Saulnier S O, Steinhoff D, Dinauer M C, Zufferey R, Trono D, Seger R A, Hossle J P
Division of Immunology/Hematology, University Children's Hospital, Zurich, Switzerland.
J Gene Med. 2000 Sep-Oct;2(5):317-25. doi: 10.1002/1521-2254(200009/10)2:5<317::AID-JGM127>3.0.CO;2-P.
Chronic granulomatous diseases (CGD) are caused by impaired antimicrobial activity in phagocytes, due to the absence or malfunction of the respiratory burst NADPH oxidase. Two-thirds of the patients have mutations in their X-linked CGD gene encoding gp91phox, the largest subunit of the NADPH oxidase.
Aimed at gene therapy of X-CGD already at the level of resting pluripotent hematopoietic stem cells, we generated an advanced HIV-1-based vector with self-inactivating (SIN2) features containing the therapeutic gp91phox gene. In this vector an internal cytomegalovirus (CMV) promoter exclusively drives transgene expression. The green fluorescent protein (GFP) served as reporter for evaluation of gene transfer and expression in the human myeloid PLB985 X-CGD cell line.
The X-CGD cells were efficiently transduced by the VSV-G pseudotyped lentivirus constructs (up to 74% GFP+ cells at 3 days post-transduction). CMV-driven GFP-expression was stable for at least 3 weeks after transduction and persisted after granulocytic differentiation of the target cells. Using the lentivector with the gp91phox transgene, 26% and 48% of the X-CGD cells expressed gp91phox at Days 2 and 20 after co-culture with 293T producer cells, respectively. Upon granulocytic differentiation of the transduced X-CGD cells with dimethylformamide (DMF), up to 63% (mean 49%, n = 7) of the cells were found to be functionally reconstituted with mean levels of superoxide production of 31% (n = 7) compared to wild-type PLB985 cells.
Lentivirus vectors expressing gp91phox are able to at least partially correct human myeloid X-CGD cells.
慢性肉芽肿病（CGD）是由吞噬细胞中抗菌活性受损引起的，这是由于呼吸爆发NADPH氧化酶缺失或功能异常所致。三分之二的患者其编码gp91phox（NADPH氧化酶最大亚基）的X连锁CGD基因发生突变。
为了在静止的多能造血干细胞水平上对X-CGD进行基因治疗，我们构建了一种具有自我失活（SIN2）特征的基于HIV-1的先进载体，其中包含治疗性gp91phox基因。在该载体中，一个内部巨细胞病毒（CMV）启动子专门驱动转基因表达。绿色荧光蛋白（GFP）用作报告基因，用于评估人髓系PLB985 X-CGD细胞系中的基因转移和表达。
VSV-G假型慢病毒构建体可有效转导X-CGD细胞（转导后3天高达74%的GFP+细胞）。CMV驱动的GFP表达在转导后至少3周内稳定，并在靶细胞粒细胞分化后持续存在。使用携带gp91phox转基因的慢病毒载体，与293T生产细胞共培养后第2天和第20天，分别有26%和48%的X-CGD细胞表达gp91phox。在用二甲基甲酰胺（DMF）诱导转导的X-CGD细胞进行粒细胞分化后，发现高达63%（平均49%，n = 7）的细胞功能得到重建，与野生型PLB985细胞相比，超氧化物产生的平均水平为31%（n = 7）。
表达gp91phox的慢病毒载体能够至少部分纠正人髓系X-CGD细胞。
