507. Down-Regulation of the Epithelial Sodium Channel ENaC through Antisense Oligonucleotides
Authors: Uta Griesenbach, Sara Escudero Garcia, Chris Kitson, Raymond Farley, Charanjit Singh, Hazel Painter, Rebecca L. Smith, Ron Scheule, Seng Cheng, Mark Edbrooke, Steve Hyde, Duncan M. Geddes, Eric W. F. W. Alton
Journal: Molecular Therapy (2004)

Sodium absorption across the airway epithelium is increased in cystic fibrosis (CF) patients and may contribute to abnormal water transport and accummulation of sticky sputum, a hallmark of the CF lung disease. Down-regulation of epithelial sodium channels (ENaC) may attenuate the CF lung disease. One approach to decrease ENaC function is the specific down-regulation of its mRNA. Single-stranded DNA antisense oligonucleotides (ssDNA) bind to mRNA sequence-specifically and trigger RNase-H mediated degradation of the ssDNA/mRNA hybrid. However, careful selection of the ssDNA sequence is required to obtain optimal mRNA reduction. Here, we compared five murine ENaC antisense DNAs (20 nucleotides each) for their ability to decrease ENaC mRNA in M1 cells, a murine kidney cell line that expresses ENaC abundantly. It has previously been demonstrated that the alpha subunit of ENaC is the most crucial for channel function and all five ssDNAs were therefore directed against this subunit. First, fluorescently labelled ssDNA was complexed with Lipofectamine 2000 and transfection efficiency determined. Interestingly, the ssDNA rapidly (after 30 min) accumulated in the nucleus of transfected cells and after 6 hrs >90% cells were transfected. The nuclear localisation of the antisense ODNs was consistent with their proposed site of action. Forty-eight hrs after transfection cells were harvested and mRNA was prepared for quantitative RT-PCR using Taqman. The relative degree of aENaC mRNA reduction varied between antisense molecules and ranged from 5 to 60% (n=6, p<0.05). The most efficient ssDNA was used for in vivo transfection. Genzyme lipid 67 (GL67) has been widely used for gene transfer in vivo, but not for antisense applications. We first assessed if GL67 complexed antisense ssDNA efficiently. Gel retardation studies showed that with a 6:8 molar ratio of cationic lipid:nucleotide almost all ssDNA was complexed (n=3). Light-scatter analysis showed that complexes had an average size of 390±24 nm, but varied in size (n=3). Nasal potential difference assays (PD) have been widely used to measure ion transport across murine nasal epithelium. A decrease in ENaC function would result in a drop in baseline PD, because sodium transport is the main component of basal ion transport in the nasal mucosa. Here, we complexed aENaC antisense or control ssDNA to GL67 (80 mg/mouse) and perfused these complexes into the mouse nose over 75 min via a thin plastic catheter. Twenty-four, 48 and 96 hrs after administration the PD was measured in individual cohorts of animals (n=10/group). After the PD measurements all animals were culled and Taqman RT-PCR carried out on nasal mRNA. We did not detect a drop in aENaC mRNA or changes in PD at any time-point. A critical factor for antisense function in the mouse nose is transfection efficiency. In preliminary experiments (n=3) using fluorescently-labelled ODNs we noted that transfection efficiency varied drastically from animal to animal, and will therefore require further optimisation.