Basic Study for Gene Therapy for the Patients with Chronic Granulomatous Disease -Construction of MND-gp91/PAM51-

慢性肉芽肿病基因治疗基础研究-MND-gp91/PAM51的构建-

基本信息

批准号：
11670768
负责人：
NUNOI Hiroyuki
金额：
$ 2.37万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Chronic granulomatous disease(CGD) is an inherited disorder of host defense against microbial infections caused by defective activity of the phagocyte NADPH oxidase. I had purified p47- and p67-phox protein and cloned the gene of this enzyme complex and analyzed the genes of patients with chronic granulomatous disease. Since 1994, I have involved in the development of retrovirus vector (Ha-MDR-IRES-gp91/PA317, Ha-MDR-IRES-p67/PA317 and Ha-MDR-IRES-p47/PA317) for gene therapy to the patients. The following problems in the clinical application have been raised in our retrovirus ; 1)low and short expression of the interested protein, 2)low virus titer of the retrovirus, 3)inactivaton of transcription etc. MFGS-gp91/293/SPA that had been developed in the cooperative work had a problem about inactivation of transcription etc. These low protein expression efficiency after gene introduction and unstable expression might be caused by the inactivation of the methylation of the virus promoter. B … More y these experience, we employed MND retorvirus vector which is tolerate for methylation of the virus promotor area developed. We also employed PAMP51 cell reported as a high titer retrovirus producer cell in stead of PA317 producer cell. MND-gp-91 plasmid was transfected in PAMP51 cell and several high titer retrovirus producer cell were cloned. In this cloning procedure, we could only use FACS analysis by 7D5 and gp91-phox monoclonal antibody because this vector does not have the selection marker. In more than 200 MND-gp-91/PAM51 clones, the highest virus titer was 1 2 X 10^5/ml which was just 2-3 times higher than MND-gp91/PA317. This titer was 50〜100 times lower than MFGS-gp91/293/SPA vector(1-2X10^7/ml). MND-gp91/PAM51 retrovirus recover superoxide generating activity only 4 5% of normal after transduction to the gp91-phox deficient patient B cell. It was not possible to confirm the tolerate effect on MND vector against inactivation by methylation.Apart from this gene therapy study, we reported additional kindred in whom an IFN-γ-dependent increase in neutrophil superoxide production was observed in three affected patients. The defect in the CYBB gene for gp91-phox was identified as an otherwise silent mutation adjacent to the third intron of CYBB gene that alters mRNA splicing. By molecular analysis, we found significant differences in the splicing pattern of CYBB gene transcripts in patient neutrophils between 1 and 25 days after administration of INF-γ. Furthermore, a complete transcript containing the missing exons could be detected in all specimens after the treatment. The changes in the splicing pattern of the transcripts and the prolonged effect on superoxide generating ability of patient neutrophils indicate that INF-γ induced a partial correction of the abnormal splicing of CYBB gene transcripts in myeloid progenitor cells. Less

慢性肉芽肿病（CGD）是一种由吞噬细胞NADPH氧化酶活性缺陷引起的宿主防御微生物感染的遗传性疾病。我纯化了p47-和p67-phox蛋白，克隆了该酶复合物的基因并分析了患者的基因。患有慢性肉芽肿病，自1994年以来，我参与了逆转录病毒载体的开发。（Ha-MDR-IRES-gp91/PA317、Ha-MDR-IRES-p67/PA317 和 Ha-MDR-IRES-p47/PA317）对患者进行基因治疗时，我们在临床应用中提出了以下问题。逆转录病毒；1）目的蛋白表达低且短；2）逆转录病毒滴度低；3）转录失活等。合作开发的MFGS-gp91/293/SPA存在转录失活等问题。基因导入后蛋白表达效率低、表达不稳定可能是由于病毒启动子B的甲基化失活造成的。 ……根据这些经验，我们采用了 MND 逆转录病毒载体，该载体能够耐受所开发的病毒启动子区域的甲基化，我们还采用了据报道为高滴度逆转录病毒生产细胞的 PAMP51 细胞。 PA317生产细胞。将MND-gp-91质粒转染到PAMP51细胞中，并克隆了几个高滴度逆转录病毒生产细胞。在该克隆过程中，我们只能使用7D5和gp91-phox单克隆抗体进行FACS分析，因为该载体没有。在 200 多个 MND-gp-91/PAM51 克隆中，最高病毒滴度为 1 2 X。 10^5/ml，仅比 MND-gp91/PA317 高 2-3 倍，该滴度比 MFGS-gp91/293/SPA 载体（1-2X10^7/ml）低 50-100 倍。 /PAM51逆转录病毒在转导至gp91-phox缺陷患者B细胞后，仅恢复正常的4±5%的超氧化物生成活性。不可能确认 MND 载体对甲基化失活的耐受作用。除了这项基因治疗研究之外，我们还报告了其他亲属，在三名受影响的患者中观察到 IFN-γ 依赖性中性粒细胞超氧化物产生的增加。 CYBB 基因中的 gp91-phox 被鉴定为邻近 CYBB 基因第三个内含子的沉默突变，它改变了 mRNA 剪接。给予INF-γ后1至25天患者中性粒细胞中的CYBB基因转录本此外，在治疗后的所有样本中都可以检测到包含缺失外显子的完整转录本，以及转录本剪接模式的变化和长期效应。对患者中性粒细胞超氧化物生成能力的影响表明，INF-γ 诱导了骨髓祖细胞中 CYBB 基因转录物异常剪接的部分纠正。