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

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells

“…Previous work performed on lymphoblastoid cells from two other erythropoietic porphyrias, ie congenital erythropoietic porphyria and hepato-erythropoietic porphyria, demonstrated the feasibility of a fluorescence-based selection of genetically corrected cells. 25,26 We applied this system to the BM cells of the EPP mouse model. Because of the specific fluorescence of porphyrins, the development of an efficient strategy of selecting transduced cells depends on a clear difference in porphyrin accumulation between deficient and corrected cells.…”

“…Fluorescence-based selection of retrovirally transduced cells and BM transplantation Forty-eight hours after the infection of deficient BM cells, medium was replaced by ␣-MEM medium containing 7% FCS, 1 mmol/l ALA, 2 mmol/l melatonin 25 (protecting agent) and 50 mol/l FeSO 4 (Sigma, Saint-Quentin, France) at pH 7.4. Cell sorting experiments were performed 24 h after addition of ALA.…”

Cellular and Gene Therapy for Erythropoietic Porphyrias

“…Retrovirus-mediated expression of UROD was successfully used in lymphoblastoid cells from HEP patients. 89 Mixed-culture experiments demonstrated the absence of metabolic cross-correction of deficient cells by normal cells. However, cellular expansion in vitro and in vivo in immunodeficient mice suggested that genetically corrected cells have a competitive selective advantage.…”

“…70,102 Deficient HSCs were transduced with a bicistronic lentiviral vector expressing EGFP and FECH. 70 EGFP-positive or -negative cells were isolated by flow cytometry and transplanted into three groups of lethally irradiated EPP recipient mice.…”

“…Previous work performed on lymphoblastoid cells from HEP and CEP patients demonstrated the feasibility of a fluorescence-based selection of corrected and non-corrected cells. 89,91,102 Fontanellas et al 103 performed an ex vivo gene transfer of human FECH with a retroviral vector to deficient HSCs, followed by re-injection in EPP mouse of the transduced cells with or without fluorescence-based selection. As observed in the study by Pawliuk et al, preselection of corrected cells based on the disappearance of porphyrin-induced fluorescence greatly improved the metabolic correction efficiency and was associated with full correction of the photosensitivity.…”