Correction of uroporphyrinogen decarboxylase deficiency (hepatoerythropoietic porphyria) in Epstein-Barr virus-transformed B-cell lines by retrovirus-mediated gene transfer: fluorescence-based selection of transduced cells.
Hepatoerythropoietic porphyria (HEP) is an inherited metabolic disorder characterized by the accumulation of porphyrins resulting from a deficiency in uroporphyrinogen decarboxylase (UROD). This autosomal recessive disorder is severe, starting early in infancy with no specific treatment. Gene therapy would represent a great therapeutic improvement. Because hematopoietic cells are the target for somatic gene therapy in this porphyria, Epstein-Barr virus-transformed B-cell lines from patients with HEP provide a model system for the disease. Thus, retrovirus-mediated expression of UROD was used to restore enzymatic activity in B-cell lines from 3 HEP patients. The potential of gene therapy for the metabolic correction of the disease was demonstrated by a reduction of porphyrin accumulation to the normal level in deficient transduced cells. Mixed culture experiments demonstrated that there is no metabolic cross-correction of deficient cells by normal cells. However, the observation of cellular expansion in vitro and in vivo in immunodeficient mice suggested that genetically corrected cells have a competitive advantage. Finally, to facilitate future human gene therapy trials, we have developed a selection system based on the expression of the therapeutic gene. Genetically corrected cells are easily separated from deficient ones by the absence of fluorescence when illuminated under UV light.
Fontanellas
A
A
Laboratoire de Pathologie Moléculaire et Thérapie Génique, FR 60 Biologie des Greffes, Université Victor Segalen Bordeaux 2, Bordeaux, France.
Mazurier
F
F
Moreau-Gaudry
F
F
Belloc
F
F
Ged
C
C
de Verneuil
H
H
D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology* / Q000637:transplantation; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D018920:Coculture Techniques; D005434:Flow Cytometry; D015316:Genetic Therapy; D004854:Herpesvirus 4, Human; D006801:Humans; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence; D017121:Porphyria, Hepatoerythropoietic / Q000201:enzymology* / Q000235:genetics / Q000628:therapy; D012641:Selection, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D014575:Uroporphyrinogen Decarboxylase / Q000172:deficiency* / Q000235:genetics
D014575:Uroporphyrinogen Decarboxylase