Generation of a CpG-Free Clinical Trial Plasmid for Cystic Fibrosis Lung Gene Therapy.

Pringle IA, Lawton AE, Green A-M, Varathalingam A, Coles RL, Painter H, Davies LA, Nunez-Alonso G, Cheng SH, Yew NS, Gill DR, Hyde SC

Molecular Therapy, 15 S389

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The American Society of Gene Therapy Annual Conference, Seattle, 2007

We are undertaking clinical trials for the development of a gene therapy for Cystic Fibrosis (CF) lung disease. Clinical studies are planned for the aerosol delivery of single- and multiple-doses of Genzyme lipid (GL67)/plasmid DNA (pDNA) complexes with a view to achieving clinical benefit in the lungs of patients with CF. The ideal clinical plasmid would be safe, and efficacious in generating high level, persistent gene expression in airway epithelial cells.

Previously we (and others) have observed a CpG-dependent induction of host pro-inflammatory cytokines following delivery of GL67/pDNA complexes to the mouse lung. Thus to maximise safety and to minimise a host CpG response, we constructed a novel, small (1100bp), 4th generation, CpG-free plasmid backbone. We then generated a series of modular plasmids to test a variety of CpG-free enhancer/promoter sequences for the required high-level persistent reporter gene expression. Compared with several promoters, the novel hCEFI (human Cytomegalovirus enhancer/elongation factor 1alpha) promoter, demonstrated persistent high level Luciferase activity for at least 6 weeks following aerosol delivery to the mouse lung and was selected as a suitable candidate.

A synthetic, CpG-free, codon-optimised version of the cDNA for CFTR (Cystic Fibrosis Transmembrane conductance Regulator) was inserted into the 4th generation plasmid backbone to generate a CFTR expression plasmid suitable for use in the clinic (designated pGM169). Following transient transfection of HEK293T cells in culture, and the subsequent preparation of RNA and protein extracts 48 hours later, TaqMan RT-PCR and Western blotting were used to confirm the efficient expression of CFTR from this plasmid. During the scale-up from routine production of small (2mg) quantities of plasmid DNA, to larger (>100mg) quantities required for in vivo studies, an unexpected, small (190bp) deletion was generated in the promoter region (position 19 to 210).

Careful manipulation of the culture growth conditions, including selection of small bacterial 'micro-colonies' and monomeric forms of the plasmid, was required to permit successful production of large quantities of plasmid DNA; this was confirmed by routine sequencing of all plasmid batches. Pre-clinical studies following aerosol delivery into the lungs of mice and sheep are now underway to examine the CFTR expression profile in vivo. In addition we are working with the commercial supplier Advisys Inc. to manufacture GLP and GMP material for the planned clinical studies..