Reducing Immunogenicity and Enhancing Efficacy: Advancements in dsRNA Removal Technologies for Therapeutic mRNA
Background
The success of COVID-19 mRNA vaccines has established messenger RNA (mRNA) technology as a key player in the development of biopharmaceuticals. Its rapid production speed, high efficacy, and excellent cost-effectiveness have made it a promising next-generation therapeutic platform. However, challenges remain. One significant hurdle is the presence of impurities generated during the synthesis process.
In vitro transcription (IVT) processes inevitably produce various byproducts in addition to the desired single-stranded mRNA. These include unreacted caps, nucleoside triphosphates (NTPs), template DNA, and unpurified enzymes. Among these, double-stranded RNA (dsRNA) is a major contributor to reduced therapeutic efficacy. It can trigger unwanted immune responses, leading to inflammation, and also inhibits the process by which mRNA produces proteins. Consequently, the removal of dsRNA has become a critical research area for the development of safe and effective mRNA therapeutics.
Key Findings
dsRNA removal strategies can be broadly divided into two categories: post-synthesis separation methods, which remove dsRNA from the synthesized mixture, and pre-emptive inhibition methods, which prevent dsRNA formation during synthesis. This review provides a systematic comparison of the principles and advantages/disadvantages of each technology.
Post-Synthesis Separation: Chromatography and Cellulose Filters
Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) and cellulose-based purification are representative technologies for separating dsRNA from completed IVT reaction mixtures. RP-HPLC separates single-stranded mRNA and dsRNA with high resolution by detecting differences in hydrophobicity. However, it has the drawbacks of consuming large amounts of organic solvents during purification and being difficult to scale up for large-scale production.
Cellulose-based purification, on the other hand, is a cost-effective alternative. It is based on the property of cellulose, which selectively binds to dsRNA under specific buffer conditions. This technology allows for the purification of large quantities of mRNA in an environmentally friendly manner without the use of toxic organic solvents, making it a highly valuable technology for industrial applications.
Pre-emptive Inhibition: Enzyme Modification and Reaction Condition Optimization
Efforts are also being made to minimize dsRNA formation during the synthesis process. One of the most prominent areas is the modification of DNA polymerase enzymes, which are used to transcribe DNA into RNA. For example, designing mutant T7 RNA polymerases can suppress aberrant transcription activity and prevent dsRNA synthesis.
The addition of chaotropic agents to the reaction solution is also gaining attention. These agents weaken hydrogen bonds, thereby naturally preventing the formation of complementary RNA strands. Magnetic beads are also being actively used to selectively collect template DNA or target mRNA. Furthermore, molecular optimization techniques are being introduced, such as fine-tuning the 3' terminal sequence of the DNA template or designing the base sequence of the mRNA molecule to disrupt the formation of secondary structures.
Significance and Future Prospects
The development of dsRNA removal technologies is a key to significantly improving the safety and efficacy of mRNA therapeutics. By enabling the production of high-purity mRNA, it can expand the application of the mRNA platform to areas beyond vaccines, such as cancer therapies and gene editing. Reducing unnecessary immune-inducing side effects will allow for safer increases in dosage, reducing the physical burden on patients.
However, there are many challenges to overcome before large-scale commercialization. It is difficult to directly transfer existing purification technologies, which are primarily laboratory-based, to large-scale production lines. For example, chromatography processes have the drawbacks of high equipment investment costs and slow purification speeds. Therefore, the development of continuous purification processes that achieve low cost and high efficiency, and the establishment of standardized guidelines, are key priorities.
Messenger RNA (mRNA) technology has emerged as a cornerstone in vaccine development and therapeutic applications, offering key benefits such as high potency, rapid scalability, and cost-effectiveness. The success of COVID-19 mRNA vaccines has underscored their efficacy and safety. However, residual byproducts generated during mRNA synthesis, such as unincorporated caps, nucleoside triphosphates (NTPs), DNA templates, enzymes, abortive transcripts, and double-stranded RNA (dsRNA), pose significant challenges to the clinical application of the RNA therapy. Among these, dsRNA is particularly problematic as it can activate various innate immune responses, suppress mRNA translation and potentially compromise the therapeutic efficacy of mRNA. Therefore, effectively removing dsRNA from in vitro synthesized mRNA is essential before its used in preclinical or clinical settings. In this review article, we provide a comprehensive overview of current mRNA development pipelines and ongoing clinical trials, and recent advances in mRNA purification techniques. Specifically, we focus on strategies for dsRNA removal, which can be broadly categorized into two approaches: (1) separating or removing dsRNA from in vitro transcription (IVT) mRNA products using methods such as RP-HPLC chromatography and cellulose-based purification; and (2) minimizing dsRNA formation during IVT by employing engineered RNA polymerase mutants, chaotropic agents, and magnetic beads, as well as modifying/optimizing DNA templates or RNA molecules to reduce dsRNA generation. We also discuss the advantages and limitations of these purification methods, the factors influencing the selection of purification strategies, and explore potential future directions for improving dsRNA purification technologies and their applications in mRNA-based therapeutics.
This research provides insights into addressing the major challenges faced by mRNA drug development companies during the commercialization phase: 'quality control (QC) and production cost.' Specifically, the establishment of a high-purity dsRNA removal process will enable contract development and manufacturing organizations (CDMOs) to meet consistent purity standards for mRNA drugs, significantly reducing defect rates. In clinical settings, it is expected to contribute to patient safety. By alleviating concerns about adverse effects caused by excessive immune responses, it may be possible to expand the scope of clinical trials to chronic diseases that require repeated administration of high doses of mRNA, such as cardiovascular diseases or rare genetic diseases.