Zinc-finger nucleases (ZFNs) are a powerful tool that can be used to edit the human genome ad libitum. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to generate zinc-finger proteins. As a result, two phase I clinical trials aimed at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these lymphocytes from infection with the virus have been initiated. Moreover, ZFNs have been successfully employed to knockout or correct disease-related genes in human stem cells, including hematopoietic precursor cells and induced pluripotent stem cells. Targeted genome engineering approaches in multipotent and pluripotent stem cells hold great promise for future strategies geared toward correcting inborn mutations for personalized cell replacement therapies.
Short-lived ZFN expression from episomal DNA-based expression vectors—such as plasmid DNA, integrase-deficient lentiviral vectors, adenoviral vectors, and vectors based on adeno-associated virus—can only be achieved in mitotic cells, which ensures rapid dilution of the vectors during cell divisions. Because DNA-based vector systems have a tendency to integrate into the host genome, it will be important to closely follow the fate of the ZFN expression vectors in the target cells. An alternative way of delivering ZFNs is the transfer of ZFN-encoding mRNA, which ensures rapid but transient ZFN expression and avoids the issue of illegitimate integration.
Microinjection of ZFN-encoding mRNA has been performed in zebrafish and rat single-cell embryos, and the ZFN-mediated gene disruption frequency was comparable to plasmid DNA delivery. Moreover, delivery of ZFNs by mRNA transfection has been used to target the integration of a transgene into the AAVS1 locus in human iPSCs.
If direct in situ correction of a disease locus is not an option, an important consideration will be to determine where to integrate a therapeutic transgene cassette into the human genome. The AAVS1 site on chromosome 19 is thus far the most promising candidate for such a safe harbor, as a native insulator region appears to both protect transgene expression from position-effect variegation and silencing and prevent the transgene promoter from affecting the host transcriptome.
The fact that ZFNs can be used to create knockout animals is especially encouraging and emphasizes the high specificity the technology has reached in the last 3 years. Moreover, the development of alternative designer nucleases, such as TALENs and meganucleases, has further spurred interest in targeted genome engineering approaches. Conversely, studies reporting ZFN off-target activities in zebrafish and human cells must not be overlooked and should serve as the basis for further improvement of the technology. The employment of highly specific designer nucleases is especially important when DSB-based genome engineering is applied to multipotent or pluripotent stem cells, such as HSCs or iPSCs, with their high proliferative potential. Even so, the remarkable progress achieved in the last few years demonstrates that ZFNs represent a tool that allows researchers and clinicians for the first time to rationally edit the genome of human cells and to take this technology from the bench to the bedside for therapeutic applications.