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

ABSTRACT: Disorders involving ?-globin gene mutations, primarily ?-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 ?-globin expression in red blood cells. Because models involving erythroid differentiation of CD34⁺ cells have limitations in assessing ?-globin reactivation, we focused on human ?-globin locus-transgenic (?-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 ?-globin promoter. We transduced HSPCs from ?-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 ?-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human ?- to ?-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.

SUBMITTER: Li C 

PROVIDER: S-EPMC6024639 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature