Efficient gene transfer and high-level expression are the major goals of any gene therapy strategy. We have selected retroviral insertion of the ferrochelatase gene (FC) in erythropoietic protoporphyria (EPP) in hematopoietic stem cells as our model system. The ferrochelatase gene and cDNA have previously been described, and are amenable to PCR and manipulation leading to the development of gene therapy for EPP. The molecular basis of EPP is firmly established, in that a large number of mutations in FC have been described which are causally related to the pathoetiology of EPP. Cultured EPP fibroblasts can be transduced with recombinant FC, and enzyme levels are restored to normal, demonstrating in vivo phenotypic reversion. Thus, demonstration of in vitro phenotypic reversion would represent the critical step in a preclinical study aimed at applying gene therapy to EPP. The relatively small FC cDNA is only 1.6 kb in size, and can therefore be easily cloned and manipulated in insert-size restricted retroviral vectors. Second, the gene product itself is labile, and is ubiquitously expressed in all tissues examined, thereby obviating the need for targeted cell-type specific gene expression. Third, techniques for selection, enrichment and transduction of hematopoietic stem cells are already well-established. Fourth, phenotypic reversion of EPP can be monitored in vivo not only by levels of expression of the FC gene, but importantly, by demonstrating the functionality of the gene product by an FC enzyme activity assay. Fifth, partial gene expression may be sufficient for phenotypic reversion, since we know from some clinically unaffected EPP heterozygotes that achieving 100% gene replacement efficiency may be unnecessary, as many individuals with significantly reduced enzyme levels are asymptomatic. Collectively, these features underscore the feasibility of developing gene therapy for EPP, which could potentially lead to therapeutic strategies for the other forms of porphyria, as well.
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