RESUMO
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.
Assuntos
Técnicas de Transferência de Genes , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Lentivirus/genética , Glicoproteínas de Membrana/genética , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Genes Reporter , Vetores Genéticos , Granulócitos/citologia , Granulócitos/metabolismo , Doença Granulomatosa Crônica/enzimologia , Proteínas de Fluorescência Verde , HIV-1/genética , Humanos , Proteínas Luminescentes/genética , Mutação , NADPH Oxidase 2 , NADPH Oxidases/genética , Fenótipo , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.
