Inflammation-free Human and Murine Promoters for Non-viral CFTR Lung Gene Therapy.

Hyde SC, Green AM, Davies LA, Lawton AE, Yew NS, Cheng SH, Gill DR, Pringle IA

Pediatric Pulmonology, Vol 31, 307


The North American Cystic Fibrosis Conference, Orlando, 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 Inc., San Diego, CA, USA), utilising the hCEFI (human Cytomegalovirus enhancer/elongation factor 1alpha) promoter (Hyde et al., 2008 Nature Biotechnology 26: 549). 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 number of CpG-free promoters currently available, severely restricted the design of these plasmids.

To extend the available repertoire of CpG-free promoters we searched freely accessibly DNA sequence databases with custom designed search algorithms to identify naturally occurring promoters that are CpG-free from at least -500bp to +10bp relative to their transcription start sites. Of the 6 promoters identified, 5 were human (hTSB, hAPO2A, hCBOX, hREG1B, hTDOX) and one was murine (mFABP4) and none to our knowledge have been studied previously in the context of gene therapy. The promoter sequences were isolated and inserted into our CpG-free plasmid backbone in either Native pDNA form (promoter only) or Enhanced pDNA form (including the CpG-free human CMV enhancer upstream). Native and Enhanced plasmids were complexed with GL67A and delivered to mouse lung by nasal instillation (80 µg pDNA/100 µl, 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 (for example the widely used CpG-containing CMV promoter results in Lux activity that are only 0.1% hCEFI levels at d14).

Native promoters at both d1 and d14 performed poorly, with Lux activity substantially lower than those observed with the hCEFI promoter (P<0.001), at levels only slightly higher than naive animals. However, activity from Enhanced hREG1B, hTDOX and mFABP4 promoters was similar to hCEFI (Mann-Whitney, P>0.05). In particular, the Enhanced mFABP4 promoter performed well, generating lung reporter gene activity of 129% and 115% hCEFI levels at d1 and d14 respectively.

This study demonstrates that CpG-free promoters can be readily identified in freely accessibly DNA sequence databases and that they can direct significant levels of lung transgene expression. The efficacy of these novel CpG-free promoter constructs is currently being evaluated in our models of aerosol mediated gene transfer.