THE EFFECT OF MUCOLYTIC AGENTS ON GENE-TRANSFER ACROSS A CF SPUTUM BARRIER IN-VITRO

Trials of gene transfer for cystic fibrosis (CF) are currently underwa y. However, direct application to the airways may be impeded by the pr esence of airway secretions. We have therefore assessed the effect of CF sputum in the expression of the reporter gene beta-galactosidase co mplexed with the cationic liposome DC-Chol/DOPE in a number of cell li nes in vitro. Transfection was markedly inhibited in the presence of s putum; the effect was concentration dependent and was only partially a meliorated by removal of sputum with phosphate-buffered saline (PBS) w ashing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 mu g/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant algina se (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adeno- virus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfec tion significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrat ed similar inhibition to that observed with sputum and was also amelio rated by pre-treatment of DNA-covered cells with rhDNase. In a separat e series of experiments performed in the absence of added sputum or ge nomic DNA, increasing concentration of rhDNase resulted in a concentra tion-related decline in transfection efficiency. However, even at the highest concentration (500 mu g/ml of rhDNase), transfection efficienc y remained more than 50% of control. Thus pretreatment of CF airways w ith rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.