Introduction of Two Simultaneous Mutations By Genome Editing Greatly Enhances the Accumulation of the Endogenous Fetal Hemoglobin in Human Normal Erythroid Cells

Thalassemia or sickle cell patients with increased fetal hemoglobin have an ameliorated clinical picture up to transfusion independency. Genome editing with targeted nucleases of the γ-globin silencer BCL11a or of the HBG promoter has been shown to reenact an HPFH-like phenotype by significantly increasing the endogenous fetal globin (HbF) expression. In order to achieve higher efficiency in HbF reactivation we developed an innovative approach, mediated through a highly customizable helper dependent-adenoviral vector (HD-Ad5/35++) with nuclease activity. This vector bares the Cas9 gene and two sequential gRNAs, permitting simultaneous targeting of two different DNA loci, specifically, the erythroid enhancer of BCL11a and the HBG promoter to recreate the -114 to -102 HPFH deletion. This double targeting vector was compared to the respective single gRNA vectors. We observed that disruption of the BCL11a-enhancer increased Gγ globin, whereas disruption of the HBG promoter had a greater impact on the Αγ globin expression. Simultaneous disruption of both loci in human erythroid progenitors increased the overall HbF expression from less than 1% in the control to more than 20%, through a possibly synergistic effect of the two mutations (HbF:2.2% in BCL11a-enhancer only knock-out, 10.8% in HBG-only knock out) by affecting both gamma globin chains. The editing efficiency per locus was similar between the single gRNA and double gRNA vectors. Erythroid cell morphology and phenotypic profile of the double knock-out cells did not differ compared to the single knock-out cells. In addition to our in vitro experiments, we observed that mobilized peripheral blood CD34+ cells transduced and edited by the double gRNA vector, can efficiently engraft in NSG mice. Furthermore, the engrafted edited cells after erythroid differentiation expressed significantly higher levels of gamma globin compared to the control. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders.