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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 1

In vitro studies with HDAd-ABE8e vectors in CD34+ cells from healthy donors.

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In vitro studies with HDAd-ABE8e vectors in CD34+ cells from healthy don...
(A) Reactivation of γ-globin by HBG1/2 base editor. In adult erythroid cells, expression of γ-globin is inhibited by a number of repressor proteins, including BCL11A, which binds within the promoter of the 2 copies of the HBG gene (HBG1/Gγ and HBG2/Aγ). This directs the action of the strong β-globin locus control region (LCR) toward expression of β-globin. LCR activity can be switched back to the γ-globin by targeting the BCL11A binding motif using an adenine base editor (31). Expected outcomes of editing the –118 to –113 TGACCA BCL11A binding motif by ABE8e are shown on the bottom. In addition to the target –113 A>G conversion, bystander editing of neighboring adenines was observed. The –116 A>G conversion would further destroy the BCL11A binding motif (underlined blue). In the absence of the –110 A>G bystander conversion, a GATA motif would be created (underlined red). (B) The vectors contain an HBG1/2 sgRNA and the ABE8e gene linked to miRNA regulatory elements. The ABE8e gene is either under the control of the PGK promoter (HDAd-PGK.ABE8e) or the EF1α promoter (HDAd-EF1α.ABE8e). The vectors also contain a PGK-mgmtP140K cassette for O6-BG/BCNU selection. The IR and frt sequences are remnants from previous vectors that were integrated by SB100x transposase. They are irrelevant for this study, which does not employ the SB100x integrating system. (C) Editing rate in CD34+ cells from 3 healthy, G-CSF–mobilized donors. Cells were transduced at an MOI of 2,000 viral particles (vp)/cell, and 3 days later, genomic DNA was analyzed by Sanger sequencing for the –113 A>G conversion. ABEmax is an early adenine base editor version (12). (D) Sequences of the top 12 edited alleles in CD34+ cells from donor #1 after transduction with HDAd-EF1α.ABE8e. The target site –113 A (A8 in the sequence shown in the lower panel) is marked by a green box. Editable window in the spacer by ABE8e is highlighted in orange. (E and F) In vitro transduction studies with donor #1 CD34+ cells that were subjected to erythroid differentiation (ED) and 1 cycle of O6-BG/BCNU selection. (E) Schematic of the experiment. (F) Percentage of –113 A>G conversion at different time points without or with O6-BG + 15, 25, or 35 μM BCNU added at day 3. The SD was less than 10% for all time points. n = 3 technical replicates.

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