Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood. 2018 Jun 28;131(26):2915-2928. doi: 10.1182/blood-2018-03-838540. Epub 2018 May 22.


Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from β-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into β-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human β- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Erythrocytes / metabolism
  • Female
  • Gene Editing / methods*
  • Gene Expression*
  • Hematopoietic Stem Cell Mobilization
  • Hematopoietic Stem Cell Transplantation
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Promoter Regions, Genetic
  • beta-Globins / genetics*
  • gamma-Globins / genetics*


  • beta-Globins
  • gamma-Globins