The p53 challenge of hematopoietic stem cell gene editing

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 -deficient chronic granulomatous disease

“…Our data are in line with reports on the use of AAV6 for template delivery in HDR-based editing, 55 , 56 , 57 indicating that the observed genotoxicity may be due to activation of p53-mediated DNA damage response upon concomitant exposure of HSPCs to CRISPR-induced DSBs and prolonged persistence of AAV genomes (ITRs) in the edited cells. 58 , 59 We cannot judge on the potential toxic effects mediated by IDLV template delivery in HSCs as no correction was achieved with our construct. Another potential challenge with our approach could arise from the fact that the HDR mechanism specifically targets cycling cells (S/G2 phase), raising the possibility that a large fraction of dormant, long-term reconstituting HSCs will not be targeted.…”

Enrichment strategies to enhance genome editing

“…Depending on the desired edit and target locus, numerous factors and challenges can impair genome modification efficiencies, some of which include: (1) inefficient delivery of the gene editing system [ 12 , 13 ], (2) toxicity caused by delivery modality and exposure to gene editing components [ 14 – 16 ], (3) restriction to a narrow target sequence window with inefficient nuclease target sites, which could be caused by complex target sequences with repetitive elements, unusual GC content, or a dense chromatin state [ 17 , 18 ], and (4) quiescent cells without an active endogenous repair machinery [ 11 , 19 ]. Consequently, suboptimal gene editing efficiencies may hamper the use of gene editing technologies for some applications [ 1 , 20 ].…”

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi

“…The presence of foreign nucleic acids triggers DNA damage response (DDR) pathways, leading to cell-cycle arrest, transcriptional blockage, reduced proliferation, and potentially apoptosis. 28 , 29 Ensuring safety in the various aspects described is of utmost importance, particularly as Cas9-based therapeutic GE has entered clinical trials. Several talks focused on establishing a standardized protocol for the measurement and evaluation of on- and off-target activity, which included generating big data to develop models that better predict off-target activity.…”