Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC309
Correction of the Cystic Fibrosis Transmembrane Conductance Regulator in Cystic Fibrosis Epithelial Cells using Zinc Finger Nuclease Homology-Directed Repair
J. A. Hollywood1,2, C. M. Lee1,2, R. Flynn3, K. Kaschig1,2, M. F. Scallan2, P. T. Harrison1
1. Physiology, University College Cork, Cork, Ireland. 2. Microbiolgy, University College Cork, Cork, Ireland. 3. Pediatrics, University of Washington, Seattle, Washington, United States.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP regulated ion channel, allows Cl- flux across the apical membrane of epithelial cells. Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in CFTR. The most common mutation a CTT deletion (ΔF508) disrupts CFTR channel activity. Cl- flux can be restored following the addition of CFTR cDNA to a model CF cell line (Rich et al, 1990). However, attempts to deliver CFTR cDNA to the lungs of patients to restore function have not resulted in any clinical benefit (Davies and Alton, 2010). An alternative approach is to correct the mutations in the genome. Gene correction involves introduction of a donor DNA molecule containing the correct sequence to cells which then triggers homology-directed repair (HDR) of the mutant gene. Advantages include permanent correct temporal spatial expression and restoration of any splice variants. However, the efficiency of HDR can be as low as 1 in 106 treated cells. Double stranded breaks (DSBs) in the target gene created by synthetic restriction enzymes zinc finger nucleases (ZFNs) can increase the rate of HDR using a donor sequence to 1 in 5 treated cells (Urnov et al, 2005). We have previously described ZFNs that can create a specific DSB near to the ΔF508 mutation (Lee et al, 2008). Here, we describe the use of these ZFNs to repair the ΔF508 mutation in a CFTE cell line. A 4.3 kb donor was amplified from the CFTR gene of a human bronchial epithelial cell-line using high-fidelity PCR such that the missing CTT sequence was near the centre of the sequence. The donor sequence was cloned into a plasmid between the inverted terminal repeat sequences of the adeno-associated virus genome, pCFTR-Donor. A modified version of pCFTR-Donor (pCFTR-Donor-XC) containing XhoI and ClaI restriction sites was created by site directed mutagenesis to assist quantification of gene correction efficiency. A tracheal epithelial cell line (CFTE) derived from a CF patient homozygous for the ΔF508 mutation was transfected with a total of 4 µg DNA and 10 µl Lipofectamine with the following plasmids: pZFNs only, pCFTR-Donor only, pCFTR-Donor-XC only, pZFNs and pCFTR-Donor, or pZFNs and pCFTR-Donor-XC. Seventy-two hours post transfection, total RNA was extracted and cDNA synthesis performed. To detect wild-type CFTR mRNA (which can only be produced if the missing CTT has been repaired in CFTR), cDNA was amplified by RT-PCR using primers that will produce a 675 bp product. A 675 bp band was seen only when cells were transfected with pZFNs and pCFTR-Donor, or with pZFNs and pCFTR-Donor-XC. Band densitometry analysis revealed that wild-type CFTR mRNA was produced at 6-13% of normal levels. Immunofluorescence analysis will be used to determine the level of CFTR protein present at the apical membrane.
Where applicable, experiments conform with Society ethical requirements