Zinc-finger nucleases for somatic gene therapy: the next frontier - PubMed (original) (raw)

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Zinc-finger nucleases for somatic gene therapy: the next frontier

Shamim H Rahman et al. Hum Gene Ther. 2011 Aug.

Abstract

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. This review describes how ZFNs have been applied to models of gene therapy, discusses the opportunities and the risks associated with this novel technology, and suggests future directions for their safe application in therapeutic genome engineering.

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Figures

FIG. 1.

FIG. 1.

Zinc-finger nuclease-mediated DNA cleavage. (A) Architecture of a zinc-finger nuclease (ZFN) subunit. A ZFN subunit encompasses three to six zinc-fingers arranged in a tandem array (depicted here is a three-finger array) and the catalytic domain of the _Fok_I endonuclease. A short linker connects the two domains. (B) ZFN target site. The target site is composed of the two target half-sites, which are separated by a short DNA spacer. (C) After dimerization of the two ZFN subunits, the nuclease is activated and cuts the DNA within the spacer sequence, leaving a 5′-overhang. (D) ZFN off-target activity. Insufficient specificity of DNA-binding permits ZFN binding to and cleaving at unintended DNA sites. Color images available online at

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FIG. 2.

FIG. 2.

Zinc-finger nuclease mediated genome editing. (A) Targeted gene disruption. A DNA double-strand break (DSB) introduced by a ZFN is repaired by the error-prone nonhomologous end-joining (NHEJ) pathway. The resulting insertions and deletions at the cleavage site can disrupt the coding sequence by inserting frameshift or nonsense mutations (m), leading to a functional knockout. (B) Targeted deletion. A targeted chromosomal deletion can be achieved if two DSBs are created simultaneously by two pairs of ZFNs at two adjacent sites on a chromosome. (C) Gene correction. A genetic defect in the genome is restored by co-introducing ZFN with a donor DNA that encompasses wild-type sequences homologous to the mutated gene (Xm). The ZFN-induced DSB stimulates homology-directed repair (HDR) between the donor DNA and the defective gene. (D) Gene complementation by targeted integration into a safe harbor. In order to restore the phenotype of a cell harboring a genetic defect, a therapeutic expression cassette (V) flanked by sequences homologous to the safe harbor locus is embedded into a donor DNA. The ZFN-induced DSB stimulates HDR and results in targeted integration of the therapeutic gene. Color images available online at

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