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.
- Copyright © 2015 American Society of Hematology