Preclinical studies of gene therapy for Chronic Granulomatous Disease

慢性肉芽肿病基因治疗的临床前研究

基本信息

批准号：
09470178
负责人：
NUNOI Hiroyuki
金额：
$ 3.46万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Chronic granulomatous disease (CGD) is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp9lphox and p22phox, and four cytosolic components, p47phox, p67phox, rac p21 and p40phox, which translocate to the membrane upon activation.In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as I out of 250,000 births. The expected life span of the COD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10% vs. 23%) and that of gp9lphox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to de … More velop the gene therapy technology for CGD patients as more promising treatment.In the current study we constructed three retrovirus vectors ; MFGS-gp91/293 SPA which contains only the therapeutic gp91phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp9lphox gene connected with an internal ribosome entry site (IRES), and a pHa-gp9l-IRES-Neo/PAMP51 which carries downstream ('3) neomycin phosphotransferase gene (Neo^r) in place of the farmer upstream MDR1 gene. We demonstrate high efficiency transduction of gp9lphox to COD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp9l or pHa-gp9l-JRES-Neo by an appropriate selection with vincristine or G418, respectively. We also demonstrate sufficient transduction of gp9lphox to CD34+heamatopoietic stem cell from the patients with gp9lphox deficiency by MFGS-gp9l/293 SPA.Our current studies suggest that the combination of the 293-SPA or PAMP5I packaging system and the bicistronic retrovirus system inserted MDR1 gene make our COD gene therapy more feasible for clinical application. Less

慢性肉芽肿病（CGD）是由编码产生超氧化物的吞噬细胞NADPH氧化酶亚基的以下四种phox基因中的任何一种突变引起的遗传性免疫缺陷，它由gp9lphox和p22phox组成的膜细胞色素b558和四种胞质成分p47phox组成。、 p67phox、rac p21 和 p40phox，它们易位到膜上在我们的小组研究中，已招募了超过 220 名 CGD 患者。根据 Kaplan Meier 分析，CGD 患者的发病率估计为 25 万名新生儿中的 COD 患者的预期寿命为 25 至 30 岁。与美国和欧洲CGD亚型的比例相比，p47phox缺陷型的比例较低（小于10% vs. 23%），gp9lphox的比例较低缺陷率更高（超过 75% vs. 60%）。我们的治疗策略已成功采用了 ST 抗生素和 IFN-γ 的预防性给药和骨髓移植。但是，有必要开发基因治疗技术。在当前的研究中，我们构建了三种逆转录病毒载体 MFGS-gp91/293 SPA，其中仅包含治疗性 gp91phox 基因，一种双顺反子；逆转录病毒 pHa-MDR-IRES-gp91/PA317 携带多重耐药基因 (MDR1) 和与内部核糖体进入位点 (IRES) 连接的 gp9lphox 基因，以及 pHa-gp9l-IRES-Neo/PAMP51 携带下游 ( '3) 新霉素磷酸转移酶基因 (Neo^r) 代替农民上游 MDR1 基因，我们证明了高效转导。 gp9lphox 至 COD EB 病毒建立的细胞系，通过 MFGS-gp91 和 pHa-MDR-IRES-gp9l 或 pHa-gp9l-JRES-Neo 分别用长春新碱或 G418 进行适当选择，对氧化酶进行高水平的功能校正。证明 gp9lphox 能够充分转导来自 gp9lphox 缺陷患者的 CD34+造血干细胞MFGS-gp9l/293 SPA。我们目前的研究表明，293-SPA或PAMP5I包装系统与插入MDR1基因的双顺反子逆转录病毒系统相结合，使我们的COD基因治疗更有可能应用于临床。