Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [RNA-seq]

By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs. Examination of the transcriptional profile in HSPCs treated with 5 different experimental conditions or untreated (UT). The experiment was performed in 3 replicates. Three-days cultured HSPCs were treated as follows: electroporation without adding any further component beside electroporation solution (UT_electro); electroporated with Cas9 RNP targeting the AAVS1 locus and transduced with the cognate AAV6 for HDR (RNP + AAV6); edited as in RNP + AA6 adding to the electroporation solution an mRNA encoding for the p53 inhibitor GSE56 (GSE56); edited as in RNP + AA6 adding to the electroporation solution an mRNA encoding for Ad5-E4orf6/7 (Ad5-E4orf6/7); edited as in RNP + AA6 adding to the electroporation solution an mRNA encoding for GSE56 and Ad5-E4orf6/7 (GSE56/Ad5-E4orf6/7). Transcriptomic analysis was performed 12 hours after treatments.