The European Society of Gene and Cell Therapy Conference, Prague, 2005
Non-viral gene therapy is being considered as novel approach to treat Cystic Fibrosis (CF) lung disease.
However, the level and duration of expression achieved from these vectors are generally very low and have been shown to be associated with toxicity and an inflammatory cytokine response in mice and in human clinical studies. This response is due in part to the detection by toll-like receptor 9 (TLR9) of unmethylated CpG dinucleotides (CpGs) present in plasmid DNA (pDNA).
Typically, pDNA vectors contain sequences of bacterial DNA (bDNA) such as antibiotic resistance genes and origins of replication which have a high number of CpGs, Even eukaryotic sequences such as promoters and cDNAs can contain high numbers of CpGs which when delivered to cells in the context of a non-viral vectors can be recognised by TLR9 and elicit an inflammatory response.
The most direct approach to overcome this response is to reduce or completely remove CpGs from plasmid sequences. We have constructed two series of modular pDNA vectors. The first series of plasmids were constructed from modular elements (origin, kanamycin resistance gene, promoter, poly A and transgene) with reduced CpG content and reduced the total numbers of CpG by 40 - 50 %.
The second series of plasmids were constructed from elements that contained no CpGs and the resulting plasmids therefore contain 0 CpGs. The plasmids pCIKLux (pCI backbone, CMV promoter, 369 CpGs), pGM104 (Genzyme ΔCpG backbone, CMV promoter, 125 CpGs) and pGM124 (ΔCpG R6K origin, ΔCpG KanR, Murine CMV enhancer, human EF1a promoter, 0 CpGs) expressing the luciferase reporter gene were complexed with liposomes of Genzyme lipid 67 and delivered to the mouse lung by nasal instillation (80 µg pDNA/100 µl). At 24 hours post dosing mice were killed and bronchoalveolar lavage fluid (BALF) was collected for analysis of neutrophils, IL-12, IFN-g and TNF-a levels. Compared to untreated controls, the mice that received pCIKLux complexes had high levels of neutrophils, IL-12, IFN-a and TNF-g in the BALF. Delivery of pGM104 to the mouse lung resulted in no significant difference in any of the four markers of lung inflammation. However, following delivery of pGM124, levels of the three cytokines and levels of neutrophils were indistinguishable from untreated control mice. Luciferase activity from pGM124 was 10 fold higher than pCIKLux and pGM104 at 24 hours post dosing and expression persisted for at least 60 days post-dosing.
Taken together, these results suggest that 0 CpG plasmids present a significant advance in terms of safety and efficacy of non-viral delivery to the lung,