Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.

Zinc finger nucleases (ZFNs) are chimeric, engineered transcription factors fused to the nonspecific endonuclease domain of restriction enzymes such as FokI. These recombinant proteins can be designed to recognize virtually any DNA sequence in the human genome and to stimulate homologous recombination by creating a targeted double-strand break (DSB).1,2 This strategy can be used to permanently repair a pathogenic nucleotide sequence and thus has significant potential for the treatment of genetic diseases. ZFNs bind DNA by virtue of their zinc finger transcription factor DNA-binding domain and induce a DSB through their nuclease domain (Fig. 1A). To induce a DSB, two FokI nuclease domains must dimerize in close proximity. Therefore, two separate ZFNs are required to generate a DSB. Given that each ZFN may contain, for example, three “fingers” that recognize a total of nine nucleotides, a pair of ZFNs can recognize 18 base pairs. An 18-nucleotide sequence is sufficient to promote a unique binding event and, hence, a single cleavage within the human genome (3 × 109 bp) because there are 418 (6.9 × 1010) unique 18-mers.3 Figure 1. ZFN design. (A) Schematic illustration of ZFN pair binding to DNA and FokI domains. The FokI cleavage domain is fused to the DNA-binding domain via (Gly3Ser)4 linker. An SV40 nuclear localization signal (SV40 NLS) located at 5′- of ZFN sequence. ... Although homologous recombination naturally occurs at a rate of 10−6, a DSB is expected to increase the frequency of specific homologous recombination events by up to 1000-fold or more, thereby approaching recombination rates with therapeutic potential.4–8 This approach has been invoked at an endogenous locus in the human genome, resulting in permanent modification of up to 20% of chromatids in vitro.1 Although mature neurons generally do not divide, there is evidence that double-strand break repair does occur by homologous recombination.9–11 Retinitis pigmentosa (RP) is a group of retinal degenerative diseases that are characterized primarily by the loss of rod photoreceptor cells. RP is the most common cause of genetic blindness and affects approximately 1 in 3000 people in the United States.12 Patients with RP experience night blindness and progressive loss of peripheral visual fields and eventually lose central vision as cone-cell viability is compromised by rod-cell death. There is no cure for RP. More than 200 different mutations in the human rhodopsin gene are known to be associated with RP.13–16 Although some of the mutations in the rhodopsin gene cause recessive RP and congenital stationary night blindness, most are implicated in autosomal-dominant RP. In this study, a pair of ZFNs was designed to bind and cleave a sequence uniquely found in the human rhodopsin gene. We demonstrate that the engineered ZFNs induce a DSB at the rhodopsin locus and stimulate homologous recombination when delivered concurrently with an ectopic donor DNA fragment homologous to the human rhodopsin gene sequence. We go on to further quantify the relative frequency of homologous recombination based on several in vitro conditions. Delivery of these rhodopsin-targeting zinc finger nucleases and an appropriate donor fragment to photoreceptors, either separately in an AAV or concomitantly in a gutted adenovirus vector, would enable potential clinical application for RP patients with mutations in the human rhodopsin gene.