Genetic Correction And Analysis Of Induced Pluripotent Stem Cells (ipscs) From A Patient With Gyrate Atrophy

Abstract

Purpose: : To correct the ornithine-Δ-aminotransferase (OAT) gene defect in iPSCs derived from a patient with gyrate atrophy (GA), and to examine the genomic integrity of the gene-corrected iPSCs.

Methods: : GA-iPSCs free of transgene sequences were generated using an episomal reprogramming strategy. Gene correction was performed using a BAC-based vector containing the complete OAT coding region and a loxP-flanked puromycin selection cassette. Correctly targeted iPSC clones were identified by PCR, DNA sequencing, FISH and high-density array comparative genomic hybridization (aCGH). Cytogenetic analysis, aCGH and exome sequencing were also performed to assess the genomic integrity of an iPSC line after reprogramming, gene correction and removal of the selection cassette.

Results: : 20 puromycin resistant colonies were obtained following electroporation of the gene-targeting construct into GA-iPSCs. The desired gene-targeting event was confirmed in 10% of clones. Although no abnormalities were observed with G-band metaphase analysis of the correctly targeted iPSC lines, aCGH and exome sequencing identified various subkaryotypic alterations. However, the vast majority of these were associated with reprogramming, consistent with previous reports. Besides correction of a single nucleotide in the OAT locus, no additional protein coding changes or copy number variation was identified in a gene-corrected iPSC line following gene targeting and cassette removal.

Conclusions: : We used BAC-mediated homologous recombination to correct a single base pair mutation in GA-iPSCs. Clonal events, drug selection and prolonged iPSC culture did not appear to cause a substantial increase in mutational load, which is crucial when considering the use of gene-corrected iPSCs for downstream therapeutic applications.

Keywords: gene transfer/gene therapy