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.