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Methotrexate selectable retroviral vectors for Gaucher disease
M J Havenga1, A B Werner, D Valerio
1Department of Molecular Cell Biology, Leiden University Medical Centre, The Netherlands.
Gene Therapy
|February 4, 1999
Summary
New gene therapy vectors for Gaucher disease enable selection of modified cells using methotrexate (MTX). This approach shows promise for in vivo selection, potentially eliminating the need for harsh pre-treatment.
Area of Science:
- Gene Therapy
- Retroviral Vectors
- Gaucher Disease
Background:
- Gaucher disease is a benign genetic disorder requiring effective gene therapy.
- Current gene therapy protocols may necessitate marrow ablative treatments.
- Developing selectable gene therapy vectors is crucial for improved treatment strategies.
Purpose of the Study:
- To develop bicistronic retroviral vectors for gene therapy of Gaucher disease.
- To enable methotrexate (MTX)-based selection of transduced cells.
- To assess the feasibility of in vivo selection for gene therapy.
Main Methods:
- Two bicistronic vectors were engineered, differing in retroviral enhancer elements.
- Vectors were used to transduce human cell lines, Gaucher fibroblasts, and murine bone marrow cells.
- Methotrexate (MTX) selection was applied to assess resistance and glucocerebrosidase (GC) expression.
Main Results:
- Transduced cells exhibited MTX resistance and expressed human glucocerebrosidase (hGC).
- Increasing MTX concentrations led to higher proviral copy numbers and GC activity, demonstrating in vitro selection.
- Murine bone marrow cells were protected from lethal MTX doses, and flow cytometry confirmed increased hGC-positive cells.
Conclusions:
- The developed bicistronic vectors facilitate MTX-based selection of gene-modified cells.
- This vector system is suitable for investigating in vivo selection strategies.
- In vivo selection could potentially reduce the need for high initial transduction efficiency and marrow ablation.