CONGENITAL ERYTHROPOIETIC PORPHYRIA - PROLONGED HIGH-LEVEL EXPRESSIONAND CORRECTION OF THE HEME BIOSYNTHETIC DEFECT BY RETROVIRAL-MEDIATEDGENE-TRANSFER INTO PORPHYRIC AND ERYTHROID-CELLS

Congenital erythropoietic porphyria (CEP) is an autosomal recessive di sorder resulting from the deficient activity of the heme biosynthetic enzyme uroporphyrinogen III synthase (UROS). Severely affected patient s are transfusion dependent and have mutilating cutaneous manifestatio ns. Successful bone marrow transplantation has proven curative, provid ing the rationale for stem cell gene therapy. Toward this goal, two re troviral MFG vectors containing the UROS cDNA were constructed, one wi th the wild-type sequence (MFG-UROS-wt) and a second with an optimized Kozak consensus sequence (MFG-UROS-K). Following transduction of CEP fibroblasts, the MFG-UROS-wt and MFG-UROS-K vectors increased the endo genous activity without selection to levels that were 18- and 5-fold g reater, respectively, than the mean activity in normal fibroblasts. No tably, the MFG-UROS-wt vector expressed UROS activity in CEP fibroblas ts at these high levels for over 6 months without cell toxicity. Addit ion of either delta-aminolevulinic acid (ALA) or ferric chloride did n ot affect expression of the transduced UROS gene nor did the increased concentrations of uroporphyrin isomers or porphyrin intermediates aff ect cell viability. Similarly, transduction of CEP lymphoblasts with t he MFG-UROS-wt vector without G418 selection increased the endogenous UROS activity by 7-fold or almost a-fold greater than that in normal l ymphoblasts. Transduction of K562 erythroleukemia cells by cocultivati on with the MFG-UROS-wt producer cells increased their high endogenous UROS activity by 1.6-fold without selection. Clonally isolated K562 c ells expressed UROS for over 4 months at mean levels 4.7-fold greater than the endogenous activity without cell toxicity. Thus, the prolonge d, high-level expression of UROS in transduced CEP fibroblasts and lym phoblasts, as well as in transduced K562 erythroid cells, demonstrated that the enzymatic defect in CEP cells could be corrected by retrovir al-mediated gene therapy without selection and that the increased intr acellular porphyrin intermediates were not toxic to these cells, even when porphyrin production was stimulated by supplemental ALA or iron. These in vitro studies provide the rationale for ex vivo stem cell gen e therapy in severely affected patients with CEP. (C) 1998 Academic Pr ess.