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RK Shuler, JH Boatright, E Stodulkova, K Rengarajan, SA Padove, JM Nickerson; Initial Investigation of Gene Repair in Ocular Tissues . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4591.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Gene therapy utilizing oligonucleotides to repair targeted mutant genes (genoplasty) has many advantages over current gene therapy strategies mediated by viral or plasmid vectors. Genoplasty, which corrects the native gene, eliminates the complications associated with host immune response, gene regulation, and cell targeting. To determine if gene repair can occur in RPE and ocular melanocytes, we tested genoplasty in a mouse model of human tyrosinase-negative albinism. Methods: We synthesized a RNA-DNA oligonucleotide (RDO) and a single-stranded DNA oligonucleotide (SDO) targeting the mutation coding for a nonfunctional tyrosinase protein in BALB/C and FVB/N albino mouse strains. Albino mouse eye explants and three different albino mouse cell lines derived from tumors of RPE and ocular melanocytes (J Y Niederkorn) were transfected with either the RDO or the SDO. Gene conversion was assessed by allele-specific real time PCR (ASRT-PCR), Fontana-Masson staining (a melanin specific stain), and western analysis using tyrosinase-specific antibodies. Results: Transfection of explants and the three cell lines with fluorescently-tagged SDO resulted in significantly increased fluorescence in treated nuclei compared to untreated controls. ASRT-PCR data was consistent with a 0.3% rate of gene repair to wild type compared with negative controls after transfection with the SDO in one cell line. Parallel experiments performed in the rd1/rd1 mouse model of Retinitis Pigmentosa demonstrated a gene repair rate of 2.5±0.77%. Fontana-Masson staining of BALB/C mouse eye explants treated with RDO demonstrated positive staining as opposed to negative controls. Western analysis of RDO and SDO treated cell lines showed increased amounts of tyrosinase protein versus untreated. Conclusion: Fluorescence microscopy suggested that high levels of oligonucleotide were delivered to nuclei in ocular tissues. ASRT-PCR data revealed a small percentage of endogenous mutant genes were repaired by SDO treatment. The Fontana-Masson staining and western analyses indicated functional tyrosinase was produced in albino ocular explants and albino cell lines following RDO or SDO treatment. These initial, limited results suggest that genoplasty can repair the tyrosinase Cys 85 Ser mutation, resulting in functional tyrosinase activity in vivo and in vitro.
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