Influence of the Human and Murine CMV Enhancer on the Duration of Expression from CpG-Free pDNA Vectors in the Mouse Lung. (2007)

Green, A.-M., Pringle, I. A., Lawton, A. E., Nunez-Alonso, G. A., Davies, L. A., Yew, N. S., Cheng, S. H., Gill, D. R. & Hyde, S. C.

Molecular Therapy, 15, S385

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Later this year we intend to commence a clinical trial in Cystic Fibrosis (CF) patients, which will utilise a novel CpG-free plasmid DNA (pDNA) vector that we have developed. Pre-clinical studies predict that this CpG-free pDNA will reduce the levels of inflammatory symptoms observed in previous non-viral trials with CF patients (Alton et al 1999, Lancet 353 947), which is believed to be due to the high CpG content of the pDNA used. Previous pDNA vectors used in clinical trials for CF have also suffered from limited duration of expression in vivo. Therefore we conducted several studies in a mouse lung model to assess the duration of expression from a number of different CpG-free pDNA vectors. In particular we have examined the effect of the human cytomegalovirus (CMV) immediate early enhancer (hCMVenh) and the murine CMV enhancer (mCMVenh) on the duration of expression in the mouse lung. We constructed a plasmid (pGM148) that contains an 1100bp backbone, a luciferase (Lux) gene, synthetic intron and the Elongation Factor 1 (EFI) promoter (Invivogen). From this we created two further plasmids, (1) pGM141 by addition of the mCMVenh (Invivogen) and (2) pGM144 by addition of the hCMVenh. The persistence of Lux activity from each plasmid was measured following aerosolisation to the mouse lung with Genzyme Lipid 67 (2.5mg/ml pDNA, 6mM GL67) in a whole body exposure chamber. Lux activity was measured at 1, 2, 7, 14 & 28 days post-aerosol (n=6 per time-point). Lux activity from pGM148 (no enhancer, EFI) was below the level of detection throughout the time-course. Therefore in the absence an enhancer, the promoter alone was unable to generate significant levels of Lux activity. At day 1 and 2, pGM141 (mCMVenh, EFI) had the highest levels of activity (500 RLU/mg) but these levels were not maintained. By day 7 Lux activity had fallen (137 RLU/mg) and by day 28 it was 10-fold lower than at day 1. Therefore when the mCMVenh was used, high levels of expression were possible but due to the decline in expression over time it offered no advantage over plasmids used in previous clinical trials for CF. At day 1 Lux activity from pGM144 (hCMVenh, EFI) was lower than pGM141 (130 RLU/mg) (P=0.04, Mann-Witney U-test) but at day 7 activity had almost doubled (217 RLU/mg). Lux activity from pGM144 continued to rise and by day 14 was 5-fold higher than day 1 (716 RLU/mg). This high level of expression persisted to at least day 28 (654 RLU/mg) (P=0.02). These results indicate that the hCMVenh can play a crucial role in determining the level and duration of expression from these pDNA vectors in the mouse lung. Only when the hCMVenh is combined with the EFI promoter have we been able to observe such sustained high levels of reporter gene expression. Our clinical trial plasmid is a CFTR expressing version of pGM144 and we are currently finalising a range of pre-clinical studies with this vector in advance of initiating clinical studies.

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