Background

Gene editing technologies for treating the root causes of genetic diseases are continuously evolving. Prime editing (PE), which precisely modifies genetic information without cleaving the DNA double helix, is considered an alternative that overcomes the safety limitations of existing gene scissors. However, its low in vivo delivery efficiency has hindered its application in actual research and clinical settings.

To safely deliver genetic material into cells, scientists have developed engineered virus-like particles (eVLPs). eVLPs mimic the appearance of viruses but do not contain genetic information, reducing the risk of genomic insertion side effects and off-target mutations. In high-throughput applications such as large-scale genomic screening, design strategies to maximize eVLP delivery efficiency are still lacking. Existing single-dose, high-concentration administration methods have proven difficult to achieve effective editing concentrations and have exposed problems such as cytotoxicity and gene silencing.

Key Findings

Recent research has revealed PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), a new gene delivery method that involves sequentially administering sub-saturating concentrations of eVLPs at optimal time intervals. This strategy, which involves infecting cells multiple times, has proven to be the key to dramatically increasing gene editing efficiency.

Experimental results have confirmed that the PRIME-VLP method increases editing efficiency by 1.5 to 2.9 times compared to conventional single-dose administration in various cell lines and genomic targets. In this process, the increase in off-target mutations, which was a concern, did not occur, and no side effects such as decreased cell viability or disruption of gene transcripts were observed. This is the result of selectively increasing only the precision correction efficiency while ensuring safety.

One of the reasons for this success is the flexible design structure that separates the delivery of prime editing guide RNA (pegRNA) and the editor protein. The researchers devised a dual-structure that first injects empty eVLPs that do not contain pegRNA and then independently delivers pegRNA. This effectively avoids the gene silencing phenomenon, which frequently occurs in conventional lentivirus-based delivery methods.

The researchers verified the performance by using drug resistance research as a model. They created a library of 6,000 pegRNAs targeting TP53, an anti-cancer gene, and conducted a large-scale screening. As a result, PRIME-VLP achieved an editing efficiency 2.8 times higher than conventional lentivirus administration. The experimental repetition also showed a significant reduction in errors, demonstrating excellent reproducibility. Based on this data, the researchers were able to precisely identify specific loss-of-function mutations in TP53 that confer resistance to the anti-cancer drug Nutlin-3.

Significance and Prospects

This achievement is significant in that it expands the use of eVLPs, which have been mainly studied for in vivo therapeutic delivery, to the field of high-throughput functional genomics screening. Large-scale gene variation screening is a core technology of modern biomedical research that elucidates gene function and discovers new drug candidates. The PRIME-VLP platform is expected to be useful in elucidating the mechanisms by which unknown gene variations cause disease in the future.

However, in order for this technology to be fully established in the industry and clinical practice, it is necessary to secure a technology for mass production of eVLPs. The development of a process for uniformly manufacturing and purifying high-quality virus-like particles in large quantities is an additional challenge. Furthermore, follow-up studies should be conducted to verify whether the same high efficiency and safety can be ensured in in vivo environments and animal models such as mice.