title: Lentivirus-mediated gene transfer of gp91phox corrects chronic granulomatous disease (CGD) phenotype in human X-CGD cells.
abstract: 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.
  
  
  CONCLUSIONS
  Lentivirus vectors expressing gp91phox are able to at least partially correct human myeloid X-CGD cells.
authors.affiliation: Division of Immunology/Hematology, University Children's Hospital, Zurich, Switzerland.
authors.forename: S O
authors.initials: SO
authors.lastname: Saulnier
authors.forename: D
authors.initials: D
authors.lastname: Steinhoff
authors.forename: M C
authors.initials: MC
authors.lastname: Dinauer
authors.forename: R
authors.initials: R
authors.lastname: Zufferey
authors.forename: D
authors.initials: D
authors.lastname: Trono
authors.forename: R A
authors.initials: RA
authors.lastname: Seger
authors.forename: J P
authors.initials: JP
authors.lastname: Hossle
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics* / Q000628:therapy*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics; D010641:Phenotype; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases