In a recent study published in Cell, a research team led by Li Wei and Zhou Qi from the Institute of Zoology at the Chinese Academy of Sciences has developed a novel gene-writing technology using retrotransposons. This breakthrough allows for all-RNA-mediated targeted gene integration in human cells.
Challenges in Gene Integration
Achieving efficient and precise integration of gene-sized DNA remains a significant challenge in genome engineering. Current technologies primarily use DNA templates as donors for gene integration, but these DNA donors present several obstacles in biomedical applications. High immunogenicity, delivery difficulties in vivo, and the risk of random genomic integration are among the major issues associated with DNA donors.
In contrast, RNA donors exhibit lower immunogenicity compared to exogenous DNA donors, can be effectively delivered using non-viral vectors, and are rapidly degraded in cells without risking random integration. Despite these advantages, very few technologies currently utilize RNA donors for targeted gene-sized DNA integration in human cells.
Retrotransposons and the R2 System
R2 retrotransposons are mobile elements that use RNA intermediates to specifically integrate into the host 28S rDNA genomic site, a location deemed a "safe harbor" due to its distance from protein-coding genes. Despite its discovery in the 1980s, the potential of R2 retrotransposons for integrating large-fragment genes into human cells had not been fully explored.
Through systematic analysis and screening, the research team identified the avian genome-derived R2Tg system as active in human cells, though with low efficiency. By employing several engineering approaches, they created an optimized version, en-R2Tg. This enhanced system, delivered by lipid nanoparticles (LNP), a non-viral vector used clinically, achieved a 25% gene integration efficiency in human liver cells and over 60% site-specific gene integration efficiency in mouse embryos.
High Specificity and Safety
The en-R2Tg system exhibits high gene integration specificity at the 28S rDNA safe harbor site, minimizing the risk of mutagenesis caused by random gene integration, which is a common issue with technologies like retroviruses. This level of specificity and efficiency marks a significant advancement in gene integration technology.
"This technology opens a door for the development of novel gene therapeutics. When it comes to a disease-related gene, there can be many different mutations that cause the same disease. Our technology offers a more general approach in which we can integrate a normal gene directly into the genome to restore function, regardless of the type of mutation," said Li Wei, the corresponding author of the study.
Potential Applications
The en-R2Tg system holds promise for various therapeutic applications. "We may even be able to use LNP to deliver our gene-writing tool and create CAR-T cells directly in our bodies to treat cancer. This could make the whole process as easy as getting a vaccination. We're excited about the potential for further development and application of this new technology in the future," added Li.
Original Publication
Chen, Y., Luo, S., Hu, Y., Mao, B., Wang, X., Lu, Z., Shan, Q., Zhang, J., Wang, S., Feng, G., Wang, C., Liang, C., Tang, N., Niu, R., Wang, J., Han, J., Yang, N., Wang, H., Zhou, Q., & Li, W. (2024). All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons. Cell. https://doi.org/10.1016/j.cell.2024.06.020