Correction of the enzyme defect in cultured congenital erythropoietic porphyria disease cells by retrovirus-mediated gene transfer.

Congenital erythropoietic porphyria (CEP) is a genetic disease characterized by an overproduction and accumulation of porphyrins in bone marrow. The enzyme defect concerns uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the heme biosynthetic pathway. It is the most severe porphyria and the treatment is largely symptomatic: gene therapy would represent a great therapeutic improvement. As a step toward the development of an effective gene therapy, we have constructed two retroviral vectors, LUSN and pMFG-US (with and without the selectable marker Neo), containing a full-length human cDNA for UROIIIS. Recombinant retroviruses were obtained by transfection of the LUSN or pMFG-US plasmid into the amphotropic packaging cell line psi CRIP. For each construct, three different producing clones were selected for their high titer (LUSN) or for their ability to express the message at a high level (pMFG-US). In vitro amplification of genomic DNA from target tissue demonstrated the presence of vector sequences. Murine fibroblasts infected in vitro expressed the human enzyme efficiently, as indicated by RNA and enzymatic studies. Retroviral-mediated gene transfer was then used to introduce the UROIIIS cDNA into human deficient cells. Enzyme activity was increased from 2% (deficient fibroblasts) to 121-274% of the normal value for the different clones. Transduced cells selected with G418 presented an 18-fold increase in enzyme activity compared to the normal cells. Furthermore, high gene transfer rate into peripheral blood progenitor cells (PBPB) was documented by in vitro amplification (PCR). These results demonstrate the potential usefulness of somatic gene therapy for the treatment of CEP.