Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene
✍ Scribed by Antonio Fontanellas; Frédéric Mazurier; Francis Belloc; Laurence Taine; Patrice Dumain; Carine Morel; Cécile Ged; Hubert de Verneuil; François Moreau-Gaudry
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 240 KB
- Volume
- 1
- Category
- Article
- ISSN
- 1099-498X
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✦ Synopsis
Background Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a de®ciency of uroporphyrinogen III synthase, the fourth enzyme of the haem biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor with death occurring in early adult life and available treatments are only symptomatic and unsatisfactory. In vitro gene transfer experiments have documented the feasibility of gene therapy via haematopoietic stem cells to treat this disease. To facilitate future ex vivo gene therapy in humans, the design of ef®cient selection procedures to increase the frequency of genetically corrected cells prior to autologous transplantation is a critical step.
Methods An alternative selection procedure based upon expression of a transferred gene was performed on a lymphoblastoid (LB) cell line from a patient with congenital erythropoietic porphyria to obtain high frequencies of genetically modi®ed cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB de®cient cells. Porphyrins exhibit a speci®c ¯uorescent emission and can be detected by cyto¯uorimetry under ultraviolet excitation.
Results
In genetically modi®ed cells, the restored metabolic ¯ow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the de®cient cells by ¯ow cytometry cell sorting.
Conclusion
This selection process represents a rapid and ef®cient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.