Reactivation Of Gamma-Globin In Adult Beta-Yac Mice After Ex Vivo And In Vivo Hematopoietic Stem Cell Genome Editing

Disorders involving beta-globin gene mutations, primarily beta-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 CD341 cells aimed toward the reactivation of fetal gamma-globin expression in red blood cells. Because models involving erythroid differentiation of CD341 cells have limitations in assessing gamma-globin reactivation, we focused on human beta-globin locus-transgenic (beta-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 gamma-globin promoter. We transduced HSPCs from beta-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 beta-YAC/CD46-transgenicmice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human beta- to gamma-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.