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Magdalena Staniszewska, Mark Consugar, Michael Farkas, Eric Pierce; Oligonucleotide-mediated gene correction in human primary fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2475.
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© ARVO (1962-2015); The Authors (2016-present)
The use of stem cell transplantation has great potential for the treatment of patients with inherited and other retinal degenerative disorders. One approach to therapy currently being evaluated is the use of gene-corrected induced pluripotent stem (iPS) cells. To test efficacy of gene editing induced by single-stranded oligodeoxynucleotides (ODNs), we evaluated the ODN-mediated correction of the pathogenic mutation in fibroblasts from a patient with gyrate atrophy. Next-generation sequencing (NGS) was utilized to address the challenge of identifying cells with the corrected gene in the absence of a selection system.
Human skin fibroblasts isolated from patient with gyrate atrophy due to a p.Ala226Val (c.C677T) mutation in OAT gene were lipofected with correcting (WT) or control (mutant) 59-nucleotide ODNs. The transfected cells were plated in a 96-well plate at a density of 100 cells/well. The targeted OAT region was PCR-amplified from each well of the 96-well plate with barcoded primers, and the purified amplicons were pooled. Illumina NGS was used to identify wells containing corrected cells, which where enriched by sub-cloning. Diagnostic digestion of the PCR-amplified targeted OAT region was used to verify gene editing events in the sub-cloned fibroblasts.
NGS of the pooled PCR products generated an average of 363,000 sequence reads per well. A total of 25 wells from the plate of cells treated with the correcting ODN had the C677 nucleotide detected above the background rate. Twenty one wells from the control plate were also identified using this approach. Sub-clonal enrichment and diagnostic digestion of the amplified targeted OAT region showed that 22% (7/31) of viable sub-clones transfected with correcting ODN contained WT sequence. In contrast, none of the viable sub-clones from the cells receiving control ODN showed a WT digestion pattern. We were not able to obtain clonal populations of ODN-corrected cells due to senescence of the fibroblasts.
These results suggest that ODNs can be used to correct single point mutations in primary cells, and that corrected cells can be detected using NGS without introduction of a selection cassette. The experiments also demonstrate that fibroblasts are not the optimal cell type for therapeutic gene correction, since they have a limited growth capacity. Testing of similar approaches in iPS cells may be warranted.
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