Chapter 2 —— 18 —— adoptive immunotherapy field as a potential alternative to CAR-T cells owing to their intrinsic tumor-cell killing activity and fewer adverse effects in patients [13]. Yet, regardless of the target cell type, in what the genetic modification procedures are concerned, the adoptive immunotherapy field is moving from randomly integrating retroviral vector and DNA transposon systems to targeted transgene insertion approaches using programmable nucleases [3,10]. In contrast to unpredictable CAR or TCR donor construct integration, programmable nuclease-assisted genome editing assures stable and homogeneous transgene expression while minimizing insertional mutagenesis risks inherent to randomly integrating vehicles. In fact, in contrast to random, targeted TCR transgene insertion leads to predictable Tcell function in vivo [14]. In this context, genomic loci generically dubbed ‘safe harbor’ are particularly appealing endogenous landing pads for CAR and TCR transgenes as insertions at these sites minimize the chances for gene silencing or variegated transgene expression while preserving the endogenous transcriptome of engineered cells [15,16]. Main limitations of CRISPR nuclease-assisted genome editing As aforementioned, programmable nucleases and HDR-tailored donor DNA constructs yield precise gene knock-ins. However, a major limitation regarding the use of programmable nucleases is the fact that, in mammalian cells, DSBs are prevalently repaired via mutagenic non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) processes instead of accurate HDR [17,18]. Moreover, in contrast to HDR, end-joining processes take place throughout the cell cycle. As a result, amongst cells exposed to donor constructs and programmable nucleases, the vast majority contains one or both target alleles disrupted by NHEJ- or MMEJ-derived small insertions and deletions (indels). This mutagenic burden, in the form of indel ‘footprints’, can lead to target protein imbalances and cell fitness losses [19]. In addition, on-target DSB formation can also yield translocations and gross chromosomal rearrangements [19–22]. Recent studies have further uncovered that on-target DSBs are capable of triggering extensive
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