Pediatric Pulmonology, 33 S1 Abstract 315
The North American Cystic Fibrosis Conference, Baltimore, 2010
Clinical studies are underway for the aerosol delivery of Genzyme lipid (GL67A)/plasmid DNA (pDNA) to the lungs of patients with CF. A key requirement for successful non-viral gene therapy is a plasmid with the ability to direct long-term hCFTR gene expression in target cell types. To achieve this, we developed a series of novel, fourth generation plasmids that were free of CpGs to minimise inflammatory responses after delivery, and tested the utility of a range of promoter, enhancer and terminator sequences in various models. We evaluated the plasmids for the expression of CpG-free, codon-optimized human CFTR (soCFTR2). Ultimately, a synthetic CpG-free promoter, hCEFI, was selected for long-term soCFTR2 expression. Successful expression of functional hCFTR protein was confirmed following transient transfection of HEK293T cells, using Western blotting, iodide efflux and patch clamp studies [Hyde 2008 Nature Biotechnology 26:549]. To evaluate the performance of pGM169 in vivo we complexed the plasmid with GL67A, and aeroslised to the lungs of mice. To quantify plasmid deposition and hCFTR mRNA expression in the lungs we used TaqMan PCR and RT-PCR assays with sensitivities in the near single copy number range. After aerosol delivery, bulk pGM169 DNA levels in the lung fell by approximately three orders of magnitude from a peak at day 1 (27.8±12.0 x10(3) copies per lung cell genome) to a steady state level by d14 onwards (P<0.05). Importantly, hCFTR mRNA was expressed in the lung at >5% endogenous (%VE) levels for at least 2 months (range 9.04-20.64%) and was not significantly different at d1, d14, d28 and d56 (P>0.33). Expression was also measured in human ex vivo cultures of nasal cells grown at an air liquid interface (Epithelix, Switzerland) (pGM169: 8/8 samples positive at d2; 5/8 at day 7), at higher levels (median 28.6 %VE at day 2) than first generation clinical plasmids (pCF1-CFTR: 3/7 samples positive at day 2, 0/7 at day 7). For clinical use, GMP production of high purity plasmid was established (less than 2% contaminating RNA, DNA, Protein; less than 5 EU/mg endotoxin). Gram scale production of pGM169 was achieved via host cell fermentation at the 100L scale, coupled with bubble lift lysis, anion exchange membrane capture, HIC chromatography purification and TFF formulation and concentration (VGXI, Houston Texas). Reproducibility was assured via validated master and working cell banks. Pharmaceutical stability of pGM169 was assessed via real-time and accelerated ICH conforming stability studies; the current shelf-life of pGM169 using our production process exceeds 4 years. The ability of the GMP lots to express hCFTR mRNA in target human airway cells was confirmed following ex vivo transfection of human nasal brushings (mean 24 %VE; n=5). The performance of a single aerosol dose of plasmid pGM169 is currently being evaluated in the lungs of CF patients.