Background

In the field of gene editing therapeutics, bacteria-derived enzymes have long been considered a double-edged sword. CRISPR–Cas proteins, derived from bacterial immune systems, boast precise target-binding capabilities but are easily recognized as foreign antigens by the human immune system. This has consistently posed risks of inducing neutralizing antibody responses and cytotoxic T-cell responses in patients, particularly during systemic delivery or treatments for chronic diseases requiring repeated administration.

To overcome this, RNA editing technology, which temporarily regulates the transcript level without permanently modifying genomic DNA, has emerged as an alternative. RNA editing holds high expectations for safety, as it can reduce concerns regarding cancer caused by permanent genetic damage or off-target cleavage. However, existing dCas13 (deactivated Cas13)-based RNA base editors also remained trapped in the structural limitation of being bacterial-derived proteins. Increasing the enzyme expression period to enhance therapeutic effects resulted in facing limitations due to human immune attacks, leading to cytotoxicity or a sharp decline in correction efficiency. Ultimately, developing a programmable corrector that operates solely with human proteins remained a long-standing challenge to simultaneously achieve efficient in vivo delivery and safety.

Key Findings

Researchers focused on Argonaute 2 (AGO2), a key factor in the human gene silencing pathway. AGO2 is a protein that searches for and degrades target RNA by binding to microRNA (miRNA) or small interfering RNA (siRNA). The research team designed a new platform called ALTER (All-human Lead-based Transcript Editor for RNA), which combines human endogenous base editing domains, such as adenosine deaminase (ADAR), with a catalytically dead or precisely controlled human AGO2 scaffold.

ALTER is assembled entirely from pure human-derived proteins, without a single foreign bacterial peptide. Synthetic oligonucleotides acting as guides bind to the AGO2 complex to precisely target specific transcript regions within the cell. Upon reaching the target, the fused deaminase domain converts adenosine (A) to inosine (I); during translation, the ribosome recognizes inosine as guanine (G), reverting the target mutation back to the normal sequence.

In comparative tests conducted on human cell lines, ALTER demonstrated superior target editing activity across a wider range of transcript regions compared to existing dCas13-based RNA editors. In terms of molecular size, it is more compact than dCas13-family complexes, significantly improving the loading efficiency into adeno-associated virus (AAV) or lipid nanoparticles (LNP). When co-cultured with patient-derived immune cells, it was confirmed that the interferon secretion and T-cell proliferation responses observed in the bacterial-derived protein complexes were suppressed to baseline levels. The frequency of off-target editing, which indiscriminately modifies unnecessary transcripts, was also maintained at a lower level compared to existing Cas13 variants.

Significance and Outlook

This achievement, demonstrating that the transcriptome can be precisely edited using only a human protein scaffold, represents an advancement that broadens the safety landscape for gene therapy development. By fundamentally blocking the immunogenic side effects that patients may experience upon exposure to bacterial proteins, it significantly lowers the barriers to clinical adoption for metabolic or neurodegenerative diseases requiring repeated drug administration. The possibility has opened to expand the therapeutic window of existing gene editing therapies, which were limited to single-dose treatments due to immune responses in the body, to multi-dose regimens.

However, challenges to be resolved before commercialization are clear. The intracellular competition between endogenous AGO2 and the administered editing complex must be precisely controlled. Follow-up work is required to optimize the binding affinity of the injected modified complex so as not to interfere with the cell's inherent RNA interference pathway or disrupt the normal microRNA network. Verifying safety upon long-term administration in various organ-specific disease models and advancing formulations to extend the in vivo half-life of chemically modified guide RNAs are expected to be the practical watershed for clinical translation.