Chapter 1 —— 13 —— functions including osmotic regulation. Interestingly, specific point mutations in ATP1A1 conferring ouabain resistance are naturally found in the human population without disrupting the regular physiological functions of the Na+/K+ ATPase. Hence, aiming at improving the performance of AAV-based gene editing procedures, we sought to investigate AAV donor constructs harboring marker-free co-selection components (selector AAV vectors) permitting ouabain-dependent enrichment for genome-edited cells. We demonstrate that combining selector AAV vectors with ouabain treatments, in addition to enriching for genome-edited cell populations, eliminates imprecise on-target edits and off-target and/or random donor DNA insertions from said populations. Importantly, selector AAV vector titration experiments showed that the highest fold-enrichment factors for genomeedited cell fractions are associated with the lowest vector input amounts. This finding is expected to be beneficial for alleviating AAV vector production costs, off-target donor insertions and P53-dependent activation of the DNA damage response linked to AAV DNA, which is known to be particularly deleterious in stem cells with scientific and therapeutic relevance, e.g., induced pluripotent stem cells and hematopoietic stem cells.
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