A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression

Robert A. Sokolic, Sudhir Sekhsaria, Yoshikazu Sugimoto, Narda Whiting-Theobald, Gilda F. Linton, Fei Li, Michael M. Gottesman, Harry L. Malech

Research output: Contribution to journalArticle

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Abstract

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O 2/ - ·), 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-COD), gp91(phox), 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 (neo(r)) 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 neo(r)). Using a chemiluminescence assay of O 2/ -· 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 O 2/ -· 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.

Original languageEnglish
Pages (from-to)42-50
Number of pages9
JournalBlood
Volume87
Issue number1
Publication statusPublished - 1996 Jan 1
Externally publishedYes

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Picornaviridae
Chronic Granulomatous Disease
Retroviridae
Viruses
Genes
Cells
Human Herpesvirus 4
B-Lymphocytes
Gene therapy
Oxidoreductases
Neomycin
Genetic Therapy
Tissue culture
Encephalomyocarditis virus
Population
MDR Genes
Chemiluminescence
Internal Ribosome Entry Sites
Cell growth
Vincristine

ASJC Scopus subject areas

  • Hematology

Cite this

Sokolic, R. A., Sekhsaria, S., Sugimoto, Y., Whiting-Theobald, N., Linton, G. F., Li, F., ... Malech, H. L. (1996). A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. Blood, 87(1), 42-50.

A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. / Sokolic, Robert A.; Sekhsaria, Sudhir; Sugimoto, Yoshikazu; Whiting-Theobald, Narda; Linton, Gilda F.; Li, Fei; Gottesman, Michael M.; Malech, Harry L.

In: Blood, Vol. 87, No. 1, 01.01.1996, p. 42-50.

Research output: Contribution to journalArticle

Sokolic, RA, Sekhsaria, S, Sugimoto, Y, Whiting-Theobald, N, Linton, GF, Li, F, Gottesman, MM & Malech, HL 1996, 'A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression', Blood, vol. 87, no. 1, pp. 42-50.
Sokolic, Robert A. ; Sekhsaria, Sudhir ; Sugimoto, Yoshikazu ; Whiting-Theobald, Narda ; Linton, Gilda F. ; Li, Fei ; Gottesman, Michael M. ; Malech, Harry L. / A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. In: Blood. 1996 ; Vol. 87, No. 1. pp. 42-50.
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abstract = "Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O 2/ - ·), 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-COD), gp91(phox), 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 (neo(r)) 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 neo(r)). Using a chemiluminescence assay of O 2/ -· 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 O 2/ -· 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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