Two strategies are under active investigation for therapeutic correction of genetic diseases such as cystic fibrosis (CF). In the gene addition approach, a normal copy of a CFTR cDNA is delivered to airway epithelia using a viral or non-viral vector. A second approach, termed gene targeting, delivers a donor DNA fragment (repair template) with the correct gene coding sequence and uses the cell's homologous recombination machinery to repair the mutation. Recently, major advances have made the possibility of gene repair by gene targeting more promising as a therapeutic approach. The first is the recognition that introduction of a double strand break (DSB) into the target gene to be repaired can stimulate the efficiency of gene targeting as much as 50,000 fold. The second is the discovery that synthetic zinc finger nucleases (ZFNs) can be engineered to introduce site-specific DSBs in mammalian cells, thereby permitting stimulation of gene targeting at specific genomic loci. We propose to study the process of gene targeting and the use of ZFNs in airway epithelial cells. The goal of these studies is to better understand the biology of gene targeting in epithelia and to use this knowledge toward developing DSB-enhanced gene targeting as a therapeutic tool for CF. We hypothesize that the combined delivery of paired zinc finger nucleases designed to efficiently bind to the human CFTR locus and of a donor DNA fragment will facilitate highly efficient repair of the ?F508 mutation by homologous recombination. We propose three aims:
Aim 1. Design zinc finger nucleases that specifically cleave the F508 mutant allele of the human CFTR locus on chromosome 7. These studies, performed in the laboratory of Co-Investigator Dr. Keith Joung, will employ a selection-based engineering strategy to build ZFN pairs with high affinities and specificities for genomic sequence unique to the CFTR F508 allele. A novel human cell-based gain-of- function screening assay will be employed to identify ZFN pairs with favorable activity and toxicity profiles.
Aim 2. Develop AAV-based vectors to deliver ZFN pairs and homologous donor DNA to human airway epithelia. Dr. McCray's group will build AAV vector constructs to express the ZFN pairs and engineer CFTR repair substrate donor DNA containing the wild type CFTR exon 10 sequence, with or without a selectable marker and bacterial plasmid rescue elements. A transfection-based approach will be employed to document function of the reagents in human airway epithelia in vitro.
Aim 3. Evaluate the efficiency of ZFN-mediated gene targeting to repair the ?F508 CFTR mutation in human airway epithelia. AAV1 vectors will be generated with the optimal ZFN pairs and homologous donor DNA fragments. We will use the human CF airway epithelial cell line CuFi8 (?F508/?F508) to evaluate the efficiency of gene targeting. Southern blot, PCR, and a bacterial plasmid rescue strategy will be used to document the efficiency and fidelity of gene targeting in human airway epithelia. Completion of these proof-of-principle studies will provide important, new data towards the long-term goal of repairing the ?F508 CFTR mutation in human airway epithelia. In addition, the project will generate novel reagents (ZFNs) specific to the human F508 CFTR genomic locus that will be made readily available to interested members of the CF research community.
