The nuclear pore dilating Agent TCHD increases gene transfer into differentiated airway epithelium ex vivo, but has no efect in vivo.

Griesenbach U, Wilson KM, Farley R, Meng C, Scheule RK, Cheng SH, Alton EW

Molecular Therapy, Vol 19, S1, Abstract 165

The American Society of Gene and Cell Therapy Annual Conference, Seattle, 2011

The nuclear membrane is a significant barrier to non-viral gene transfer into non-dividing differentiated airway epithelial cells. Trans-cyclohexane 1,2 diol (TCHD) is an amphipathic alcohol which has been shown to collapse the hydrophobic nuclear pore core and allow nuclear localization of macromolecules that would otherwise be to large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. Here, we aimed to reproduce these in vitro studies using the cationic lipid GL67A which we are currently assessing in cystic fibrosis gene therapy trials and, more importantly, to assess the effects of TCHD on transfection efficiency in fully differentiated airway epithelium ex vivo and in vivo. TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells up to 2 logs. Encouragingly exposure to 8% TCHD, but not 0.5 or 2.0%, increased gene expression in fully differentiated human air liquid interface (ALI) cultures approximately 20-fold, although this was accompanied by significant cell damage. We then assessed the effects of TCHD in vivo. GL67A/pCIKLux complexes were administered to the lungs of mice by nebulisation and various concentrations of TCHD (0.4 to 10 %) were administered by nasal sniffing immediately pre-and post-nebulisation to ensure maximal exposure of the mice (n=8/group). In a separate set of experiments mice (n=8/group) were nebulised with GL67A/pCIKLux complexes followed immediately by nebulisation of 10% TCHD (maximum concentration that could be dissolved) for 1 hr. In a third set of experiments GL67A/pCIKLux was administered to the nasal epithelium by nasal perfusion over 15 min followed by nasal perfusion of 100 ml of 10% TCHD over 15 min (n=8 mice/group). This protocol ensured prolonged contact time between the airway epithelium and the compounds. None of the TCHD treated mice had higher gene expression when compared to no TCHD controls. In conclusion, although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights, that promising in vitro findings often cannot be translated into in vivo applications.