Chapter 2 —— 19 —— chromosome fragmentation followed by haphazard reassembly (chromothripsis) [23,24] and the partial or entire loss of chromosomes (aneuploidy) [25]. Notably, the chromothripsis and aneuploidy phenomena were readily detected in T cells and hematopoietic progenitor cells subjected to CRISPR-Cas9 nuclease reagents used in clinical trials [23,25]. Notwithstanding these phenomena, recent findings are more reassuring in that, contingent upon gRNA target site selection, chromosomal losses in particular can be substantially minimized by inducing DSB formation before, as opposed to after, the activation/stimulation of the primary T-cell populations [26]. Finally, on-target DSBs trigger P53-dependent cell cycle arrest and apoptosis which limits the efficacy of HDR-mediated genome editing in regular P53positive cells [27,28], and creates selective pressure for the emergence of mutations associated with tumorigenesis. Related to the latter matter, during sub-culturing, pluripotent stem cells can acquire ‘spontaneous’ tumorassociated P53 mutations in a recurrent fashion [29] which, by virtue of being more resistant to DSBs, are in principle more prone to expansion than their wild-type counterparts once exposed to programmable nucleases. Indeed, CRISPR-Cas9 nuclease activation of certain signaling pathways can lead to the selection of cells with potentially harmful loss-of-function or dominantnegative mutations in the tumor-suppressor P53 transcription factor or gainof-function mutations in the KRAS oncoprotein [27,30]. Furthermore, recent mouse modelling experiments indicate that p53 mutant cells, rather than proceeding to malignancy via a haphazard route, are instead subjected to an unexpectedly more deterministic set of genetic instability events [31]. Together, these cytotoxic and genotoxic effects raise tangible concerns on the use of programmable nucleases for the optimal generation of autologous genetically-corrected cell products.
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