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Development of new and safer gene therapy method for severe combined immunodeficiency.

开发新的、更安全的基因治疗方法治疗严重联合免疫缺陷。

基本信息

批准号：
15390320
负责人：
KUMAKI Satoru
金额：
$ 7.81万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15390320/
关键词：
primary immunodeficiency severe combined immunodeficiency X-SCID γc chain gene therapy suicide gene 原発生免疫不全症

项目摘要

Gene therapy of SCID was efficient in correcting immunodeficiency. However, a severe adverse event, leukemia due to insertional mutagenesis by the retroviral vector, was reported in the X-SCID gene therapy clinical trial. Therefore, we performed studies aiming at developing safer gene therapy method. The main results are listed below.1. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. Cells from X-SCID patients were transduced with bicistronic retroviral vector carrying human γc chain cDNA and HSVtk gene. After confirmation of functional reconstitution of the γc chain, the cells were treated with ganciclovir (GCV). The γc chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells, and have not reappeared at least for 6 months. Furthermore, the γc chain transduced cells were still sensitive to GCV after six months. These results demonstrated the efficacy of the suicide gene therapy.2. To minimize insertional mutagenesis, vector integration was targeted towards neutral DNA regions using phage φC31 integrase system. We demonstrate two preferable integration sites in human chromosomes 18p11.2 and 13q14.1 in hematopoietic cells. No integration in or close to known proto-oncogenes was observed.3. We continue to perform molecular diagnosis of SCID using blood samples from all over Japan and South Korea. We have identified mutations in the genes encoding γc chain, Jak3, IL7Rα, RAG1 and Artemis.Remarks : Preliminary experiment of "Gene therapy Clinical Trial of X-SCID" which had been approved by the Japanese government, was performed using a patient's bone marrow cells. However, the clinical trial has been postponed due to a report of the severe adverse event by the gene therapy.

SCID基因治疗对纠正免疫缺陷是有效的。然而，在X-SCID基因治疗临床试验中报道了一个严重的不良事件，即由逆转录病毒载体插入突变引起的白血病。因此，我们开展了旨在开发更安全的基因治疗方法的研究。主要结果如下：1。为了抵消副作用，我们将自杀基因植入治疗性逆转录病毒载体中，以选择性消除转导细胞。用携带人γ - c链cDNA和HSVtk基因的双链逆转录病毒载体转染X-SCID患者细胞。在确认γ - c链的功能重构后，用更昔洛韦（GCV）处理细胞。γc链阳性细胞在低浓度下被清除，对未转导细胞无细胞毒性，至少6个月未出现。此外，γ - c链转导的细胞在6个月后仍对GCV敏感。这些结果证明了自杀基因治疗的有效性。为了减少插入突变，利用噬菌体φC31整合酶系统将载体整合到中性DNA区域。我们在造血细胞中发现了人类染色体18p11.2和13q14.1中两个较好的整合位点。未观察到整合或接近已知的原癌基因。我们继续使用来自日本和韩国各地的血液样本进行SCID的分子诊断。我们在编码γc链、Jak3、IL7Rα、RAG1和Artemis的基因中发现了突变。备注：已获日本政府批准的“X-SCID基因治疗临床试验”的初步实验使用患者的骨髓细胞。然而，由于基因治疗的严重不良事件的报道，临床试验已被推迟。