Sokolic R A, Sekhsaria S, Sugimoto Y, Whiting-Theobald N, Linton G F, Li F, Gottesman M M, Malech H L
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Blood. 1996 Jan 1;87(1):42-50.
Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.
慢性肉芽肿病（CGD）是一种遗传性血液系统疾病，涉及吞噬细胞氧化酶无法产生超氧化物（O2-.），导致反复感染。逆转录病毒基因疗法治疗血液病的成功将受到靶细胞体外转导效率以及校正后的细胞在体内替代未校正细胞能力的限制。使用含有氧化酶基因的基于MFG的逆转录病毒载体，我们之前已在体外证明对CGD的校正，但转导率较低。在本研究中，我们探索了一种策略，为转导细胞提供选择性生长优势，同时保留MFG负责高病毒滴度和增强蛋白质产生的单一启动子特征。我们构建了一种双顺反子逆转录病毒，其产生单个mRNA，编码X连锁型CGD（X-CGD）的治疗基因gp91phox和可选择的人类多药耐药基因MDR1，它们通过脑心肌炎病毒内部核糖体进入位点（IRES）连接在一起。作为对照，我们构建了一个带有脊髓灰质炎病毒IRES元件并使用细菌新霉素抗性基因（neor）作为选择元件的双顺反子载体。在来自X-CGD患者的爱泼斯坦-巴尔病毒转化的B（EBV-B）细胞（CGD的组织培养模型）中，我们使用这些载体中的任何一种并进行适当选择（MDR1用长春新碱，neor用G418），显示出CGD缺陷的校正和细胞群体的完全正常化。使用O2-.产生的化学发光测定法，用任何一种载体转导的细胞群体显示出初始校正水平为正常EBV-B细胞氧化酶活性的不到0.1%至2.7%。使用任何一种构建体，在适当选择下的细胞生长富集了转导细胞群体，导致X-CGD EBV-B细胞校正到O2-.产生水平等于或超过正常EBV-B细胞。这些研究表明，治疗基因可以通过IRES元件与抗性基因连接，从而允许选择性富集表达治疗基因的细胞。此外，在我们的研究中使用MDR1作为选择元件验证了一种重要的基因治疗方法，该方法可以允许体内选择并且可推广到多种治疗环境。
