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

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，并分析了慢性肉芽肿病患者的基因。从1994年开始，我参与了逆转录病毒载体（Ha-MDR-IRES-gp91/PA317， Ha-MDR-IRES-p67/PA317和Ha-MDR-IRES-p47/PA317）对患者基因治疗的开发。我们的逆转录病毒在临床应用中提出了以下问题：1)感兴趣蛋白的低表达和短表达，2)逆转录病毒的低病毒滴度，3)转录失活等。合作开发的mggs -gp91/293/SPA存在转录失活等问题。基因导入后蛋白表达效率低，表达不稳定可能与病毒启动子甲基化失活有关。在这些经验的基础上，我们采用了对所开发的病毒启动子区甲基化耐受的MND反转录病毒载体。我们还使用了高滴度逆转录病毒产生细胞PAMP51细胞来代替PA317细胞。用MND-gp-91质粒转染PAMP51细胞，克隆出多个高滴度逆转录病毒产生细胞。在本次克隆过程中，由于该载体没有选择标记，我们只能使用7D5和gp91-phox单克隆抗体进行FACS分析。在200多个MND-gp-91/PAM51克隆中，病毒滴度最高为1 2 × 10^5/ml，仅为MND-gp91/PA317的2-3倍。该滴度比mggs -gp91/293/SPA载体（1 ~ 2x10 ^7/ml）低50 ~ 100倍。MND-gp91/PAM51逆转录病毒转导到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。不可能证实甲基化对MND载体失活的耐受作用。除了这项基因治疗研究外，我们还报道了另外三名受影响患者中观察到IFN-γ依赖性中性粒细胞超氧化物产生增加的亲属。gp91-phox的CYBB基因缺陷被鉴定为与CYBB基因的第三个内含子相邻的一个沉默突变，该突变改变了mRNA剪接。通过分子分析，我们发现在给予INF-γ后1至25天，患者中性粒细胞中CYBB基因转录物的剪接模式存在显著差异。此外，在处理后的所有标本中都可以检测到包含缺失外显子的完整转录本。转录物剪接模式的改变和对患者中性粒细胞产生超氧化物能力的长期影响表明，INF-γ诱导髓系祖细胞中CYBB基因转录物异常剪接的部分纠正。少

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