Chapter 4 —— 52 —— genotoxic drugs (e.g. hydroxyurea and etoposide) equally fosters productive AAV vector transduction [49]. Moreover, RNA interference screens retrieved an overrepresentation of hits whose down-regulation stimulates both DNA damage and AAV transduction, although top-scoring hits act on DDR pathways only indirectly [50]. These cumulative findings support the hypothesis that genomic lesions recruit DNA damage-sensing factors that would otherwise inhibit productive AAV transduction via vector DNA binding [50]. To investigate in a more direct manner a causal relationship between productive AAV transduction and specific DNA lesions, namely programmable nuclease-induced DSBs, HeLa cells were exposed to CRISPRCas9 complexes endowed with either highly specific or promiscuous gRNAs, i.e. gRNACALM2 and gRNAVEGFA, respectively [29,51,35,36]. Real-time livecell imaging of these cells transduced with an EGFP-expressing AAV vector or with its isogenic scAAV counterpart revealed a clear DSB-dependent increase in transgene expression regardless of the AAV vector structure (Figure 3A – C). scAAV vectors, assembled by using AAV transfer constructs in which one of the two ITR terminal resolution sites is mutated, promptly hybridize upon uncoating to form transcription-competent doublestranded genomes owing to their internal inverted repeat arrangement (Supplementary Figure S7) [12,52,53]. Monitoring of AAV vector transduction via real-time live-cell imaging established the expected higher transgene expression kinetics of scAAV-HRS1 over that of conventional single-stranded AAV-HRS1 (Figure 3D and E). Notably, regardless of AAV genome arrangement, productive transduction was highest in cells initially subjected to the highest number of DSBs induced by the promiscuous Cas9:gRNAVEGFA complexes (Figure 3A – C). This data is consistent with the aforementioned hypothesis whereby DNA damage, in this case CRISPRCas9-induced DSBs, recruits DDR proteins known to bind palindromestructured AAV genomes and, in doing so, leave said genomes unhindered for transgene expression.
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