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

C Li, N Psatha, P Sova, S Gil, H Wang… - Blood, The Journal …, 2018 - ashpublications.org
C Li, N Psatha, P Sova, S Gil, H Wang, J Kim, C Kulkarni, C Valensisi, RD Hawkins
Blood, The Journal of the American Society of Hematology, 2018ashpublications.org
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 …
Abstract
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.
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