743. Minicircles Are Similar to Plasmids in Providing High Level, Long-Term Expression in the Lung

Background: Many gene therapy applications require persistent transgene expression to treat chronic disease. We have previously developed CpG-free plasmids capable of robust, long-term transgene expression in the mouse lung (Hyde et al; 2008 Nat. Biotech. 26:549) and confirmed their utility in clinical trials, where monthly aerosol delivery of plasmid/liposomes to patients with Cystic Fibrosis resulted in a stabilisation of their lung function over a 12-month period (Alton et al 2015 Lancet Respir. Med. 3:684). The removal of all CpGs from a plasmid, including the selection of a CpG-free promoter, can be time-consuming, therefore we evaluated which aspects of plasmid design were crucial for long-term transgene expression in the lung, including the use of minicircles instead of conventional plasmids. Results: First, we measured luciferase (Lux) expression in the lungs of BALB/c mice from a CpG-free transgene expression cassette (hCEFI-soLux) compared with a standard CpG-rich (hCEFI-Lux) cassette. Plasmid DNA was complexed with 25 kDa branched polyethylenimine (PEI) and delivered as an aerosol to the lungs of BALB/c mice (n=6 per time-point). Lux activity from the CpG-free hCEFI-soLux cassette was greater than its CpG-rich counterpart (p u003c 0.005) at every time-point (up to 28 days), with no loss of activity over the course of the study. Expression from the expression cassette containing CpGs, however, was not persistent, but declined to between 30% and 2% of respective day 1 levels by day 28 (p u003c 0.05). Next, we performed similar studies using minicircles, where the CpG-free and CpG-rich expression cassettes were manufactured as minicircles with minimal backbone sequences (209bp with 11 CpGs). When the CpG-free cassette was used there was no significant difference between Lux activity obtained from the plasmid and minicircles; in both cases robust expression persisted beyond the duration of the study (28 days). Finally, we investigated whether the detection of CpGs by the Toll-like receptor-9 (TLR9) signalling pathway played a role in the loss of expression from the CpG-rich cassette. The experiments were repeated in TLR9-deficient mice and the results showed that Lux levels were similar to those obtained in BALB/c mice. This indicated that the effect of CpGs on the in vivo expression profile (whether in the transgene cassette or backbone) is independent of the TLR9 pathway. Conclusion: These studies indicate that a CpG-free transgene cassette is crucial to achieving high levels of persistent expression in the murine lung, when delivered as a plasmid or minicircle. Minicircles have not yet been evaluated clinically, but they have several advantages over plasmids, including their reduced overall size such that a higher effective dose is delivered. These findings could also be applicable to other organs, such as muscle and liver.