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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 7

In vivo HSC transduction of β-YAC/CD46 mice to achieve γ-globin reactivation by HDAd-EF1α.ABE8e.

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In vivo HSC transduction of β-YAC/CD46 mice to achieve γ-globin reactiva...
(A) Experimental procedure. β-YAC/CD46 mice (n = 5 animals) were mobilized by G-CSF/AMD3100 and in vivo transduced by i.v. injection of HDAd-EF1α.ABE8e. In vivo selection with O6-BG/BCNU was started at day 2 after HDAd injection and repeated at days 12 and 26 at the indicated doses (30 mg/kg O6-BG + 5, 10, and 10 mg/kg BCNU). The mice were euthanized at week 16-P. The data from primary in vivo transduced mice are sown in this figure. Lin– cells were isolated from BM and i.v. injected into lethally irradiated C57BL/6J mice. The secondary transplanted mice were followed for another 16 weeks (week 16-S; see Supplemental Figure 10). (B) Loss of vector genomes in PBMCs and BM MNCs. Vector copy number (VCN) was measured by qPCR with human mgmtP140K primers. ND, not detectable. n = 5 animals for d3 and wk16; n = 3 animals for wk4 and wk6. (C–E) Target base conversion measured by Sanger sequencing. Each dot represents 1 animal. n = 5 animals. (C) Percent conversion in DNA from PBMCs. (D) Percent conversion at week 16 in PBMCs and MNCs in the spleen and BM. (E) Percent conversion at week 16 in lineage (CD3+, CD19+, Gr-1+, Ter-119+) cells and in Lin– cells in the BM. (F) Comparison of editing rates at the 4 adenines in BM MNCs at week 16 after in vivo transduction. Shown are percentages of reads. n = 5 animals. (G) Representative NGS results showing target base conversion and indels at week 16 (mouse #446). (H) Summary of all indel reads in week 16-P mice and week 16-S mice. n = 5 animals. (I) Editing on a single-cell basis. Week 16 BM Lin– cells were plated for progenitor assays, and individual colonies were subjected to NGS. Shown are sequencing data of the HBG1 and HBG2 sites of a representative colony with mono- and biallelic conversions, as well as indels (<1%, not visible).

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