Prime Editing Efficiency Increased 2.9-Fold with Sequential Delivery of Virus-Like Particles, Enabling Large-Scale Screening
Background
Prime Editing (PE) is a precise gene editing technology that can induce various genetic variations without cleaving the DNA double strand. It has the advantage of minimizing off-target effects and unintended sequence damage compared to conventional gene editing tools. However, its large molecular size has limited its intracellular delivery. Adeno-Associated Virus (AAV) and Lipid Nanoparticles (LNP) have been used for delivery, but they can cause adverse effects due to immune responses or long-term retention.
Recently, engineered Virus-Like Particles (eVLPs), which transiently deliver the editing machinery in protein form, have emerged as an alternative. eVLPs are rapidly degraded inside cells, making them safe, but their single-dose delivery efficiency is low, limiting their use in large-scale screening.
Key Findings
The research team led by Professor Pierre Billon at the University of Calgary, Canada, developed PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), which improves PE efficiency by optimizing the eVLP delivery method. To mitigate the toxicity of high-concentration single-dose delivery to cells, they applied a sequential delivery strategy, administering low-concentration (sub-saturating) eVLPs multiple times at 3-day intervals.
This sequential delivery model increases the frequency of cell exposure to the editing tool, thereby increasing the overall cumulative editing rate. Analysis of various cell lines, including human kidney cells (HEK293T) and lung cancer cells (A549), showed that PRIME-VLP exhibited 1.5 to 2.9 times higher gene editing efficiency compared to a single high-dose delivery. No adverse effects, such as decreased cell viability, were observed, ensuring safety.
To expand to large-scale genomic screening, the research team attempted to separate the complex structure. They designed a 'pegRNA-depleted eVLP' that delivers the editing protein and the guide sequence, prime editing guide RNA (pegRNA), separately. By sequentially administering pegRNA-free eVLPs to cells containing the pegRNA library, they completed stable gene editing. Using this system, they initiated validation of a library of approximately 6,000 pegRNAs targeting TP53, a tumor suppressor gene. By treating cancer cell lines with the drug Nutlin-3 to track TP53 loss-of-function mutations, they completed a mutation map with 2.8 times higher editing efficiency and excellent reproducibility compared to the existing lentiviral delivery method.
Significance and Prospects
PRIME-VLP solves the problem of transgene genomic insertion, which was a limitation of gene editing tool delivery using viral vectors. In conventional lentivirus-based screening, gene expression is inhibited over time due to the silencing phenomenon, reducing efficiency. In contrast, eVLP-based transient delivery eliminates the editing machinery at the protein level, eliminating concerns about long-term dysfunction. By combining this with a fractional delivery method to maintain high editing efficiency, it has opened up new avenues for disease model design.
However, to advance to the clinical stage, research on in vivo delivery technology needs to be supplemented. The technology has primarily been validated at the cell culture level, and there is a lack of a control system to implement sequential delivery in the actual in vivo environment. There is also a concern that the immune system may recognize eVLP proteins as foreign and attack them. In the future, the fusion with nanocarriers or the incorporation of engineering technologies to enhance tissue selectivity will be necessary to develop it into an effective therapeutic modality.
Prime editing could resolve gene variant function at scale, but the editing machinery needs to be delivered efficiently and reproducibly. Langley, Baudrier, et al.
The PRIME-VLP system is expected to directly contribute to increasing the research productivity of bio companies that are pursuing new drug development. In large-scale gene library screening, if the target editing efficiency increases by more than 2.8 times, the candidate substance validation period can be shortened by several months. Specifically, in the field of anticancer drug development, it can be immediately applied to an evaluation platform that simultaneously induces thousands of mutations in cancer-causing genes such as TP53 and verifies the drug responsiveness of each mutation. The scenario in which rare mutations that show resistance to specific anticancer drugs are identified in advance, allowing for the development of patient-specific treatment strategies or the design of combination drug regimens to overcome resistance, is now within reach. Ultimately, it is expected to reduce the toxicity of gene therapy materials and maximize efficacy, thereby contributing to increasing the success rate of clinical entry in new drug pipelines.