Background

Fundamental treatment for rare genetic diseases begins with genome editing technologies that accurately and safely correct pathogenic mutations. The 1st generation CRISPR-Cas9 system opened a new chapter in genome research with its ability to recognize and cut target sequences, but it possessed the fatal flaw of inducing DNA double-strand breaks (DSBs). This was because it was accompanied by random base insertions and deletions, followed by risks such as chromosomal rearrangements.

Developed to overcome these limitations, Prime Editing (PE) is a technology that combines reverse transcriptase with specially engineered prime editing guide RNA (pegRNA). It employs a method of cutting only one strand of DNA at the target site and directly inserting the desired genetic information. Academics predict that this technology could correct approximately 89% of known human pathogenic genetic variants.

However, the high precision proven in laboratories and at the cellular level has not directly translated into patient treatment. The absence of in vivo delivery technology to fully deliver editing tools to target organs within the human body and the lack of large-scale manufacturing processes that meet pharmaceutical quality standards have been the primary obstacles preventing clinical entry.

Key Findings

A recently published review study closely analyzed the accumulated in vivo PE therapeutic research achievements, focusing on retinal, metabolic, and hematologic genetic diseases. In models of congenital retinal diseases, PE complexes delivered directly into the eye led to the recovery of retinal pigment epithelium cell function, and in liver-targeted metabolic disease models, the normalization of target protein expression was confirmed. In the field of blood diseases, efforts to directly correct hematopoietic stem cells in vivo are ongoing, with preclinical efficacy data being reported successively.

The researchers categorized the technological barriers encountered during the clinical translation stage into three areas, despite successes in animal models:

First is the immunogenicity and cargo capacity limits of viral vectors, including Adeno-Associated Virus (AAV). Because the prime editor is large, as it combines the Cas9 protein and reverse transcriptase, it is difficult to fully contain within a single AAV vector. Although the dual-vector method, which involves splitting the delivery into two parts, is primarily used, this approach leads to reduced delivery efficiency and an increased risk of immune responses in patients upon repeated administration.

Second is the risk of off-target effects and the generation of unintended byproducts observed in non-dividing cells. In mature tissues where cell division has ceased, such as hepatocytes or neurons, concerns remain regarding unexpected genomic mutations due to the non-specific activity of reverse transcriptase or unintended binding of pegRNA.

Third is the bottleneck of large-scale production with uniform quality. The purification and formulation standardization required to stably produce complex proteins and long-length pegRNA at a commercial scale while maintaining biological activity remains in its early stages.

Significance and Outlook

In vivo prime editing represents a key step toward moving beyond the limitations of existing ex vivo approaches—which involve extracting cells, manipulating them outside the body, and re-injecting them—to achieve the ultimate therapeutic goal of directly correcting disease causes within the patient. It is widely regarded as significantly expanding the scope of treatment for various rare diseases with complex genetic defects.

To accelerate clinical translation, the research team proposes a shift away from dependence on viral vectors toward delivery systems based on Lipid Nanoparticles (LNP) or cell-free protein-RNA complexes (RNP). Non-viral delivery systems can serve as a realistic alternative that reduces immune rejection, enables repeated administration, and lowers the cost and complexity of large-scale production processes.

However, ligand engineering to precisely control in vivo tissue selectivity, along with the accumulation of genetic stability verification data through long-term tracking, must still be prioritized. The clinical translation roadmap toward curing genetic diseases will be completed only when innovations in delivery engineering and manufacturing processes work in tandem.