Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell (HSPC) gene therapies have recently moved beyond gene-addition approaches to encompass targeted genome modification or correction, based on the development of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas technologies. Advances in ex vivo HSPC manipulation techniques have greatly improved HSPC susceptibility to genetic modification. Targeted gene-editing techniques enable precise modifications at desired genomic sites. Numerous preclinical studies have already demonstrated the therapeutic potential of gene therapies based on targeted editing. However, several significant hurdles related to adverse consequences of gene editing on HSPC function and genomic integrity remain before broad clinical potential can be realized. This review summarizes the status of HSPC gene editing, focusing on efficiency, genomic integrity, and long-term engraftment ability related to available genetic editing platforms and HSPC delivery methods. The response of long-term engrafting HSPCs to nuclease-mediated DNA breaks, with activation of p53, is a significant challenge, as are activation of innate and adaptive immune responses to editing components. Lastly, we propose alternative strategies that can overcome current hurdles to HSPC editing at various stages from cell collection to transplantation to facilitate successful clinical applications.

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques.

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.