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Lentivirus‐mediated gene transfer of gp91 phox corrects chronic granulomatous disease (CGD) phenotype in human X‐CGD cells
Author(s) -
Saulnier Sandra O.,
Steinhoff Dirk,
Dinauer Mary C.,
Zufferey Romain,
Trono Didier,
Seger Reinhard A.,
Hossle Johann P.
Publication year - 2000
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/1521-2254(200009/10)2:5<317::aid-jgm127>3.0.co;2-p
Subject(s) - chronic granulomatous disease , genetic enhancement , transduction (biophysics) , viral vector , biology , microbiology and biotechnology , transgene , nadph oxidase , haematopoiesis , lentivirus , green fluorescent protein , cell culture , stem cell , gene , virology , immunology , virus , genetics , recombinant dna , reactive oxygen species , biochemistry , viral disease
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 gp91 phox , 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 gp91 phox 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 gp91 phox transgene, 26% and 48% of the X‐CGD cells expressed gp91 phox 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 gp91 phox are able to at least partially correct human myeloid X‐CGD cells. Copyright © 2000 John Wiley & Sons, Ltd.