Abstract
To develop a gene therapy protocol suitable for the treatment of a benign disease such as Gaucher disease, we have developed two bicistronic vectors that allow transduced cells to be selected for with methotrexate (MTX). The two vectors differ in the presence or absence of a mutant polyoma enhancer (ΔMo+PyF101) replacing the wild-type retroviral enhancer in the LTR. Infection of human TF-1 and K562 cells, Gaucher type II fibroblasts and murine hemopoietic bone marrow cells conferred MTX resistance and glucocerebrosidase (GC) expression. Upon increasing MTX concentrations, the number of proviral copies and GC activity increased, demonstrating in vitro selection of retrovirus-transduced cells. At high MTX selection pressure, up to 140 μM for infected Gaucher type II fibro- blasts, no endogenous wild-type DHFR amplification could be detected, indicating that both retroviral constructs provide sufficient DHFR protein levels. Upon transduction, murine bone marrow cells were protected against otherwise lethal MTX concentrations (range 1–5 μM MTX). Flow cytometry specific for human GC (hGC) demonstrated that in vitro selection resulted in increased percentages of hGC-positive murine cells. In conclusion, the generated bicistronic vectors are ideally suited to investigate whether an in vivo selection approach for retrovirus-transduced cells is feasible. Such a strategy might abolish the need for a high initial transduction efficiency and might result in a gene therapy protocol devoid of the undesirable need for marrow ablative treatment of the recipient.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Havenga, M., Werner, A., Valerio, D. et al. Methotrexate selectable retroviral vectors for Gaucher disease. Gene Ther 5, 1379–1388 (1998). https://doi.org/10.1038/sj.gt.3300735
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3300735
Keywords
This article is cited by
-
Hematopoietic stem cell gene therapy with drug resistance genes: an update
Cancer Gene Therapy (2005)
-
Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells
Gene Therapy (2002)
-
In vivo methotrexate selection of murine hemopoietic cells transduced with a retroviral vector for Gaucher disease
Gene Therapy (1999)