Chapter 4 —— 46 —— together with the AAVS1-targeting gRNA GS1 (i.e. AdVP.eC94NLSGS1). The eCas94NLS protein is a variant of eSpCas9(1.1) [29] whose enhanced performance results from having two extra nuclear localization signals [30]. Importantly, both AdVP.C9KARA and AdVP.eC94NLSGS1 contained intact vector genomes (Supplementary Figure S1). Dose-response transduction experiments in hMSCs confirmed that second-generation AdVs present a strikingly higher cytotoxicity profile than viral gene-free AdVPs (Supplementary Figure S2 and Supplementary Files https://figshare.com/s /a999fdb4400aaf1828c5). These data are similar to earlier results from our laboratory showing that, amongst second-generation AdVs and viral genefree AdVPs containing the same transgene cassettes, the former induces notably higher and dose-dependent cytotoxic effects in transduced cells [20].
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