citations

Efficient prime editing in mouse brain, liver and heart with dual AAVs

Authors:

Jessie R. Davis, Samagya Banskota, Jonathan M. Levy, Gregory A. Newby, Xiao Wang, Andrew V. Anzalone, Andrew T. Nelson, Peter J. Chen, Andrew D. Hennes, Meirui An, Heejin Roh, Peyton B. Randolph, Kiran Musunuru & David R. Liu

In:

Source: Nature
Publication Date: (2024)
Issue: 42: 2

Research Area:

Gastroenterology

Neurobiology

Toxicology

Cells used in publication:

Astrocyte, mouse: Species: mouse; Tissue Origin: brain
293T: Species: human; Tissue Origin: kidney

Platform:

4D-Nucleofector® X-Unit

Experiment

Nucleofection of APOE4 murine astrocytes

Astrocytes containing APOE4 isoform of the APOE gene (originated from Taconic Biosciences) were nucleofected using program EN-150 with SF Cell Line 4D-Nucleofector X Kit (Lonza). In brief, 200,000 astrocytes were resuspended in 20?µl of buffer with 1?µg of PE2 mRNA, 90?pmol synthetic pegRNA (Integrated DNA Technologies) and 60?pmol synthetic nicking sgRNA (Synthego). For PE5 experiments, 2?µg of hMLH1dn mRNA was also included in the nucleofection. After nucleofection, the cells were diluted to 100?µl of pre-warmed media and recovered for 10?min at 37?°C, followed by plating in 12-well plates. Seventy-two hours after nucleofection, gDNA was harvested.

Abstract

Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.

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