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Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses

Abstract

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann–Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.

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Fig. 1: Development of split-intein CBEs and ABEs.
Fig. 2: Optimization of split-intein base-editor AAVs.
Fig. 3: Systemic injection of v5 AAV9 editors results in cytosine and adenine base editing in heart, muscle and liver.
Fig. 4: AAV-mediated cytosine and adenine base editing in the CNS by two delivery routes.
Fig. 5: AAV-mediated cytosine and adenine base editing in the retina following sub-retinal injections of 2-week-old Rho-Cre;Ai9 mice.
Fig. 6: Base editing of Npc1I1061T in the mouse CNS.

Data availability

The data that support the results of this study are available within the paper and its Supplementary Information. All unmodified reads for sequencing-based data in the manuscript are available from the NCBI Sequence Read Archive under accession number PRJNA532891. AAV genome sequences are provided in the Supplementary Information. Key plasmids from this work will be available from Addgene (depositor: D.R.L.), and other plasmids and raw data are available from the corresponding author on request.

Code availability

The custom code used in this study is provided in the Supplementary Information.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. UG3 TR002636, U01 AI142756, RM1 HG009490, R01 EB022376 and R35 GM118062), St. Jude Collaborative Research Consortium, DARPA (HR0011-17-2-0049), Ono Pharma Foundation, Bill and Melinda Gates Foundation and Howard Hughes Medical Institute. We thank the Harvard Center for Biological Imaging for infrastructure and support. We thank F. Zhang, B. Deverman and K. Chan for equipment access, guidance and helpful discussions, M. Doan for image analysis assistance and A. Hamidi for assistance with editing the manuscript.

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Contributions

J.M.L. designed the research, constructed the plasmids, produced the AAV and performed the HEK cell, mouse systemic and CNS injection experiments. W.-H.Y. performed all of the CBE 3T3 experiments, image analysis and off-target analysis. J.R.D. performed the ABE 3T3 experiments. L.W.K. constructed the plasmids and performed the HEK cell experiments. N.P., R.B. and E.H. performed the retinal experiments. J.C. conceived of the retinal experiments and performed the data analysis. Q.L. conceived of and performed the sub-retinal injection experiments and data analysis. D.R.L. designed and supervised the research. J.M.L., W.-H.Y. and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Prime Medicine, Editas Medicine and Pairwise Plants, all of which are companies that use genome editing. D.R.L., J.M.L., W.-H.Y. and L.W.K. have filed patent applications on AAV systems for base editor delivery. The remaining authors declare no competing interests.

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Supplementary Figs. 1–11, Supplementary Tables 1 and 2, Supplementary Methods, Supplementary Code and Supplementary Notes.

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Levy, J.M., Yeh, WH., Pendse, N. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat Biomed Eng 4, 97–110 (2020). https://doi.org/10.1038/s41551-019-0501-5

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