Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.
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PMID:A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. 854 75

The coordinate expression of a marker gene and therapeutic genes in a single vector is markedly advantageous. The internal ribosome entry site (IRES) from encephalomyocarditis and that from polio viruses were used to construct a polycistronic retroviral vector pGCEN/TRI, where Neo-resistant gene was used as a marker gene and TNF-alpha cDNA, IL-2 cDNA as genes of interest, so that the LTR promoter derived the expression of a tricistronic mRNA. Amphotropic packaging cells PA317 transfected with pGCEN/TRI using LipofectAMINE was selected with G418 and the positive clones expressing the genes of interest to produce high-titer retrovirus (5x10(5) CFU/ml) were obtained. PCR confirmed the presence of proviral DNA in the positive producer cells and Northern blot analysis revealed a single, LTR promoted transcript. The transduced cells expressed TNF-alpha and IL-2 at different levels. This demonstrated that polycistronic retroviral vector containing IRES could efficiently express multiple therapeutic genes in the same target cell.
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PMID:The Construction and Expression of Polycistronic Retroviral Vector Carrying Genes of TNF-alpha, IL-2 and NeoR. 1217 76