In vivo prime editing enabled by lipid nanoparticles (LNPs)

With the ability to precisely rewrite new information into the genome, prime editing (PE) holds vast potential to revolutionize therapeutic development. However, the clinical application of PE has been hampered by a lack of effective in vivo delivery mechanisms. In recent years, lipid nanoparticles (LNPs) have gained significant attention as alternatives to viral vectors for delivering nucleic acid molecules. Now, for the first time, in vivo PE using LNPs as delivery systems is reported. Specifically, researchers at the University of Massachusetts Chan Medical School (UMass Chan) demonstrated that co-delivery of chemically modified prime editing guide RNA (pegRNA) and prime editor messenger RNA (PE mRNA) enables the desired edit in wild-type mice and that the efficacy of LNP-delivered PE is improved in an immunodeficient mouse model. TriLink’s patented CleanCap Reagent AG (3′-OMe) and N1-Methylpseudouridine-5'-Triphosphate are cited in this study. 

Chemically modified pegRNA and PE mRNA provide efficient prime editing in vitro 

The UMass Chan researchers targeted the mouse Pcsk9 gene with two PE strategies: a +3–5 GCG to TTA substitution or insertion of +1 TTAC. Each pegRNA was introduced with PE mRNA (capped with CleanCap AG 3’ OMe analog and containing N1-methyl-pseudouridine modifications) into mouse Hepa1-6 cells via electroporation, then the percentage of correct edits and indels was quantified using next-generation sequencing (NGS). Because the +1 TTAC insertion strategy exhibited substantially higher efficiency in generating the desired edit, it was selected for future studies. These initially involved designing a series of pegRNAs with diverse modification patterns to identify those that best maintained efficient PE in vitro. 

LNP delivery of chemically modified pegRNA and PE mRNA achieves in vivo prime editing 

Next, the UMass Chan researchers established a pipeline to investigate the in vivo performance of gene editors via LNP-based delivery. This was validated by co-encapsulating the Pcsk9 single-guide RNA (sgRNA) with the SpyCas9 mRNA in LNPs for delivery to mice and assessing gene editing in liver tissue. After LNP performance was confirmed by gene editing results, the LNPs were subsequently used to co-deliver chemically modified pegRNAs with PE mRNA for prime editing, the results of which were modest but aligned with the in vitro findings. 

In vivo prime editing is enhanced by appropriate re-dosing 

The ability to re-dose is an important advantage of LNP delivery that promises to improve PE efficiency in vivo. To test this theory, the UMass Chan researchers compared a single 60 µg dose with five daily 60 µg doses, again using NGS to measure prime editing in the liver. Surprisingly, daily dosing did not improve PE efficiency, but instead caused severe liver toxicity and abnormalities. These adverse effects were overcome by switching to a less frequent dosing regimen (60 µg once per week for three weeks), which also enhanced the PE efficiency by 1.8-fold. 

PE efficiency is improved in an immunodeficient mouse model 

The lack of enhanced editing efficiency in the daily repeat dosing experiment was hypothesized to be due to innate immune responses limiting the translation of RNA from successive doses. To explore this possibility, the UMass Chan researchers included a group of immunodeficient mice (NOD scid gamma, or NSG, animals) in the weekly repeat dosing experiment. Results showed that the PE efficiency of a single dose (3 mg/kg) was 2.8-fold higher in the NSG mouse than in the wild-type mouse, although further investigation is required to determine whether this finding is specifically immune-related. 

Future perspectives 

The successful use of LNPs as delivery systems for in vivo prime editing represents a major development for the field of gene engineering medicine. To realize further advances, researchers will need to consider factors including RNA design, dosing regimen, and LNP formulation, as well as understand the significance of immune-related factors. 

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Article reference: Chen, Z., Kelly, K., Cheng, H. et al. In Vivo Prime Editing by Lipid Nanoparticle Co-Delivery of Chemically Modified pegRNA and Prime Editor mRNA. GEN Biotechnology 2(6):490-502 (2023). https://www.liebertpub.com/doi/abs/10.1089/genbio.2023.0045?journalCode=genbio