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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 5

Ex vivo HSC base editing for γ-globin reactivation by HDAd-EF1α.ABE8e.

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Ex vivo HSC base editing for γ-globin reactivation by HDAd-EF1α.ABE8e.
(...
(A) Schematic of the experiment. BM Lin– cells were isolated from β-YAC/CD46 transgenic mice and transduced ex vivo with HDAd-EF1α.ABE8e at an MOI of 500 vp/cell. After 1 day in culture, 1 million cells per mouse were transplanted into lethally irradiated C57BL/6 mice, which were followed for 16 weeks (week 16 primary [week 16-P]). Data from these mice are shown in this figure. BM Lin– cells from these mice were then used for secondary transplantation and these mice were monitored for another 16 weeks (week 16 secondary [week 16-S]; see Supplemental Figure 6). (B) Engraftment of transplanted HDAd-EF1α.ABE8e–transduced HSCs measured by flow cytometry of human CD46 in PBMCs. Each symbol is an individual mouse. n = 5 animals. (C) Analysis of target site editing in PBMCs by Sanger sequencing. Shown are percentages of conversion for the –113 A>G site and neighboring adenines. n = 5 animals. (D) Analysis of target site editing in PBMCs, spleen, BM MNCs, BM Lin– cells, and CFU at week 16 after transplantation by Sanger sequencing. n = 5 animals. (E) Comparison of editing rates (by NGS) at the 4 adenines in the transplant (ex vivo transduced Lin– cells cultured for 3 days) and BM MNCs at week 16 after transplantation. Shown are percentages of reads. Note the log10 scale of the y axis. n = 3 animals. (F) NGS of the target area (222 bp amplicon; ~100 nucleotides upstream and downstream of the spacer). Left panel: base substitutions (green), deletions (blue), and insertions (red) in the target area for 1 representative mouse. Right panel: summary of all indel reads in the transplant, week 16-P mice and week 16-S mice. *P < 0.05. Statistical analyses were performed using 2-way ANOVA. (G) Editing on a single cell basis. Week 16 BM Lin– cells were plated for progenitor assay, and individual colonies were subjected to NGS. Shown is a representative mouse with 100% biallelic editing of the HBG1/2 sites. n = 36 (3 mice, 12 colonies per mouse analyzed).

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