Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo.

Thalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype.