Chapter 6 —— 156 —— through imprecise NHEJ processes [11,12,16,17]. These end-joining processes can further yield chromosomal insertion of concatemeric and subgenomic AAV species further compounding the range of HDR-independent bystander events [11,12,17,18]. Finally, possibly due to mimicking DNA lesions or repair intermediates, AAV genomes can impair cell viability through P53-dependent DDR activation whose consequences have been reported to be particularly deleterious in stem cells [19,20]. In conclusion, a growing amount of research indicates that there are distinctive genotoxic and cytotoxic effects linked to chromosomally integrated and episomal AAV DNA forms, respectively, with the build-up of these deleterious effects being strictly proportional to input AAV vector amounts. Hence, with the aim of tackling the shortcomings associated with AAV-based genome editing, in Chapter 5, a marker-free co-selection system [21], dependent on the potent specific inhibitor of the Na+/K+ ATPase pump encoded by ATP1A1, ouabain, was co-opted and investigated. To this end, AAV donor vectors endowed with a secondary ATP1A1-selectable donor module and matched gRNA units were assembled and tested. Such AAV designs, dubbed selector AAV vectors, seek enriching for cells coedited through at a primary target locus and ATP1A1 alleles that confer resistance to ouabain upon HDR-mediated acquisition of specific polymorphisms. Importantly, besides enriching for gene KI cell populations, combining selector AAV vectors with ouabain selection triggered the elimination of HDR-independent edits and off-target and/or random AAV donor DNA insertions from said populations. This selector AAV principle was successfully applied for inserting whole transgenes at safe harbor genomic loci as well as for tagging endogenous proteins. Further, selector AAV vector titration experiments revealed that the highest fold-enrichment factors of gene-targeted cell fractions were associated with the lowest vector input amounts. This feature might become beneficial for alleviating both AAV production costs and detrimental P53-dependent DDR activation. Of note, the dual viral vector platform described in Chapter 4 was put to use for
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