Correction of the sickle-cell disease mutation in human hematopoietic stem/progenitor cells

Megan D. Hoban, Gregory J. Cost, Matthew C. Mendel, Zulema Romero, Michael L. Kaufman, Alok V. Joglekar, Michelle Ho, Dianne Lumaquin, David Gray, Georgia R. Lill, Aaron R. Cooper, Fabrizia Urbinati, Shantha Senadheera, Allen Zhu, Pei-Qi Liu, David E. Paschon, Lei Zhang, Edward J. Rebar, Andrew Wilber, Xiaoyan Wang, Philip D. Gregory, Michael C. Holmes, Andreas Reik, Roger P. Hollis, Donald B. Kohn

Key points

  • Delivery of ZFNs and donor templates results in high levels of gene correction in human CD34+ cells from multiple sources, including SCD BM.

  • Modified CD34+ cells are capable of engrafting immunocompromised NSG mice and produce cells from multiple lineages.


Sickle-cell disease (SCD) is characterized by a single point mutation in the seventh codon of the beta-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells (HSCs) would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrated efficient targeted cleavage at the beta-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector [IDLV] or a DNA oligonucleotide), high levels of gene modification were achieved in CD34+ hematopoietic stem and progenitor cells (HSPCs). Modified cells maintained their ability to engraft NOD/SCID/IL2rγnull (NSG) mice and to produce cells from multiple lineages, though with a reduction in the correction levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34+ cells from the bone marrow of sickle patients resulted in the production of wild-type hemoglobin tetramers.

  • Submitted December 11, 2014.
  • Accepted February 7, 2015.