The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.
OBJECTIVE
As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production.


METHODS
To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test).


RESULTS
An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants.


CONCLUSIONS
A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.
Bellantuono
I
I
Department of Immunology, Imperial College School of Medicine, The Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. i.bellantuono@ic.ac.uk
Lashford
L S
LS
Rafferty
J A
JA
Fairbairn
L J
LJ
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D018014:Gene Transfer Techniques / Q000592:standards; D005822:Genetic Vectors / Q000737:chemistry; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D012367:RNA, Viral / Q000096:biosynthesis; D012190:Retroviridae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D014764:Viral Proteins / Q000032:analysis
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D012367:RNA, Viral; D014764:Viral Proteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases