Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [BAR-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. We performed clonal tracking of HDR-edited HSPCs by a barcoding-based strategy in xenotransplanted NSG mice. We performed deep-sequencing of the on-target barcoded region in bulk and human sorted hematopoietic subpopulations. Transplanted HSPCs were edited in the AAVS1 locus using the barcoded AAV6 vector in presence or absence of GSE56 and/or Ad5-E4orf6/7.