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-

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670768/
• 关键词: Chronic Granulomatous Disease; gp91-phox; gene therapy; MND-gp91; PAMP51; PA317; MFGS-gp91; 293; SPA; INF-γ; PAMP51 レトルウイルス・ベクター; PA317 レトルウイルス・ベクター; SPA レトルウイルス・ベクター; 慢性肉芽腫症遺伝子治療; MIND-gp91ベクター; Ha-MDR-IRES-gp91ベクター; 239・SPAベクター; 病型分類

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抵抗微生物感染的遗传性疾病。我已经纯化了p47-和p67-phox蛋白，克隆了这种酶复合物的基因，并分析了慢性肉芽肿病患者的基因。自1994年以来，我参与了逆转录病毒载体（Ha-MDR-IRES-gp 91/PA 317，Ha-MDR-IRES-p67/PA 317和Ha-MDR-IRES-p47/PA 317）的开发，用于患者的基因治疗。在我国逆转录病毒的临床应用中提出了以下问题; 1）目的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。MFGS-gp 91/293/合作开发的SPA存在转录失活等问题，这些基因导入后蛋白质表达效率低、表达不稳定的问题，由病毒启动子甲基化失活引起。B ...更多信息 根据这些经验，我们使用了对所开发的病毒启动子区域的甲基化具有耐受性的MND病毒载体。我们还采用了PAMP 51细胞作为高滴度逆转录病毒生产细胞，而不是PA 317生产细胞。将MND-gp-91质粒转染PAMP 51细胞，获得了几株高滴度的逆转录病毒产生细胞。在此克隆过程中，我们只能使用7 D5和gp 91-phox单克隆抗体的FACS分析，因为该载体没有选择标记。在200多个MND-gp-91/PAM 51克隆中，最高病毒滴度为1.2 × 10^5/ml，仅为MND-gp 91/PA 317的2-3倍。该滴度比MFGS-gp 91/293/SPA载体（1- 2 × 107/ml）低50 × 100倍。MND-gp 91/PAM 51逆转录病毒转染gp 91-phox缺陷型患者B细胞后，超氧化物歧化酶活性仅恢复正常的4 5%。目前还不能证实MND载体对甲基化失活的耐受作用，除了这项基因治疗研究外，我们还报道了另外3例受影响的患者，在这些患者中观察到IFN-γ依赖性中性粒细胞超氧化物生成增加。CYBB基因中gp 91-phox的缺陷被鉴定为邻近CYBB基因第三内含子的沉默突变，其改变mRNA剪接。通过分子分析，我们发现在INF-γ给药后1 - 25天，患者中性粒细胞中CYBB基因转录本的剪接模式存在显著差异。此外，在处理后的所有标本中可以检测到含有缺失外显子的完整转录本。转录本剪接模式的变化和对患者中性粒细胞超氧化物生成能力的长期影响表明，INF-γ诱导了髓系祖细胞中CYBB基因转录本异常剪接的部分纠正。少

项目成果

Ishibashi F.et al.: "Improved superoxide generating ability by interferon-gamma due to splicing pattern change of transcripts in neutrophils from patients with a splice site mutation in CYBB gene."Blood. (In press). (2001)

Ishibashi F.等人：“由于CYBB基因剪接位点突变患者的中性粒细胞转录本的剪接模式改变，干扰素-γ提高了超氧化物生成能力。”血液。

H.Koga: "Tetratricopeptide Repeat (TPR) Motifs of p67phox Participate in Interaction with the Small GTPase Rac and Activation of the Phagocyte NADPH Oxidase."J.Biol.Chem.. 274. 25051-25060 (1999)

H.Koga：“p67phox 的四肽重复 (TPR) 基序参与与小 GTPase Rac 的相互作用以及吞噬细胞 NADPH 氧化酶的激活。”J.Biol.Chem.. 274. 25051-25060 (1999)

Tsuchiya T.: "Uncompetitive inhibition of superoxide generation by a synthetic peptide corresponding to a predicted NADPH binding site in gp91 phox, a component of the phagocyte respiratory oxidase."Biochemi.Biophys.Res.Comm.. 257・1. 124-128 (1999)

Tsuchiya T.：“与吞噬细胞呼吸氧化酶的一个组成部分 gp91 phox 中预测的 NADPH 结合位点相对应的合成肽对超氧化物生成的非竞争性抑制。”Biochemi.Biophys.Res.Comm.. 257・1。 (1999)

H.Nunoi: "A heterozygous mutation of -actin associated with neutrophil dysfunction and recurrent infection"Proc.Natl.Acad.Sci.. 96. 8693-8698 (1999)

H.Nunoi：“β-肌动蛋白杂合突变与中性粒细胞功能障碍和复发性感染相关”Proc.Natl.Acad.Sci.. 96. 8693-8698 (1999)

H.Nunoi, et al: "A heterozygous mutation of -actin associated with neutrophil dysfunction and recurrent infection"Proc.Natl.Acad.Sci.. 96. 8693-8698 (1999)

H.Nunoi 等人：“与中性粒细胞功能障碍和复发性感染相关的 α-肌动蛋白杂合突变”Proc.Natl.Acad.Sci.. 96. 8693-8698 (1999)