Ferrochelatase cDNA Delivered by Adenoviral Vector Corrects Biochemical Defect in Protoporphyric Cells

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Abstract
Protoporphyria is generally an autosomal dominant disease characterized genetically by mutations in the ferrochelatase gene. The interaction between the wild-type and mutant ferrochelatase protein is unknown. The aim of this study was to evaluate the ability to correct the enzymatic and biochemical defects in cells from patients with protoporphyria, using a replication-defective human adenovirus for gene transfer. Over-expression of ferrochelatase was accomplished by construction of a vector in which expression of the wild-type ferrochelatase cDNA was driven by the constitutive cytomegalovirus (CMV) promoter, introduction and packaging of the cDNA into human adenovirus dl309, and transduction of normal and protoporphyric fibroblasts. Fibroblasts from controls and patients were infected with the ferrochelatase adenovirus or a control adenovirus and assayed for ferrochelatase activity and the accumulation of protoporphyrin upon challenge with the precursor δ-aminolevulinic acid (ALA). At a multiplicity of infection (moi) of 10, greater than 85% of both the wild-type and protoporphyric fibroblasts were infected. The recombinant adenovirus increased the ferrochelatase protein content and activity in the wild-type and protoporphyric fibroblasts with equal efficiency. Therefore, the presence of the mutant ferrochelatase protein did not inhibit the ferrochelatase activity expressed by the transgene. This study demonstrates that a recombinant adenovirus can be used to transfer human ferrochelatase cDNA with greater than 95% efficiency into normal and protoporphyric fibroblasts in culture. Over-expression of the ferrochelatase protein corrects the enzymatic and biochemical defect of the disease protoporphyria, and furthermore is able to overcome any possible inhibitory effect from the mutant ferrochelatase protein produced by the protoporphyric fibroblasts. These results indicate that transfer and over-expression of the ferrochelatase cDNA might be an effective therapy for patients with protoporphyria.