Lentivirus-mediated gene transfer of gp91phox corrects chronic granulomatous disease (CGD) phenotype in human X-CGD cells

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Lentivirus vectors expressing gp91phox are able to at least partially correct human myeloid X-CGD cells.