Gene therapy is an attractive treatment for many genetic diseases because rather than treat the symptoms of the disease, it has the potential to correct the underlying defect. Cystic fibrosis and hemophilia A are two monogenic disorders that are particularly well-suited to treatment with gene therapy as a relatively small increase in the function is needed to see improvement. Gene therapy has provided some correction in both diseases using a variety of vector systems but sustained expression and long term correction have yet to be demonstrated in the clinic. It is unclear in which cell type(s) correction of the underlying defect in cystic fibrosis will be most effective. Studies indicate that the majority of CFTR expression is in the submucosal glands and ciliated epithelia – a terminally differentiated cell type (Engelhardt, J.F. et al, 2004, Journal of Clinical Investigation). Therapeutic gene transfer would thus be most effective if achieved in a progenitor cell type. Additionally, the native regulation of CFTR has not been definitively elucidated. To this end, one goal of our studies is to develop a lentiviral vector system with heterologous promoters of varying strengths and cell specificity to aid in our selection of optimal reagents for appropriate CFTR expression. We show that use of novel internal promoters from the human PLUNC and WDR65 genes direct persistent expression in the airway. Additionally, disruption of the nasal epithelium with the detergent polidocanol eliminated reporter expression in mouse airway. Two weeks post-treatment, expression returned indicating targeting of a progenitor cell population with our novel vectors.
Integrating vector systems can treat chronic diseases such as cystic fibrosis because expression can persist long term from these vectors if cells with progenitor capacity are targeted (Sinn, P.L. et al, 2005, Journal of Virology). However, the potential for genotoxicity from vector-related dysregulation is a concern. Thus, a second aim of these studies was to develop a lentiviral vector that can target a specific locus in the genome. We developed a FIV vector in which the integrase was modified with a protein-binding domain that when co-delivered with a fusion consisting of the cognate protein and a DNA binding domain would tether the vector to the appropriate locus. Unfortunately, integrase modification rendered the vector catalytically inactive. Lastly, we hoped to develop a non-viral transposon vector system (piggyBac) for gene transfer applications to the liver for treatment of hemophilia A. The recent demonstration that piggyBac transposase is highly active in mammalian cells warrants further development of this vector as an alternative to other non-viral integrating vector systems currently under investigation. We showed persistent reporter and therapeutic transgene expression in the livers of mice treated with the piggyBac vector. Furthermore, we show for the first time in vivo persistence and increased expression from the recently developed hyperactive transposase. The development of integrating vectors targeted to specific tissues or genomic loci is important in for treatment of the monogenic diseases cystic fibrosis and hemophilia A.