Molecular Therapy, 18 S1 S283
The American Society of Gene and Cell Therapy Annual Conference, Washington DC, 2010
Clinical studies are underway for the aerosol delivery of Genzyme Lipid 67A (GL67A) complexed with plasmid DNA (pDNA) to the lungs of patients with cystic fibrosis (CF). In designing the clinical plasmid we wished to minimize CpG-related inflammatory responses associated with delivery of lipid/pDNA complexes, thus we generated a CpG-free plasmid based on the R6K origin of replication (Invivogen, France). We also required a promoter capable of long-lived expression in the lung.
Previously we have shown that the human polyubiquitin C (UbC) promoter could generate persistent transgene expression in the mouse lung (Gill et al 2001 Gene Therapy 8 1539-46) but unfortunately, we were unable to remove all CpGs from the UbC promoter without losing efficacy. We therefore developed the novel hCEFI (human Cytomegalovirus enhancer/elongation factor 1alpha) promoter, which has demonstrated persistent high levels of transgene expression (at least 8 weeks) following aerosol delivery GL67A/pDNA to the lungs of mice (Hyde et al 2008 Nature Biotech 26 549-51). In comparative studies the hCEFI and UbC promoters exhibit similar duration of transgene expression in the mouse lung, but the levels of expression from hCEFI are consistently higher. Here, we directly compare the duration of expression from these two promoters following hydrodynamic delivery of naked pDNA to the mouse.
Both promoters were inserted into our standard CpG-free pDNA backbone upstream of a CpG-free luciferase reporter gene. Naked pDNA (10?g/ml) was prepared in D-PBS and injected in a volume equivalent to 10% bodyweight over 5 seconds into the mouse tail vein (female BALB/c 5-6 weeks). At various time points the liver, lung, kidney, heart and spleen were harvested for luciferase activity. In the liver at d1 post injection, expression from hCEFI was 85-fold higher than UbC (P=0.004 Mann-Whitney U). However UbC-directed expression persisted at the same level for 5 weeks post injection while hCEFI-directed expression did not persist and was 4 logs lower than UbC by 5 weeks (P=0.004). Similar patterns of expression were observed in the lung, heart, kidneys and spleen with hCEFI exhibiting higher peak expression at early time points but poor persistence, while UbC exhibited steady expression from d1 to 5 weeks post injection.
Our results show that persistence of expression from the UbC promoter is consistent whether the plasmid is delivered via aerosol or via hydrodynamic delivery, but expression from the hCEFI promoter is dependent upon the route of gene delivery and the target organ. In the context of aerosol delivery to the lung hCEFI is superior to any other we have tested but when using hydrodynamic delivery of naked pDNA the profile of gene expression is less advantageous.
We are similarly testing a series of novel CpG-free promoter elements for their expression profiles. Together these results demonstrate that promoter selection should be based on the most relevant model available using a relevant delivery method and that generalised theories identifying factors affecting promoter performance need to be studied in a wide range of model systems.