709. Gene Correction and Gene Knockouts in Human Somatic Cells Using Zinc Finger Nucleases

Abstract

A potentially ideal way to treat monogenic diseases is to correct the mutation that causes the disease rather than complement the mutation by adding back an unmutated copy of the gene. One way of performing gene correction is by gene targeting. Gene targeting is the replacement of an endogenous segment of DNA with a homologous exogenous segment of DNA by homologous recombination. The rate of gene targeting is usually far too low in human somatic cells to be of therapeutic or experimental utility. If a DNA double-strand break is created in the genomic target gene, however, the rate of gene targeting increases by several thousand fold and can reach an overall rate of 3-5%. These high rates of gene targeting were achieved using the I-SceI endonuclease (Sce) and by inserting a Sce target site into a reporter gene. For gene targeting to become useful, a method to create a DSB in any target gene, without the prior insertion of a Sce target site, needs to be developed. Towards this end we have found that model zinc finger nucleases, artificial protein fusions between a zinc finger DNA binding domain and a the endonuclease domain from the FokI restriction enzyme, could stimulate gene targeting nearly as efficiently as Sce in human somatic cells. Zinc finger nucleases act as dimers in which two binding sites (“ZFN half-sites”) are in an inverted orientation separated by six basepairs (“ZFN full site”). In this work, I show that zinc finger nucleases can be designed to stimulate gene targeting at half sites from the human-β globin and the human common-γ chain genes. These half-sites had the consensus sequence 5′-GNNGNNGNN-3′. Furthermore, I was able to design zinc finger nucleases to stimulate gene targeting at the “ZFN full-site consensus sequence” 5′-nnCnnCnnC833nGnnGnnGnn-3′. The GFP gene contains a natural ZFN full site consensus sequence and the GFP zinc finger nucleases stimulated gene correction by gene targeting by 1000-fold at this site. The human CD8α gene also contains a ZFN full site consensus sequence and the CD8 zinc finger nucleases efficiently stimulated gene knockouts via gene targeting in human somatic cells. Overall, therefore, zinc finger nucleases to stimulate gene targeting in endogenous genes can be generated by to the ZFN full site consensus sequence using a relatively easy “off-the-shelf” approach. This finding raises the possibility of being able to use gene targeting as an experimental tool in human somatic cell genetics and, with further development, the possibility of treating monogenic diseases via gene targeting and zinc finger nucleases.