Harnessing DNA barcoding to enhance and sustain polyclonality in gene-edited hematopoietic stem cells

A challenge in gene editing for hematopoietic stem and progenitor cells (HSPCs) is achieving efficient editing while preserving long-term engraftment and clonal diversity. Tracking edited clones with high resolution is essential to understand the impact of editing on hematopoiesis. We developed a barcoded AAV6 donor template (BC-AAV) to precisely monitor the fate of edited HSPCs following transplantation. Our findings reveal that, despite initial barcode diversity <i>in vitro</i>, human hematopoiesis generated by edited HSPCs transplanted in immunodeficient mice is driven by a limited number of dominant clones. The engraftment of gene-edited cells follows an oligo/polyclonal pattern, indicating that editing does not alter clonal dynamics in this model. Using BC-AAV, we optimized a gene editing protocol for correcting the <i>PKLR</i> gene, responsible for pyruvate kinase deficiency, a rare disorder that causes severe anemia due to red blood energy imbalance. We implemented key improvements. GMP-grade StemSpan AOF medium and StemRegenin-1 increased clonal diversity while maintaining hematopoietic potential. NHEJ inhibitor AZD-7648, significantly boosted editing efficiency <i>in vitro</i>, and a shorter transduction period enhanced engraftment and clonal balance without compromising editing outcomes. This refined strategy for gene editing in human HSPCs optimizes both efficiency and long-term polyclonal dynamics and has important implications for clinical applications.