Abstract:
We recently published the first description of correction of a CF causing mutation(DF508) in a tracheal epithelial cell line by homology directed repair (HDR) usingzinc finger nucleases (ZFNs) and a donor plasmid (Lee et al., 2012).At present the approach is limited to correcting one mutation. To address this, we areusing our existing cftr-specific ZFNs to target a cftr mini-gene to intron 9 with theaim to correct CF mutations throughout exons 10−24 (~80% of all CF mutations)with a single ZFN pair/donor plasmid. As proof-of-principle that our ZFNs cantarget exogenous sequences to intron 9 we first sought to precisely incorporate a 7bp Tag sequence into the cftr gene at the ZFN target site by HDR using a 1.5kbdonor plasmid. Analysis of cells treated with our ZFNs and donor by nested PCRrevealed that the Tag can be successfully introduced into the cftr gene at the correctlocation.To effect gene repair, the mini-gene repair construct requires a promoterless cftrpartial cDNA (exons 10−24) with appropriate splice acceptor and poly A sites.Incorporation of the mini-gene would result in full length corrected CFTR mRNAproduction that is under the control of the endogenous promoter, a major limitationexperienced when delivering exogenous cDNA. Li et al., 2011, previously replacedexons 2−8 of the F9 gene restoring haemostasis in haemophilic mice using thisstrategy. Successful gene correction using a mini-gene would result in normalspatiotemporal expression of the corrected CFTR gene, that is permanent for thelifetime of the cell and is not subject to gene silencing. It could be of use as analternative strategy to cDNA addition for gene therapy. 60 Permanent correction of >80% of disease-causing mutations in human CF cells. Available from: https://www.researchgate.net/publication/273413986_60_Permanent_correction_of_80_of_disease-causing_mutations_in_human_CF_cells [accessed Sep 28, 2017].