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

SCID的基因治疗是纠正免疫缺陷的有效方法。然而，在X-SCID基因治疗的临床试验中，报告了一个严重的不良事件，即由于逆转录病毒载体插入突变而导致的白血病。因此，我们开展了旨在开发更安全的基因治疗方法的研究。主要研究结果如下：1。为了抵消副作用，我们已经将自杀基因整合到治疗性逆转录病毒载体中，以选择性地消除转导细胞。用携带人γc链基因和HSVtk基因的双顺反子逆转录病毒载体转导X-SCID患者的细胞。在确认γc链功能重建后，用更昔洛韦(GCV)处理细胞。γc链阳性细胞在低浓度下被清除，对未转导的细胞无细胞毒性，至少6个月内不再出现。γc链转导细胞在6个月后仍对环丙沙星敏感。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区域。我们发现人类染色体18p11.2和13q14.1在造血细胞中有两个较好的整合位点。未观察到已知原癌基因的整合或接近。我们继续使用日本和韩国各地的血液样本进行SCID的分子诊断。我们已经鉴定了γc链、JAK3、IL7Rα、RAG1和Artemis编码基因的突变。备注：日本政府批准的X-SCID基因治疗临床试验的初步实验是用患者的骨髓细胞进行的。然而，由于有报道称基因疗法发生了严重的不良事件，临床试验被推迟了。

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.