Zinc Finger Nucleases: Tailor-made for Gene Therapy

Abstract

Genome editing with the use of zinc finger nucleases has been successfully applied to variety of a eukaryotic cells. Furthermore, the proof of concept for this approach has been extended to diverse animal models from Drosophila to mice. Engineered zinc finger nucleases are able to target specifically and manipulate disease-causing genes through site-specific double strand DNA breaks followed by non-homologous end joining or homologous recombination mechanisms. Consequently, this technology has considerable flexibility that can result in either a gain or loss of function of the targeted gene. In addition to this flexibility, gene therapy by zinc finger nucleases may enable persistent long term gene modification without continuous transfection- a potential advantage over RNA interference or direct gene inhibitors. With systemic viral delivery systems, this gene-editing approach corrected the mutant factor IX in models of mouse hemophilia. Moreover, phase I clinical trials have been initiated with zinc finger nucleases in patients with glioblastoma and HIV. Thus, this emerging field has significant promise as a therapeutic strategy for human genetic diseases, infectious diseases and oncology. In this article, we will review recent advances and potential risks in zinc finger nuclease gene therapy.

Fig. 1. The components and mechanisms of Zinc Finger Nuclease (ZFN)

ZFN is composed of a three-finger binding domain and a FokI nuclease cleavage domain that are connected by a linker. Each finger binds to three base pairs. Site-specific double strand break (DSB) can be induced by ZFN at endogenous loci. The repair process leads to small gene deletions or insertions in non-homologous end joining (NHEJ) pathway. Corrected-gene or transgene (one or multiple) can be integrated into the mutant gene by an alternative mechanism, homology-directed repair (HDR), while co-expressing ZFN and donor DNA.

Fig. 2. Therapeutic applications of ZFN

Three general strategies have been used to deliver ZFN in animal models. Upper, gene-modified offspring can be generated by directly injecting ZFN encoded DNA or mRNA into the embryos followed by implantation in foster mothers. Middle, human T cells or stem cells modified by ZFN ex vivo can then be transplanted to an animal model. Lower, ZFN can be delivered to the target cells to correct mutant genes by using adeno-associated virus (AAV) as a vector.