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Vincent T. Ciavatta, Cristina Kendall, John M. Nickerson, Jeff H. Boatright; Augmenting Targeted Gene Repair in RPE Cells With Zinc Finger Nuclease-Induced Double-Stranded Breaks. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1397.
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Site directed gene repair in vivo, or ex vivo followed by autologous cell transplantation, could theoretically be used to treat retinal degenerations caused by mutations that disrupt RPE or photoreceptor function. Transfection of wildtype oligonucleotide sequences into mouse photoreceptors can induce site-specific gene repair, but rate of conversion is prohibitively low (Andrieu-Soler C, et al, Mol Vis, 2007). DNA damage in close proximity to the mutant base(s) can potentiate homologous recombination and theoretically increase gene repair events. Zinc finger nucleases (ZFNs), engineered proteins containing a DNA binding domain linked to a nuclease domain, have been shown to increase homologous recombination. Our present goal is to determine if ZFNs designed against the mouse rd12 mutation can produce a double-stranded break and potentiate homologous recombination in cell culture. Mouse Neuro-2A and fibroblasts from rd12 mouse brain were used as surrogates for rd12 mouse RPE cells.
A ZFN heterodimer pair was designed to cut in the third exon, 5’ to the rd12 C to T point mutation. ZFN heterodimer-encoding plasmids were purchased (Sigma). The Neon and Nucleofector electroporators were used for plasmid transfection of Neuro-2A cells and brain-derived fibroblasts from rd12 mice. Cotransfection with GFP encoding plasmid was used to assess transfection efficiency. Cells were maintained at 30°C or 37°C and harvested at 24, 48, and 72 h to assess site-specific ZFN endonuclease activity with a kit (Transgenomic).
Plasmid transfection efficiency was greater than 80% for Neuro-2A cells and fibroblasts. DNA from ZFN-transfected Neuro-2A cells was cut at the expected site in the RPE65 gene. Frequency of cutting increased with time of incubation and was greater at 30°C than 37°C. Site-specific cutting of DNA from ZFN-transfected fibroblasts was faint-to-undetectable.
We are investigating the usefulness of ZFNs to potentiate gene repair in RPE cells. Cutting of the RPE65 gene in Neuro-2A cells confirmed that the ZFN heterodimers are capable of inducing a double-stranded break at the expected site. Lower ZFN activity in fibroblasts was observed relative to Neuro-2A cells. A wildtype RPE65 sequence with silent mutations to disrupt the ZFN binding site and novel restriction sites was created and can be used to test whether the ZFN-induced double-stranded break will increase gene repair incidence at the rd12 locus.
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