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In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies
Chang Li, … , Evangelia Yannaki, André Lieber
Chang Li, … , Evangelia Yannaki, André Lieber
Published August 25, 2022
Citation Information: JCI Insight. 2022;7(19):e162939. https://doi.org/10.1172/jci.insight.162939.
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Research Article Hematology Stem cells

In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies

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Abstract

Individuals with β-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a –113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.c. and i.v. injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in > 60% –113 A>G conversion, with 30% γ-globin of β-globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-Seq. In vitro, in HSCs from β-thalassemia and patients with sickle cell disease, transduction with the base editor vector mediated efficient –113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.

Authors

Chang Li, Aphrodite Georgakopoulou, Gregory A. Newby, Kelcee A. Everette, Evangelos Nizamis, Kiriaki Paschoudi, Efthymia Vlachaki, Sucheol Gil, Anna K. Anderson, Theodore Koob, Lishan Huang, Hongjie Wang, Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, André Lieber

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Figure 2

In vitro editing studies with CD34+ cells from patients with β-thalassemia and SCD.

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In vitro editing studies with CD34+ cells from patients with β-thalassem...
(A) Schematic of the experiment. G-CSF/plerixafor mobilized CD34+ cells from 3 β-thalassemia (Thal) patients were used. SCD CD34+ cells from fresh peripheral blood were isolated after blood transfusion of 3 patients (1 β0/βS, 2 βS/βS patients). CD34+ cells were transduced with HDAd-EF1α.ABE8e or left untransduced (UNTD). Cells were then subjected to erythroid differentiation for 18 days. Aliquots were collected at the indicated time points. On day 3, 1 set of ED cells was treated once with O6-BG/BCNU for in vitro selection (only from second βS/βS patient, we obtained sufficient numbers of CD34+ cell to perform in vitro selection). (B) Editing rate of the –113 A target site and percentage of reads with indels analyzed by NGS for the thalassemia samples (left panel) and SCD samples (right panel). Note the different scale on the y axes for on-target editing and indels. (C) Flow cytometry analysis for γ-globin/HbF at the end of ED in total cells and in differentiated enucleated erythroid (NucRed–) cells in Thal and SCD samples. (D) Measurement of globin protein chains by HPLC. Shown is the percentage of human γ-globin relative to human β- and α-globin chains in Thal samples (n = 3), 2 SCD (βS/βS) samples without selection, and 1 SCD (βS/βS) sample with selection. Statistical analyses of the data from the Thal samples were performed using 2-way ANOVA.

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