Chapter 6 —— 154 —— length base editor proteins. This work departs from earlier studies based on dual AAV delivery of RGNs where potentially deleterious outcomes in the form of prevalent end-joining ‘capture’ of Cas9-encoding AAV genomes at nuclease target sites were detected in various murine tissues, including skeletal muscle [11,12]. It is consensual the view that a key bottleneck regarding the application of genome editing technologies concerns the difficulty in delivering the attendant tools in an effective manner, especially so into regular, nontransformed and primary cells including therapeutically relevant cell types. In this context, physical and chemical transfection methods based on, for instance, electroporation, polyplexes and polycations, permit introducing RGNs and donor DNA constructs into mammalian cells. However, achieving optimal transfection efficiencies without noticeable cytotoxic effects in the aforementioned primary cell types, remains challenging. Moreover, neither electroporation nor transfections of RGNs and/or donor DNA substrates are easily amenable to in vivo settings. And, often, these gene-editing tool delivery methods depend on cell type-specific reagents whose compositions may be unknown due to proprietary reasons. Equally of note, electroporation and transfections are further reliant on time-consuming optimizations of cell type-specific parameters whose performances can vary due to subtle experimental conditions, e.g. cell-cycle stage profiles of target cell populations. Diversely, viral vector transductions are straightforward to perform and typically offer higher reproducibility independently of the target cell type of choice [13]. These characteristics stem from the fine-tuned mechanisms evolved by the parental viruses for nuclei acid transfer into host cell nuclei. In Chapter 4, inspired by the complementary attributes of two distinct classes of viral gene-free viral vectors, namely, high-capacity adenoviral vectors (AdV) and AAV vectors (e.g., large cargo compatibility and source of recombinogenic HDR substrates, respectively), a dual viral vector system is
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