Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors

Jessie R Davis; Xiao Wang; Isaac P Witte; Tony P Huang; Jonathan M Levy; Aditya Raguram; Samagya Banskota; Nabil G Seidah; Kiran Musunuru; David R Liu

doi:10.1038/s41551-022-00911-4

Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors

Nat Biomed Eng. 2022 Nov;6(11):1272-1283. doi: 10.1038/s41551-022-00911-4. Epub 2022 Jul 28.

Authors

Jessie R Davis^{1

2

3}, Xiao Wang^{4

5

6}, Isaac P Witte^{1

2

3}, Tony P Huang^{1

2

3}, Jonathan M Levy^{1

2

3}, Aditya Raguram^{1

2

3}, Samagya Banskota^{1

2

3}, Nabil G Seidah⁷, Kiran Musunuru^{4

5

6}, David R Liu^{8

9

10}

Affiliations

¹ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
³ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁷ Laboratory of Biochemical Neuroendocrinology, Montreal Clinical Research Institute (IRCM), University of Montreal, Montreal, Quebec, Canada.
⁸ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. drliu@fas.harvard.edu.
⁹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁰ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.

Abstract

The viral delivery of base editors has been complicated by their size and by the limited packaging capacity of adeno-associated viruses (AAVs). Typically, dual-AAV approaches based on trans-splicing inteins have been used. Here we show that, compared with dual-AAV systems, AAVs with size-optimized genomes incorporating compact adenine base editors (ABEs) enable efficient editing in mice at similar or lower doses. Single-AAV-encoded ABEs retro-orbitally injected in mice led to editing efficiencies in liver (66%), heart (33%) and muscle (22%) tissues that were up to 2.5-fold those of dual-AAV ABE8e, and to a 93% knockdown (on average) of human PCSK9 and of mouse Pcsk9 and Angptl3 in circulation, concomitant with substantial reductions of plasma cholesterol and triglycerides. Moreover, three size-minimized ABE8e variants, each compatible with single-AAV delivery, collectively offer compatibility with protospacer-adjacent motifs for editing approximately 82% of the adenines in the human genome. ABEs encoded within single AAVs will facilitate research and therapeutic applications of base editing by simplifying AAV production and characterization, and by reducing the dose required for the desired level of editing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Adenine
Angiopoietin-like Proteins / genetics
Animals
Dependovirus* / genetics
Gene Editing
Humans
Mice
Proprotein Convertase 9* / genetics

Substances

PCSK9 protein, human
Proprotein Convertase 9
Adenine
ANGPTL3 protein, human
Angiopoietin-like Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding