Pediatric Pulmonology, 40 S28 280
The North American Cystic Fibrosis Conference, Baltimore, 2005
Gene therapy for Cystic Fibrosis lung disease will likely require long-term gene expression and/or repeated administration. Due to the disadvantageous immune response observed with viral vectors, we consider non-viral plasmid (pDNA) based gene transfer agents as the more appropriate system for long-term gene therapy. However, in the lung, many non-viral gene transfer agents lead to transient gene expression. For example, CMV-mediated luciferase reporter expression peaks at day 1 and falls to background by day 7 irrespective of the gene transfer agent used. pDNA vectors that contain numerous CpG motifs are associated with a host inflammatory response, which in turn, may contribute to the loss of transgene expression. We investigated the effects of CpG minimisation on the efficacy and duration of transgene expression following aerosol delivery of pDNA complexed with Polyethylenimine (PEI) to the lungs of BALB/c mice. Transgene expression from a variety of promoter/enhancer sequences was measured in the context of a pDNA backbone (origin of replication, antibiotic resistance & polyA sequence) with minimal CpG content, and compared with a standard CpG-rich backbone (as in pCIKLux). The use of a CMV enhancer/promoter, or a ?CpG CMV derivative in which all CpG motifs had been removed, resulted in luciferase activity at day1, which was similar to pCIKLux (p=0.26), falling to background within 7 days. When hybrid CMV/polyubiquitin promoters were tested in the context of a minimal CpG backbone, luciferase activity from the CUCI promoter (CMV enhancer/UbC promoter) was equivalent to pCIKLux at day1 and was detectable at 28 days post-dose (p=0.0082). Luciferase activity from the CUBI promoter (CMV enhancer/UbB promoter) directed levels of luciferase five-fold higher than pCIKLux (p=0.0001), which fell sharply at day 7, but remained detectable at 28 days post-dose. However, when the UbC promoter was tested in the context of a minimal CpG backbone, luciferase activity that was indistinguishable from pCIKLux at day 1 (p=0.7459) was sustained at this level for at least 28 days, and up to 56 days in a separate study. Importantly, when luciferase expression from the UbC promoter in a CpG-rich backbone was compared to the backbone with minimal CpG content, the latter resulted in 10-fold increased expression levels at day 28 (p=0.0001). These data suggest that it in addition to the number of CpG motifs in a pDNA, the combination of promoter and backbone can impact the expression profile. In conclusion we have selected a pDNA-backbone-promoter combination that when complexed with PEI and delivered as an aerosol, results in high and sustained levels of reporter gene expression; these properties are essential for the development of an effective gene based treatment for CF.