Preclinical studies of gene therapy for Chronic Granulomatous Disease

慢性肉芽肿病（CGD）是一种遗传性免疫缺陷病，由以下四种phox基因突变引起，这些phox基因编码超氧化物生成吞噬细胞NADPH氧化酶的亚基。它包括由gp 9 lphox和p22 phox组成的膜细胞色素b558和四种胞质组分p47 phox、p67 phox、rac p21和p40 phox，它们在激活后移位到膜上。CGD患者的发病率估计为I/250，000例出生。根据Kaplan Meier分析，COD患者的预期寿命为25 - 30岁。与美国和欧洲的CGD亚型比例相比，p47 phox缺陷型CGD亚型比例较低（小于10%比23%），而gp 9 lphox缺陷型CGD亚型比例较高（大于75%比60%）。ST抗生素和IFN-γ的预防性给药和骨髓移植已成功地用于我们的治疗策略。但是，有必要 ...更多信息 本研究构建了三种逆转录病毒载体，分别为：MFGS-gp 91/293 SPA，其仅含有治疗性gp 91 phox基因，双顺反子逆转录病毒pHa-MDR-IRES-gp 91/PA 317，其携带多药耐药基因（MDR 1）和与内部核糖体进入位点（IRES）连接的gp 91 phox基因，和pHa-gp 91-IRES-Neo/PAMP 51，其携带下游（β）新霉素磷酸转移酶基因（Neo β r）代替farmer上游MDR 1基因。我们证明了gp 9 lphox向COD EB病毒建立的细胞系的高效转导，其中分别通过长春新碱或G418的适当选择，MFGS-gp 91和pHa-MDR-IRES-gp 91或pHa-gp 9 l-JRES-Neo对氧化酶进行了高水平的功能校正。MFGS-gp 91/293 SPA可有效地将gp 9 lphox转导至CD 34+造血干细胞。本研究表明，293-SPA或PAMP 5I包装系统与插入MDR 1基因的双顺反子逆转录病毒系统相结合，可使COD基因治疗在临床上更具有可行性。少

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