|
|
 Previous Article | Table of Contents | Next Article 
A bicistronic retrovirus vector containing a picornavirus internal ribosome
entry site allows for correction of X-linked CGD by selection for MDR1
expression
RA Sokolic, S Sekhsaria, Y Sugimoto, N Whiting-Theobald, GF Linton, F Li, MM Gottesman and HL Malech
Laboratory of Host Defenses, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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.
Volume 87,
Issue 1,
pp. 42-50,
01/01/1996
Copyright © 1996 by The American Society of Hematology
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. Junemann, Y. Song, G. Bassili, D. Goergen, J. Henke, and M. Niepmann
Picornavirus Internal Ribosome Entry Site Elements Can Stimulate Translation of Upstream Genes
J. Biol. Chem.,
January 5, 2007;
282(1):
132 - 141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Mutoh, J. Mitsuhashi, Y. Kimura, S. Tsukahara, E. Ishikawa, K. Sai, S. Ozawa, J.-i. Sawada, K. Ueda, K. Katayama, et al.
A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein.
Mol. Cancer Ther.,
April 1, 2006;
5(4):
877 - 884.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Licht, M. Haskins, P. Henthorn, S. E. Kleiman, D. M. Bodine, T. Whitwam, J. M. Puck, M. M. Gottesman, and J. R. Melniczek
Drug selection with paclitaxel restores expression of linked IL-2 receptor gamma -chain and multidrug resistance (MDR1) transgenes in canine bone marrow
PNAS,
February 20, 2002;
(2002)
52712199.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hibino, K. Tani, K. Ikebuchi, M.-S. Wu, H. Sugiyama, Y. Nakazaki, T. Tanabe, S. Takahashi, A. Tojo, S. Suzuki, et al.
The Common Marmoset as a Target Preclinical Primate Model for Cytokine and Gene Therapy Studies
Blood,
May 1, 1999;
93(9):
2839 - 2848.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. M. Weil, G. F. Linton, N. Whiting-Theobald, S. J. Vowells, S. P. Rafferty, F. Li, and H. L. Malech
Genetic Correction of p67phox Deficient Chronic Granulomatous Disease Using Peripheral Blood Progenitor Cells as a Target for Retrovirus Mediated Gene Transfer
Blood,
March 1, 1997;
89(5):
1754 - 1761.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Licht, M. Haskins, P. Henthorn, S. E. Kleiman, D. M. Bodine, T. Whitwam, J. M. Puck, M. M. Gottesman, and J. R. Melniczek
Drug selection with paclitaxel restores expression of linked IL-2 receptor gamma -chain and multidrug resistance (MDR1) transgenes in canine bone marrow
PNAS,
March 5, 2002;
99(5):
3123 - 3128.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
