Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery.

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88-200 fold in human AIP fibroblasts and synthesis of PP was increased from 21-152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3-7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.