Chapter 4 —— 70 —— linear protein-capped adenovector genomes [13,26], AAV HMEJ donors do not necessarily outperform their AAV HR donor counterparts in terms of global gene knock-in proficiencies, which is consistent with data released while this work was under review [68]. It is conceivable, nonetheless, that in different cell types or genomic positions, differences in genome editing efficiencies between AAV vectors presenting HMEJ versus HR templates will arise. Possibly, incoming AAV genomes harboring intrinsically recombinogenic secondary-structured ITRs [16,17] preclude, to a large extent, the need for DSB-mediated HMEJ donor excision in order to facilitate target DNA engagement. On the other hand, earlier studies demonstrated that incoming AAV vector genomes form ITR head-to-tail concatemers in transduced cells [69,70]. Recent experiments established that such multiple copy genomes can be readily detected at CRISPR-Cas9-induced chromosomal breaks when derived from AAV HR donors [68]. These byproducts, alike head-to-tail integrase-defective lentivector donor concatemers [13], are expected to be substantially more disruptive when editing coding sequences than when targeting safe harbor loci introns or other non-coding sequences to, for instance, complement a genetic defect. Interestingly, supporting the view that the AAV ITRs drive end-to-end DNA recombination, targeted AAV donor concatemer insertions seem to be significantly diminished when using ‘double-cut’ AAV HMEJ donors [68]. However, the fate of the ITRs released upon DSB-processing of AAV HMEJ donors warrants further investigation. This is especially so in therapeutic contexts where off-target insertion of these transcriptionally competent elements can lead to insertional mutagenesis and, potentially, proto-oncogene deregulation and oncogenesis [63,66,67]. Moreover, determining whether additional DSB formation upon AAV HMEJ donor cleavage exacerbates DDR activation merits further investigation. Consistent with earlier experiments using HMEJ and other ‘double-cut’ templates embedded in plasmid DNA [7–9] or protein-capped adenovector genomes [13,26], herein tested scAAV HMEJ donors yield lower amounts of
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