Molecular Therapy, 16 S182
The American Society of Gene Therapy Annual Conference, Boston, 2008
Clinical studies are planned for the aerosol delivery of Genzyme Lipid GL67A complexed with plasmid DNA (pDNA) in the lungs of patients with cystic fibrosis (CF).
In order to minimise CpG-related inflammatory responses we generated a CpG-free clinical trial plasmid based on the R6K origin of replication (Invivogen, France), utilising the hCEFI (human Cytomegalovirus enhancer/elongation factor 1alpha) promoter.
This clinical plasmid (pGM169) expressing human CFTR and a similar luciferase (Lux) expressing version (pGM144) have demonstrated persistent high levels of gene expression (at least 8 weeks) following aerosol delivery GL67A/pDNA to the lungs of mice. However the limited choice of CpG-free promoters currently restricts the development of CpG-free plasmids for other diseases. Using a simple range of publicly available tools, an initial set of 6 promoters were identified in Genbank that are CpG-free from at least -500bp to +10bp relative to their transcription start sites. Of the 6 promoters, 5 were human (P1-P5) and one was murine (P6) and none had ever been studied previously in the context of gene therapy.
The promoter sequences were cloned into our CpG-free clinical plasmid backbone either in Native pDNA form (promoter only) or Enhanced pDNA form (including the human CMV enhancer upstream). Native and Enhanced plasmids were complexed with GL67A and delivered to mouse lung by nasal instillation (80g pDNA/100l, BALB/c, n=6). At day 1 (d1) and 14 (d14) post-dosing Lux activity in the lungs was compared with expression from the hCEFI promoter and naive samples. Typically this model is a harsh measure of duration of expression and very few promoters exhibit significant activity at d14 (the standard CMV promoter results in Lux activity that are only 0.1% hCEFI levels at d14). Native promoters at d1 and d14 performed poorly with Lux activity only slightly higher than naive levels. However, activity from Enhanced promoters P4, P5 and P6 at d1 was similar to hCEFI (Mann-Whitney, P0.05). In particular, Enhanced promoter P6 performed well with Lux activity 129% and 115% hCEFI levels at d1 and d14 respectively.
This study demonstrates that CpG-free promoters can be readily identified in Genbank and that they can result in significant levels of transgene expression compared with other CpG-free or conventional promoters.
These novel CpG-free promoters are now available for testing in other disease models to determine their specific expression profile in various tissues and cell types.