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Guardians of the genome protecting DNA from endogenous sources of damage

ABSTRACT: Analysis of mutational signatures caused by DNA repair defects in human induced pluripotent stem (iPS) cells. A reference human iPS cell line will be used for genetic manipulation to introduce homozygous knockouts of 100 genes known to be involved in or connected to DNA repair or DNA editing. Following a defined period of growth after homozygous knockout of each gene, sub clones will be generated and sequenced. The progenitor Ã¢Â€ÂœparentalÃ¢Â€Â IPS cell line will be used to generate reference sequence data, in order to determine the mutational signature acquired due to the gene knockout.

PROVIDER: EGAS00001000800 | EGA |

REPOSITORIES: EGA

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Project description:Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a novel approach to gene therapy. Yet it is unknown whether selective pressures during prolonged culture and multiple clonal events would introduce a mutational load incompatible with therapeutic use. Here we begin to assess whether the mutational load of gene-corrected iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected following standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine point mutations in protein-coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base pair change, no additional mutations or copy number variation were identified in iPS cells following the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden. Array comparative genomic hybridization (aCGH) were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. Human CGH 2.1M Whole-Genome Tiling v2.0D Array plus 4633 custom-designed probes which cover the gene targeting construct and the reprogramming plasmid vectors. Comparison of patient iPS cell with full skin fibroblast cells was performed on this design.

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Guardians of the genome protecting DNA from endogenous sources of damage

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