoR基因在从G-CSF动员的幼儿外周血和脐带血中纯化的CD 34 ^+细胞中的转导和长期表达，并表明CB或动员的PB中的CD 34 ^+细胞是逆转录病毒基因转移方案治疗先天性代谢缺陷的合适和现实的靶细胞。此外，我们构建了含CAG启动子和葡萄糖脑苷脂酶cDNA的逆转录病毒载体，为戈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.