A novel dual AAV system enables therapeutically relevant prime editing

With the ability to precisely correct pathogenic sequences at target DNA sites in living cells, without causing double-strand breaks, prime editors (PEs) have vast therapeutic potential. However, translating these benefits into clinical settings requires the development of efficient methods for in vivo delivery. To date, the large size of PEs has prohibited using adeno-associated viruses (AAVs) as delivery vectors, but this limitation has recently been overcome by researchers from the David Liu laboratory at Harvard University. By splitting PEs into two halves, each fused to a fast-splicing intein to allow for rapid in vivo reconstitution, Liu et al. achieved therapeutically relevant prime editing in mouse liver, brain, and heart. Their data, published in Nature, show optimized dual PE-AAVs to provide the highest levels of in vivo prime editing ever reported and suggest this approach to facilitate the treatment of diseases with a genetic component. 

Development of the split PE architecture 

To develop the split PE architecture, Liu et al. bisected the coding sequence of PE3 at positions 844 and 1024, which were hypothesized to be tolerant of structural modification. Eight different designs were then generated by mutating the three N-terminal amino acids of the C-terminal extein before fusing each of the halves to an intein from Nostoc punctiforme. Plasmids encoding the two candidate halves along with pegRNA and nicking sgRNA were then transfected into HEK293T cells and the prime editing efficiencies across three sites measured by high-throughput sequencing (HTS). The 1024-CFN PE split design showed the highest prime editing efficiency (96% of full-length PE activity) and was thus selected for additional optimization. This led to the production of a dual-AAV system termed v1 PE3-AAV, which proved capable of prime editing in the mammalian brain following its intracerebroventricular (ICV) delivery to mice and subsequent analysis of a +5 G-to-T transversion in the endogenous Dnmt1 locus. 

Therapeutically relevant prime editing in vivo 

To further explore the utility of split-intein PE-AAVs in vivo, Liu et al. delivered v1 PE3-AAV encoding three different Dnmt1 edits into adult C57BL/6 mice via retro-orbital injection and then harvested the liver, heart, and skeletal muscle tissues for HTS analysis. While low prime editing was observed in the liver for two of the edits, no prime editing was detected in the heart or skeletal muscle, suggesting a need for extra modification. By introducing changes which included switching from an EFS promoter to a Cbh promoter and using a PE architecture with improved reverse transcriptase expression and enhanced nickase activity, therapeutically relevant prime editing efficiencies of 46%, 42%, and 11% were attained in mouse liver, brain, and heart, respectively. These data represent the first documented evidence of prime editing in postnatal brain and heart, and substantially higher AAV-mediated in vivo prime editing efficiencies than have been previously reported in the liver. 

Installation of protective mutations 

Besides targeting endogenous genes, Liu et al. assessed the capabilities of split-intein PE-AAVs to edit mutations of biomedical interest in vivo. This included installing the APOE R136S mutation into astrocytes as a potential means of protecting against Alzheimer’s disease pathogenesis. In vitro verification of the methodology involved transcribing mRNAs from various plasmid templates using the HiScribe T7 High Yield RNA Synthesis Kit from New England Biolabs with full replacement of UTP with TriLink’s N1-methylpseudouridine-5′-triphosphate and co-transcriptional capping by CleanCap® Reagent AG. The mRNAs were then used for nucleofection of APOE4 murine astrocytes to identify optimal candidates. Administering the optimized dual AAVs to humanized APOE3 mice via ICV injection resulted in prime editing efficiencies of 9.4% in neocortex APOE cDNA and 11% in hippocampal APOE cDNA, confirming this approach to be a viable option for therapeutic PE delivery. 

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Article reference: Davis JR, Banskota S, Levy JM, et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat Biotechnol. 2023. https://www.nature.com/articles/s41587-023-01783-y