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V.T. Ciavatta, S.A. Padove, J.H. Boatright, J.M. Nickerson; Genoplasty in Mouse Retina Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2336.
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Purpose: Genoplasty is emerging as a viable method for treating a variety of genetic disorders. Our goal is to determine if genoplasty can be effective in treating diseases manifested in post-mitotic retinal cells. To this end, the present study used mouse tissue and cell lines to: 1) ask if retinal cell nuclear extracts support genoplasty and 2) help dissect the genoplasty mechanism. Methods: An in vitro reaction, in which nuclear extracts were incubated with plasmid DNA and a repair oligonucleotide, and genetic readout in E. coli were used to assess gene repair activity conferred by nuclear extracts. To define polymerase requirements, specific inhibitors of DNA polymerases were included in repair reactions. Nuclear extracts were prepared from liver (positive control), neural retina, and RPE-derived cell lines. The plasmid, pBRtetS, was a tetracycline-sensitive version of pBR322 in which a point mutation creates a premature stop codon in the tet gene. The repair oligonucleotide, either an RNA/DNA hybrid molecule or an all DNA molecule, was designed to repair the point mutation in the tet gene. After incubation, plasmid DNA was recovered from the in vitro reactions and transformed into recA1 E. coli. Aliquots of transformed E. coli were plated on ampicillin- and tetracycline-containing LB plates. Successful gene repair was indicated by restoration of tetracycline resistance and confirmed by restriction digests and sequencing of plasmids from tetracycline resistant colonies. Results: Incubation with nuclear extracts from either neural retina, RPE/choroid, or an RPE-derived cell line resulted in 1 to 10 tetracycline resistant colonies per million ampicillin resistant colonies, with neural retina extracts yielding the highest repair rates. For comparison, incubation with liver extracts resulted in 10 to 100 tet colonies/million amp colonies. Boiling the extract, excluding the extract or repair oligonucleotide, or substituting an indifferent oligonucleotide resulted in <0.01 tet/million amp, suggesting a 100 to 1,000 stimulation over background for retinal extracts. Addition of aphidicolin (100 ng/mL), an inhibitor of family B mammalian DNA polymerases, increased the ratio of tet to amp colonies by 2 to 3 fold compared to controls. Conclusions: In vitro genoplasty is supported by nuclear extracts from mouse neural retina, RPE/choroid, and an RPE-derived cell line. Sensitivity to boiling suggests one or more proteins are involved in the process. That tet:amp ratio was not decreased by aphidicolin suggests a DNA polymerase other than those in family B is used during repair. The results are encouraging for further development of genoplasty for gene therapy in the eye.
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