Chapter 5 —— 119 —— respectively, in K562 cells (Levesque et al. 2022). Hence, after assembling AAV donor constructs endowed with matched gRNA units and ouabainselectable sequences, we demonstrate that these selector AAV vectors, in addition to enriching for gene-targeted cell populations, achieve concomitant removal of imprecise HR-independent edits and off-target and/or random AAV donor DNA insertions. Interestingly, selector AAV vector titration experiments revealed that the highest fold-enrichment factors of genetargeted cell fractions are associated with the lowest vector input amounts which are expected to be beneficial for alleviating both AAV production costs and detrimental P53-dependent DDR activation. In this study, two types of selector AAV vector designs were investigated, namely, vector particles containing bipartite donor modules for ATP1A1 and target gene co-editing, and vector particles bearing an in-linkage donor DNA module for direct ATP1A1 targeting and selection. Selector AAV vectors with bipartite donors can be customized to implement distinct types of genomic edits (e.g., gene knock-ins, gene-tagging, or gene-repairing) at different loci, like the herein targeted AAVS1 safe harbour locus and LMNA alleles. The former locus is a commonly used genomic landing pad for achieving homogenous and stable transgene expression (Lombardo et al. 2011; Pavani et al. 2021); the latter encodes lamin, a product found in the nuclear lamina matrix of proteins located underneath the inner nuclear membrane and whose mutations underpin, for instance, Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome. However, although the co-delivery of selectable ATP1A1 and donor sequences in single AAV vectors assures their presence in individual cells, at low vector doses, bipartite donor availability at primary and secondary loci should become limiting. This point is consistent with the observed gradual decrease in ouabain-dependent gene editing levels at primary target sequences as a function of diminishing AAV vector amounts.
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