Fluorescence-based selection of retrovirally transduced cells in congenital erythropoietic porphyria: direct selection based on the expression of the therapeutic gene.

Background: Congenital erythropoietic porphyria (CEP) is an inherited disease caused by a deficiency 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 efficient 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 modified cells. The presence of exogeneous delta-aminolevulinic acid (ALA), a haem precursor, induces an increase in porphyrin accumulation in LB deficient cells. Porphyrins exhibit a specific fluorescent emission and can be detected by cytofluorimetry under ultraviolet excitation.

Results: In genetically modified cells, the restored metabolic flow from ALA to haem led to a lesser accumulation of porphyrins in the cells, which were easily separated from the deficient cells by flow cytometry cell sorting.

Conclusion: This selection process represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.