A platform for programmable RNA editing using protein-oligonucleotide conjugates for precise mRNA modification

Background

RNA editing is an emerging field within molecular biology and therapeutic medicine that focuses on the precise modification of RNA transcripts to correct genetic errors or modulate gene expression. Unlike genome editing, which permanently alters DNA, RNA editing offers a transient and reversible approach, making it particularly attractive for therapeutic applications where permanent changes may carry risks or ethical concerns. The ability to selectively modify messenger RNA (mRNA) sequences has the potential to treat a wide range of genetic disorders, especially those caused by point mutations. However, the field faces significant challenges in achieving efficient, specific, and safe editing of endogenous RNA molecules within living cells, necessitating the development of novel technologies that can overcome these hurdles.

Current approaches to targeted RNA editing, such as those based on endogenous adenosine deaminase acting on RNA (ADAR) enzymes or CRISPR/Cas-derived systems, suffer from several limitations. ADAR-based methods typically require the formation of double-stranded RNA structures, which can be difficult to program with high specificity and may result in widespread off-target editing throughout the transcriptome. CRISPR/Cas-based RNA editors, while programmable, often exhibit variable efficiency depending on the target sequence and can trigger undesirable immune responses or off-target effects. Additionally, most existing platforms rely on genetic delivery of editing enzymes, which can lead to prolonged enzyme expression and increased risk of unintended edits. These challenges highlight the need for more precise, efficient, and transient RNA editing solutions that minimize off-target activity and are compatible with direct delivery methods for therapeutic use.

Technology

This technology enables precise, programmable RNA editing by facilitating the conversion of adenosine (A) to inosine (I) at specific sites within messenger RNA (mRNA), which is interpreted as guanosine (G) during protein synthesis. The system is built around a covalent conjugate consisting of an evolved adenosine deaminase enzyme (such as TadA-derived deaminases) fused to a self-labeling protein tag (e.g., HaloTag), which is then chemically linked to a synthetic antisense oligonucleotide (ASO) modified with a ligand. The ASO is designed to hybridize with a complementary mRNA sequence, guiding the tethered enzyme to the precise editing site. Innovative ASO architectures, including “bulge-forming” designs, create single-stranded loops in the target mRNA to enhance editing specificity and efficiency. The conjugate can be delivered directly into cells using methods such as electroporation or lipid nanoparticles, enabling transient RNA modification without altering the genome and allowing correction of disease-causing mutations.

What differentiates this technology is its combination of direct, non-genetic delivery and highly tunable specificity, which addresses many limitations of existing RNA editing platforms. Unlike CRISPR/Cas-based or traditional ADAR systems, this approach does not require permanent genetic modification or the introduction of exogenous genes, reducing the risk of off-target effects and immune responses. The use of evolved TadA-derived deaminases expands the range of editable RNA motifs and improves efficiency over natural enzymes. The rational design of ASOs—including bulge-forming and internally conjugated variants—further enhances precision by minimizing unintended edits. Direct delivery of the protein-ASO conjugate as a ribonucleoprotein complex drastically reduces transcriptome-wide off-target events compared to plasmid-based systems. The platform’s demonstrated ability to correct pathogenic mutations, such as premature stop codons in disease-relevant genes, highlights its therapeutic potential and positions it as a versatile, next-generation solution for RNA-targeted treatments.

Benefits

• Enables precise, programmable A-to-I RNA editing to correct disease-causing mutations without altering the genome
• Uses covalent protein-ASO conjugates for targeted delivery and high editing specificity
• Employs evolved TadA-derived deaminases for enhanced editing efficiency and broader substrate scope
• Bulge-forming ASO designs improve editing precision and reduce off-target effects
• Direct delivery of recombinant protein-ASO complexes minimizes transcriptome-wide off-target editing
• Transient and tunable editing reduces risks associated with permanent genome modifications
• Demonstrated therapeutic potential by restoring protein function in disease-relevant mutations (e.g., PTEN)
• Offers improved delivery options compared to gene-based editors, enabling editing in unmodified cells

Applications

• Correction of genetic disease mutations
• Restoration of protein function in cells
• Transient therapeutic RNA editing
• Personalized RNA-based medicine development
• Functional genomics research tools

Additional info

This RNA editing system employs a covalent conjugate of an adenosine deaminase enzyme, a self-labeling protein tag, and an antisense oligonucleotide (ASO). Delivered directly to cells, the ASO guides the enzyme to specific mRNA sequences, facilitating site-specific A-to-I conversion. This enables programmable, transient RNA modification to correct disease-causing mutations without altering the genome.