Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs. [Display omitted] •Long-term engraftment with HDR-edited HSPCs is markedly impaired in an NHP model•HDR-edited HSPCs are far less clonally diverse than lentivirally transduced HSPCs•p53 inhibition improves but does not rescue engraftment of HDR-edited cells Lee and colleagues directly compare the long-term engraftment ability and clonality of CRISPR HDR gene-edited versus lentivirally transduced HSPCs in a non-human primate model. They demonstrate that both engraftment and clonal diversity of HDR-edited HSPCs are markedly reduced compared with lentivirally transduced cells. These findings have implications for safe clinical development.