Engineered Enzyme Reduces Harmful dsRNA, Paving the Way for Safer mRNA Therapeutics
Researchers from Yeasen Biotechnology and its subsidiary Molefuture Biotechnology have achieved a critical breakthrough in mRNA technology. By engineering the T7 RNA polymerase (T7 RNAP), the team significantly reduced the generation of double-stranded RNA (dsRNA) during in vitro transcription (IVT), offering a novel approach to enhance the safety and efficiency of mRNA vaccines and therapeutics. This research, titled "Engineered T7 RNA Polymerase Reduces dsRNA Formation by Lowering Terminal Transferase and RNA-Dependent RNA Polymerase Activities," was published in The FEBS Journal on March 3, 2024. The breakthrough has been applied to Yeasen’s newly launched GMP-grade Cleascrip™ T7 RNA Polymerase (Cat#10629), validated by multiple mRNA industry partners.
Frontier Challenges and Breakthroughs in mRNA Technology
mRNA technology has garnered significant attention due to its broad potential in vaccines, cancer immunotherapy, and gene therapy. However, the dsRNA byproducts generated by T7 RNAP during IVT have long been a persistent challenge for the industry. As an immune-stimulating factor, dsRNA triggers innate immune responses, inhibits protein translation, and elevates safety risks for mRNA products. Traditional methods rely on complex purification processes to remove dsRNA, but these approaches are both costly and inefficient.
Yeasen’s research team resolved this challenge through innovative methods. By combining directed evolution and semi-rational design, they successfully developed a series of highly efficient and low-toxicity T7 RNAP mutants. Among these, the combinatorial mutant M17 exhibited dsRNA levels of only 1.8% of the wild-type, significantly reducing immunogenicity while substantially improving mRNA translation efficiency.
Innovative Technologies Driving Research Breakthroughs
The research team employed fluorescence-activated droplet sorting (FADS) technology combined with molecular beacon probes to achieve ultra-high-throughput screening. They constructed random mutant libraries and single-point saturation mutant libraries, identifying key mutants through microplate screening.
Figure 1: Ultra-High-Throughput Screening via FADS and Molecular Beacon
Figure 2: Key Mutant Variants Identified Through Screening
Ultimately, the M17 mutant optimized via DNA shuffling demonstrated the best performance. Computer simulations and functional experiments further revealed that the M17 mutant effectively reduces dsRNA generation by diminishing RNA-dependent RNA polymerase (RDRP) activity and terminal transferase activity.
Figure 3: M17 Mutant Reduces dsRNA via Suppressed RDRP and Terminal Transferase Activities
Experimental validation results showed:
- In RAW264.7 cells, mRNA transcribed by M17 induced interferon-beta (IFN-β) expression at only 9.7% of the wild-type level, indicating a significant reduction in immunogenicity.
- In HEK293 cells, the number of EGFP-expressing cells increased, and fluorescence intensity remained stable, demonstrating markedly enhanced translation efficiency.
Figure 4: M17 Lowers Immunogenicity in RAW264.7 Cells and Enhances Translation in HEK293 Cells
Profound Implications for the mRNA Industry
This breakthrough marks a milestone in the development of mRNA technology. By 2030, the mRNA industry’s market size is projected to exceed $100 billion, spanning vaccines, cancer therapies, and treatments for rare diseases.
The T7 RNAP mutants developed by Yeasen reduce dsRNA generation at the source, lowering production costs and purification demands while improving the safety and efficacy of mRNA products.
A chief scientist at the company stated: “Our research demonstrates that preventing dsRNA generation during the transcription stage is more efficient than traditional purification methods. The M17 mutant sets a new benchmark for producing safer and more effective mRNA products, accelerating the translation of mRNA therapies into clinical applications.”
Yeasen’s Innovation Leadership
As a leading provider of molecular biology reagents,Yeasen and Molefuture have long focused on enzyme engineering and IVT technology development. This collaborative achievement further strengthens their leading position in mRNA technology. Yeasen has launched the GMP-grade Cleascrip T7 RNA Polymerase (Cat#10629), which significantly reduces dsRNA content and is ideal for mRNA vaccine and drug development.
Yeasen plans to integrate these mutants into its IVT reagent kits and actively pursue collaborations with global mRNA developers. A company co-founder remarked: “This is just the beginning. We will continue optimizing T7 RNAP to deliver tailored solutions for diverse therapeutic needs.”
Future Prospects: The Boundless Potential of mRNA Technology
As mRNA technology expands into chronic disease treatment and personalized medicine, Yeasen’s innovations inject fresh momentum into the industry. By minimizing the immune risks associated with dsRNA, mRNA therapies are poised to achieve broader clinical applications. Yeasen calls for collaboration between academia and industry to drive the next phase of mRNA breakthroughs based on this research.
About Yeasen Biotechnology
Yeasen Biotechnology (Shanghai) is a leading provider of molecular biology reagents, enzymes, and custom services, dedicated to advancing life sciences research and therapeutic development. With a focus on quality, innovation, and customer collaboration, Yeasen supports scientists and companies worldwide in unlocking the potential of biotechnology.