Novel CpG depleted and codon optimised CFTR cDNAs maintain the structure and fuction of CFTR protein.

Varathalingam A, Lawton AE, Munkonge FM, Chan M, Somerton L, Griesenbach U, Alton EW, Pringle IA, Hyde SC, Gill DR

British Society of Gene Therapy Conference, London, 2006

CpG dinucleotides present in plasmid DNA (pDNA) are known to contribute to the induction of an acute inflammatory response when delivered to the lungs of experimental animal models and patients with Cystic Fibrosis. Reduction in the number of CpGs within plasmid expression vectors has been shown to reduce the host inflammatory response to non-viral gene transfer. The standard Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) cDNA contains 59 CpG motifs, therefore we have generated a CFTR cDNA (termed soCFTR) which has all CpGs removed without altering the amino acid sequence. In addition this cDNA is codon optimised resulting in a sequence that is only 77% identical to the standard CFTR cDNA. To study the impact of soCFTR cDNA on CFTR protein we investigated the structure and function of the CFTR protein in cell culture. Western blotting showed that soCFTR cDNA directed the expression of abundant CFTR protein that was correctly glycosylated. SoCFTR cDNA directed CFTR function that was also confirmed by Iodide125 effllux analysis. TaqMan RT-PCR assay confirmed the presence of vector specific CFTR mRNA in vivo at day 14 following nasal perfusion (FABP CF mice, n=21, Mann Whitney p<0.05). These data confirm that CpG depletion and codon optimisation of CFTR cDNA does not alter the CFTR structure and function in cell culture and can lead to persistent CFTR expression in vivo. Non-viral mediated CFTR gene transfer with this new form of the CFTR cDNA will be investigated in a variety of model systems in preparation for clinical studies.