Background

Recently, the bio-industry has shown great interest in RNA-level base editing technology as an alternative to gene scissors. This is because DNA editing can permanently alter the genome, potentially causing off-target effects and other adverse reactions. In contrast, RNA editing temporarily modifies genetic information, making it easier to ensure safety. In particular, technologies that utilize adenosine deaminase acting on RNA (ADAR), which already exists in the human body, are considered to be advantageous because they do not require the introduction of exogenous proteins, thus reducing the risk of immune responses.

However, the design of guide RNAs (gRNAs) to direct the natural ADAR enzyme to the target site still has limitations in terms of efficiency. Existing design methods have used complementary guide RNAs that bind completely to the target RNA sequence to attract the enzyme. Analysis revealed that this complete double-stranded structure is extremely inefficient in maximizing ADAR enzyme activity. For use as a gene therapy, a new design approach that mimics the RNA structure that natural ADAR enzymes prefer within cells is considered a breakthrough.

Key Findings

The joint research team focused on identifying the natural substrates in the human genome where ADAR enzymes are most active. The analysis focused on the unique structural features of 'inverted Alu repeats,' where ADAR editing frequently occurs. The research team proposes 'MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides)' as an alternative, a new guide RNA platform that precisely mimics this natural motif.

They measured the actual correction performance of the MIRROR platform using various human cell lines. Laboratory evaluation showed that the new guide RNA structure exhibits up to 5.7-fold higher RNA base editing efficiency compared to existing design methods. In addition, both chemically modified short guide RNAs (approximately 20-40 nucleotides) and long-form guide RNAs expressed in cells via plasmids showed consistent correction activity.

The research team went further and investigated the therapeutic potential in primary hepatocytes of alpha-1 antitrypsin deficiency (AATD) mouse models, a rare genetic disease. By targeting the Z mutant RNA of the SERPINA1 gene in these hepatocytes and converting it to the normal sequence, a significant increase in the amount of normal antitrypsin protein secreted from the hepatocytes was observed. This demonstrates that the MIRROR technology can function accurately even in a complex in vivo genetic environment.

Significance and Prospects

The greatest advantage of MIRROR technology is that it does not introduce highly toxic exogenous proteins. Existing third-generation CRISPR-Cas9 technology uses bacterial-derived Cas9 proteins, which may cause immune rejection. In contrast, the MIRROR approach, which utilizes intracellular ADAR enzymes, is ideal for inducing high-efficiency targeted therapy while avoiding genome damage. Since only small guide RNA sequences need to be delivered into the body, it is also easy to ensure delivery efficiency.

However, there are also obstacles to overcome before it can become a new drug used in clinical practice. The most representative challenge is the off-target phenomenon, in which unintended RNA bases are also edited. Because the guide RNA structure highly mimics natural sequences, there is a possibility that it may loosely bind to non-target sites, potentially causing unexpected adverse effects. Therefore, a thorough screening process to verify the presence or absence of off-target adverse effects throughout the genome must follow before administration to patients. In addition, research to optimize delivery vehicles such as lipid nanoparticles (LNPs) to stably deliver the drug to the target organ is essential.