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 in vitro, 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 PKLR 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 in vitro, 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.