80-fold Increase in In Vivo Prime Editing Efficiency via High-Density RNA Modification, Achieving 70% Precise Liver Correction with a Single LNP Dose
Background
Prime editing (PE) is considered a next-generation gene therapy technology because it can precisely induce base substitutions, insertions, and deletions without causing double-strand breaks in the genome or requiring external DNA. The challenge of safely and accurately delivering the correction tools to target organs in vivo has remained the biggest obstacle to clinical entry. This is because existing adeno-associated virus (AAV) vectors have consistently raised concerns about off-target mutations due to long-term organ retention and the risk of triggering immune responses. There was also a limitation in that the capacity of viral particles made it difficult to fully accommodate large protein-RNA complexes.
To enhance safety, the combination of messenger RNA (mRNA) and lipid nanoparticles (LNPs), which acts transiently and then degrades in vivo, is emerging as an alternative. The problem, however, has been low editing efficiency upon systemic administration. Achieving therapeutic-level correction required repeated administration of high doses, which often entailed toxicity exceeding the tolerable range in humans. The decisive cause of this limitation is the structural vulnerability of prime editing guide RNA (pegRNA). Unlike standard single guide RNAs, the reverse transcription template region is extensively exposed, making it vulnerable to degradation by enzymes inside and outside the cell. Previous methods that chemically modified only the ends were insufficient to fully preserve activity in the complex in vivo environment.
Key Findings
The research team presented a new engineering strategy that maximizes degradation resistance by introducing high-density chemical modifications to specific motifs of the pegRNA. This approach moves away from past methods that only protected the ends, applying dense modifications across internal motifs vulnerable to degradation. It was confirmed that applying high-density modifications to various nucleotide sequences, including the widely used MS2 RNA motif, dramatically improved the in vivo stability of the guide RNA.
The research team conducted an experiment administering improved pegRNA and prime editor mRNA encapsulated in LNPs via systemic intravenous injection in mice. A single injection achieved editing efficiency approaching 70% across the entire mouse liver tissue, successfully correcting the majority of hepatocytes. This represents an approximately 80-fold increase in editing efficiency compared to conventional terminal modification pegRNAs. At clinically applicable safe LNP dose levels, the in vivo expression suppression effect of the target protein was also clearly demonstrated. In vivo data confirmed that suppression of disease target protein expression is achievable without excessive dosing or repeated injections.
The scalability of this technology is not limited to prime editing. When the research team applied the high-density RNA motif modification method to Base Editing (BE) and split-platform editing, an increase in base editing efficiency of up to 11-fold was observed. This demonstrates its value as a universal technology applicable across different editing platforms.
Significance and Outlook
This research serves as a milestone showing that high-efficiency precise gene editing is possible in vivo without relying on viral vectors that carry the risk of permanent expression. It is regarded as a significant advancement in gene therapy development, as it achieves therapeutic concentrations while leveraging the advantage of mRNA that disappears after transient action. The achievement of correcting 70% of liver tissue through intravenous injection of lipid nanoparticles is expected to serve as a driving force that significantly accelerates the commercialization of liver disease treatments. It is expected to provide an immediate breakthrough in the treatment of congenital metabolic diseases originating in the liver, such as dyslipidemia or hereditary amyloidosis.
Practical challenges remain before reaching commercialization. Current LNP technology tends to concentrate in liver tissue due to interactions with apolipoprotein E in the bloodstream; therefore, developing new targeted delivery vehicles will be necessary to expand targeting to organs other than the liver. Optimization of processes to increase the synthesis yield and reduce the cost of long pegRNAs with dense chemical modifications is also essential. The academic and industrial sectors plan to focus their research efforts on precisely verifying the safety profile of therapeutics through preclinical validation in primates and off-target analysis targeting the human genome.
Prime editing holds promise for therapeutic applications. However, viral delivery of the prime editor presents challenges for clinical translation due to concerns regarding long-term expression. Meanwhile, systemic delivery using non-viral vectors has been limited by low efficiency, the need for repeated injections and reliance on doses that exceed clinically translatable levels. Here we develop engineered prime editing guide RNAs (pegRNAs) with densely modified RNA motifs and demonstrate their application for efficient in vivo prime editing. By systemically delivering the prime editor in RNA format via a single injection of lipid nanoparticles, we achieved nearly 70% editing efficiency in the bulk mouse liver, indicating successful editing of the majority of hepatocytes. Notably, a single injection at a clinically translatable lipid nanoparticle dose was sufficient to suppress target protein expression in vivo, resulting in a near 80-fold increase in editing efficiency compared with conventional end-modified pegRNAs. Furthermore, incorporating densely modified RNA motifs, including the widely used MS2 motif, proved broadly applicable across various RNA sequences and split RNA-guided genome editing platforms, resulting in up to an 11-fold increase in base editing efficiency. These findings present a generalizable approach for enhancing the therapeutic potential of prime editing and expanding the utility of RNA-based therapeutics.
In clinical settings, a 'one-shot' treatment scenario could materialize for patients with hereditary liver diseases who previously had to take medication for life or receive periodic injections, by correcting the root cause of the disease with a single intravenous injection. For example, in patients with dyslipidemia or congenital metabolic diseases, a single administration of an LNP formulation within clinically permissible limits in an outpatient setting precisely corrects genetic mutations in the liver to approximately 70%, thereby permanently blocking the production of pathogenic proteins in the blood. From an industrial perspective, an economic path has opened to mass-synthesize complex gene-editing medicines by utilizing existing mRNA vaccine manufacturing facilities without relying on complex viral production facilities. The ability to achieve high production yields and rapid process conversion leads to practical benefits by curbing the development costs of expensive orphan drugs and accelerating the pace of clinical pipeline advancement.