Long-term multi-lineage engraftment of genome-edited hematopoietic stem cells after autologous transplantation in nonhuman primates

Christopher W. Peterson, Jianbin Wang, Krystin K. Norman, Zachary K. Norgaard, Olivier Humbert, Colette K. Tse, Jenny J. Yan, Richard G. Trimble, David A. Shivak, Edward J. Rebar, Philip D. Gregory, Michael C. Holmes and Hans-Peter Kiem

Key points

  • We are the first to show that genome editing approaches can modify multi-lineage, long-term repopulating cells in a large animal model.

  • We demonstrate that genome-edited hematopoietic stem cells engraft with similar kinetics as observed for lentivirus-modified stem cells.


Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether disruption of the CCR5 locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early post-transplant, and 3–5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multi-lineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome editing therapies for HIV and potentially other diseases.

  • Submitted September 28, 2015.
  • Accepted February 26, 2016.