The approval and widespread use of messenger RNA (mRNA)-based vaccines for SARS-CoV-2 represented a significant milestone in the use of mRNA as a therapeutic agent. These products reified the incredible potential of in vitro transcribed (IVT) mRNA theoretically capable of producing nearly any functional protein or peptide in the body.
mRNA-based therapeutics were first conceptualized more than three decades ago with the discovery that naked mRNA injected into mice could be translated into proteins of interest. This led researchers to further explore IVT mRNA as a potential tool for protein substitution and vaccines for cancer and infectious diseases. In the years since mRNA has been positioned as a promising therapeutic modality for numerous indications, but we’ve only scratched the surface of its full potential.
Several features make mRNA advantageous for therapeutic applications. Because mRNA transcripts do not need to enter the nucleus to be functional, they have low toxicity and relatively high transfection efficiency. Additionally, mRNA can also provide greater therapeutic efficacy over traditional transient protein and peptide drugs because its continuous translation triggers a more lasting expression of the protein or peptide of interest.
However, mRNA therapeutic development has faced numerous challenges, impeding growth in the field. Advancements that overcome the instability, immunogenicity, and delivery issues of therapeutic mRNA have been vital to progress thus far and will continue to drive the future of mRNA therapeutics.
Accelerating therapeutic progress
The development of an mRNA-based therapeutic demands far more than a nucleic acid sequence alone. For an mRNA drug or vaccine to be viable, it must be stable, tolerable, and able to enter a cell for effective protein translation in the cytoplasm. Effective delivery of therapeutic mRNA remains a substantial challenge in drug development. Because mRNA is negatively charged, passage through the anionic cell membrane presents a significant obstacle in therapeutic design. Moreover, the intracellular half-life of mRNA is only about seven hours, limiting its potency even after successful cellular entry.
Advancements in mRNA loading mechanisms enable effective cellular entry, high translation efficiency, evasion of ribonuclease degradation, and low toxicity and immunogenicity. Lipid nanoparticle (LNP) carriers were used in the two approved mRNA-based vaccines for SARS-CoV-2 and have been leveraged in an increasing number of drugs and vaccines currently in clinical trials. LNPs demonstrate great promise but still require careful design and formulation to effectively reach target cells and tissues and escape endosomal compartments after cellular uptake. Other delivery methods explored in the development of mRNA therapeutics include polymeric nanoparticles, cationic nanoemulsions, extracellular vesicles, exosomes, and more.
Innovations in mRNA capping methods have also been instrumental to progress in therapeutic development. The 5’ cap is an evolutionarily conserved hallmark of eukaryotic mRNA consisting of an inverted 7-methylguanosine linked to the first nucleotide of the mRNA via a triphosphate bridge. Several forms of the 5’ cap occur naturally: Cap0, Cap1, and Cap2. These forms differ based on the presence of methylation at the 2’-hydroxyl group of the first and second nucleotides following the cap, with Cap0 being an intermediate form that lacks these methyl groups entirely. The 5’ cap is an essential component of mRNA function, regulating nuclear export, mRNA splicing, turnover and decay, and self/non-self-recognition by the immune system. It also serves as an anchor for the recruitment of factors that initiate protein translation. Capping is thus essential for the creation of functional mRNA therapeutics and vaccines.
Capped synthetic mRNA was initially created using post-transcriptional enzymatic processes. RNA created in an IVT reaction would undergo a dedicated enzymatic capping reaction using capping enzymes, such as those from the Vaccinia virus, and their required substrates. Co-transcriptional capping is a second option wherein cap analogs are added directly to the IVT reaction. Overall, co-transcriptional capping offers several significant advantages over enzymatic capping. Whereas enzymatic capping requires multiple reaction and purification steps, co-transcriptional capping can be performed in a single bioreactor, curbing manufacturing costs and saving time.
Anti-reverse cap analog (ARCA), a legacy co-transcriptional capping approach, results in an inferior and immunogenic Cap0 structure and is comparatively inefficient, yielding only ~70% capped mRNA. Further advancements in cap analog technology have improved capping efficiency, stability, and immunogenicity. Continued innovation in mRNA capping technology will be instrumental in increasing manufacturing efficiency, reducing manufacturing costs, and making safer and more effective mRNA-based therapeutics a reality.
Over the last thirty years, mRNA-based vaccines and therapeutics have made the journey from concept to nascent reality. Over 100 clinical trials of mRNA products were initiated as of 2022, and two FDA-approved mRNA vaccines are currently available. mRNA therapeutics offer a path to treating previously undruggable targets and present new potential for treating cancers, curing genetic disorders, preventing infectious diseases, and more.
Continued momentum in the field will undoubtedly have a massive impact on global health, but such progress is contingent on further innovation in technology used to develop these drugs. Novel techniques for mRNA delivery, capping and more will be the key to producing more effective vaccines and lifesaving therapeutics safely, efficiently and on a wider scale than ever before.