Dr Pirkko Muhonen, Senior Field Application Scientist, Nucleic Acid Therapeutics, at Thermo Fisher Scientific, shares what’s next for RNA-based medicine post-pandemic with DDW.
With SARS-CoV-2 circulating the globe and leading to over five million deaths worldwide1, all eyes are on the critical role messenger RNA (mRNA) vaccines are playing in helping to fight the ongoing Covid-19 pandemic. While this novel immunisation strategy has shown great promise in reducing the spread of disease and protecting individuals from serious infection 2, decades of research and recent advances in RNA-based therapies point to exciting clinical possibilities across many areas of medicine—from innovative vaccination strategies for fast, expanded disease management and prevention to new options for treating common, life-threatening acute and chronic conditions.3
As researchers discover more about RNA’s diverse functions within cells, they are also fostering the development of engineered RNA molecules with powerful therapeutic properties. By addressing the illness at the source, RNA-based formulations have the potential to improve upon the historical approach to treatment of conditions like cancer, which often has unreliable efficacy and involves cytotoxic drugs. In the infectious disease realm, RNA vaccines offer multiple benefits over traditional live, attenuated, or inactivated options, including improved safety, better efficacy, and faster, easier production.
The recent focus on synthetic RNA’s therapeutic potential has laid the foundation for the development of novel, targeted treatment options that, once refined, could significantly improve clinical care and patient outcomes. Much of the RNA research fueling these efforts revolves around three technologies: messenger RNA, self-amplifying RNA, and circular RNA.
Molecules at the cutting edge of RNA research
Helping to control the Covid-19 pandemic with linear mRNA
mRNA-based Covid-19 vaccines made the theoretical promise of mRNA therapeutics a clinical reality for the first time. The rapid development of these novel formulations to meet the unprecedented pandemic demand highlights the many advantages of this vaccine technology.
Unlike traditional vaccines which work by administering a modified version of a pathogen to generate an immune response, mRNA vaccines offer patients protection without using the virus itself.4 mRNA transcripts designed to encode for a pathogen-specific protein are produced in vitro for subsequent administration. In the specific case of Covid-19, the vaccines lead to production of the coronavirus spike protein characteristic of SARS-CoV-2. The body then responds to the immunogen as it would to a natural infection, resulting in both humoral and cell-mediated immunity.4 While the mRNA vaccines for Covid-19 aren’t 100% effective in preventing infection, the data now strongly shows that immunisation prevents severe disease, hospitalisation and death.2
Traditional vaccine development takes many years and is extremely costly.4 Manufacturing relies on cell culture technologies, dedicated production facilities, and rigorous contamination control.5 Alternatively, mRNA vaccine production uses cell-free processes and is generally faster, more flexible and cost effective. Workflows designed for mRNA vaccine production can be switched to produce vaccines targeting different infectious diseases with relatively little effort. Additionally, administration of polyvalent vaccines containing mRNA transcripts coding for immunogens against multiple pathogens has the potential to increase the efficiency of vaccination programs.
As the Covid-19 vaccine effort has shown, the straightforward, rapid processing behind mRNA vaccines is a benefit to public health.6 Manufacturers are able to respond to market needs and health crises by efficiently developing and distributing a large number of doses for populations in need, which could prove vital in fighting and controlling future pandemics. The value of this capability was brought to light once again after the World Health Organization announced Omicron as a variant of concern in November 2021.7 Pfizer, BioNTech and Moderna subsequently responded with statements that a booster targeting this new variant could be ready to enter the regulatory approval process in a few short months.8
The success of the Covid-19 vaccines has accelerated the use of mRNA technology to target other infectious pathogens, such as influenza, Zika virus, Ebola, HIV and rabies, as well as non-infectious diseases like cancer, autoimmune conditions, and allergies. With hundreds of millions of doses of mRNA vaccine now successfully administered in the United States and worldwide9, scientists are looking at different ways to leverage the power of mRNA to create new, even more efficient and effective RNA-based immunisation strategies.
Development of self-amplifying RNA vaccines to combat infectious diseases and cancer
Another area of RNA research that’s garnering enthusiasm with vaccine developers is self-amplifying RNA (saRNA). Like mRNA, saRNA is a single-stranded and linear RNA molecule that enters the cytoplasm to generate an antigen of interest and promote an immune response.5 When compared to current mRNA vaccines, self-amplifying RNA molecules contain an additional sequence element to encode for an enzyme that can amplify the replication of the mRNA for extended periods of time. This allows the mRNA to remain active in target cells and produce more proteins for a longer time.
With conventional mRNA vaccines, the amount of antigen generated correlates to the amount of mRNA delivered to the cells in the vaccine dose.5 As we have seen with the mRNA vaccines for Covid-19, this direct dose-effect relationship can lead to waning immunity over time and the need for multiple doses and boosters.10 Because saRNA vaccines offer robust and lasting protein expression with a 10 to 100 times smaller dose of RNA, a lower dose of saRNA vaccine is needed to get the same robust immune response.5 Lower dose requirements result in a lower cost per dose, fewer doses needed and lower overall administration costs. The lower doses could also reduce the risk of vaccine-associated adverse side effects.10
Like mRNA, saRNA vaccine formulations can also be made with promisingly simple manufacturing workflows.5 This versatile RNA platform technology is being considered for treatment of infectious disease as well as certain types of cancer, including melanoma and colon carcinoma.
Novel circular RNA constructs against infectious diseases and cancer
Circular RNA (circRNA) is another RNA technology under development for use as a therapeutic or vaccine. These enigmatic molecules are single-stranded pieces of RNA where the ends of the RNA strand are connected, resulting in a closed loop.11 When first identified, the primary role of circRNA was thought to be limited to interactions with microRNA, a type of RNA. microRNA had been found to either degrade or block translation of associated mRNA, so the binding of circRNA to microRNA can result in increased expression of that mRNA.
It is now known that circRNA is capable of regulating transcription and translation, moving and holding proteins within certain cellular compartments, facilitating protein interactions, and even coding for protein translation.11 Research into circRNA is moving quickly, as scientists work to understand the role these elusive molecules play in normal cell function and in pathogenic disease. Recent research findings suggest circRNA molecules could be used as biomarkers of disease, drug targets, or even therapies or vaccines for prevention or treatment of infectious diseases and cancer.11
One approach to using circRNA as a therapeutic molecule is to target disease-associated endogenous circRNA and mitigate its activity through the use of an RNAi therapeutic. Another potential clinical pathway is to use engineered exogenous circRNAs to produce therapeutic proteins within target cells. circRNA has also been used to upregulate advantageous endogenous circRNA expression in both cultured cells and animal models.11 circRNA can also produce large quantities of protein for a longer duration than linear mRNAs, making these molecules a potential alternative to mRNA or saRNA for the stable expression of a therapeutic protein or antigen.12
Challenges in designing safe and effective RNA therapeutics
The mechanisms backing RNA therapy and vaccine research offer great promise, but challenges related to safety, optimised molecular design and effective delivery to target cells remain key hurdles in realising their full clinical potential.
Leveraging RNA modifications
Protecting therapies from breakdown and reducing immunogenicity are both essential when designing RNA therapeutics or vaccines. Due to multiple degradation mechanisms and enzymatic pathways, naturally occurring mRNA is notoriously unstable with a very short half-life in vivo. Synthetic RNA molecules are affected by these same enzymatic pathways. RNA is also highly immunostimulatory and can provoke undesired detrimental effects when administered to patients. To be an option for clinical treatment, RNA-based drugs should be designed to limit immunostimulatory effects while also remaining stable and pharmacologically active long enough to reach target cells and promote the desired response.13
To increase RNA drug half-life and reduce immunogenicity, researchers have fortunately found ways to modify the molecular structure of RNA to improve the stability, efficiency, and safety of therapeutic formulations. These modifications often involve optimising the codons used in the RNA drug, changing the ends of the RNA strands (capping and tailing) or substituting parts of the molecule with chemically modified nucleotides, such as pseudouridine and 1-methylpseudouridine. Each of these changes can impact and modulate the stability, translation efficiency and immunostimulatory effects of RNA.
Both Pfizer/BioNTech and Moderna’s mRNA Covid-19 vaccines use the modified mRNA nucleotide 1-methylpseudouridine.14 CureVac initially tested a Covid-19 vaccine, CVnCoV, using non-modified nucleosides and silent sequence changes that did not affect protein synthesis. This particular formulation, while delivered at a lower dose than Pfizer/BIONTech or Moderna’s vaccines, did not generate an immune response that was deemed sufficient enough to bring the drug to market. 14 CureVac has subsequently produced a second generation re-engineered CV2CoV mRNA vaccine that has shown improved protection against SARS-CoV-2 in animal models and comparable levels of neutralising antibodies to the Pfizer/BIONTech and Moderna vaccines.15
Developing effective delivery mechanisms
Getting synthetic RNA to target cells requires a carrier that is safe and effective while also being biologically inert. Given these requirements, designing an optimised carrier is a challenging step in developing patient-ready RNA-based therapies.
A major achievement in RNA therapeutics was the development of nanoparticle carriers. These carriers can be made from a range of materials, but the most widely used and studied type thus far is lipid nanoparticles (LNPs).11,16 To work effectively, LNP-mRNA formulations need to overcome multiple barriers. The lipid formulation of the LNP needs to protect mRNA from nuclease degradation and the LNP-mRNA complex also needs to be internalised into target cells, ensuring that the RNA is released intracellularly.
Both currently authorised Covid-19 mRNA vaccines, and several other RNA-based drugs both in the research pipeline and in clinical use, rely on LNP delivery mechanisms.17 One size does not fit all when it comes to the LNP drug delivery vehicle, however. Different RNA structures and doses will have varying sizes and charge distributions and different target cells, which affect an LNP carrier’s suitability for a given RNA molecule. Another important consideration for an LNP-RNA drug formulation is having the selectivity to reach a specific target cell or cell type.
Like RNA itself, LNPs can be modified to properly encapsulate different types of RNA and be able to deliver therapeutic molecules to target cells, achieving the desired treatment result., Researchers are working to achieve selectivity by adjusting the proportion of certain lipid components to target only certain cell types for drug delivery. This is especially an area of focus for saRNA, since the RNA sequence is significantly larger than today’s single strand mRNA vaccines.
Lastly, some commonly used LNP components activate host immune responses.16 Polyethylene glycol (PEG)-lipids, for example, may induce hypersensitivity reactions and accelerated blood clearance. Continued investigation into alternative natural and synthetic polymers to replace PEG for mRNA therapies is ongoing, and sure to pave the way for the delivery of future mRNA-based treatments and vaccines.
Reducing the risk of adverse effects with RNA
mRNA-protein interactions and the genetics that drive cellular processes are complex, intertwined, and, in some cases, not yet fully understood, making predicting and limiting treatment-associated adverse events especially challenging with RNA therapies. When synthesising mRNA formulations for patient administration, it is critically important that researchers use all the analytical tools available to identify and address the range of possible downstream translation and protein effects. For example, immune responses toward in vitro transcribed (IVT) mRNA may lead to suppressed antigen expression and, hence, impact vaccine efficacy.
Even though synthetic RNA does not directly affect a patient’s DNA, other measures can be taken to help reduce potential adverse effects of RNA-based drugs. RNA sequence design should be optimised to ensure the therapeutic mRNA encodes only for desired proteins in specific tissues. Once that protein has been generated, the inherent mRNA degradation pathways eliminate synthetic mRNA in a relatively short time. These processes and limitations, inherent to the physiological mechanism driving RNA therapies, help to limit unwanted adverse effects.
In vitro synthesis of RNA can, however, result in undesired shorter, longer, or double-stranded RNA byproducts, and these impurities are capable of causing severe immune reactions contributing to unwanted side effects.18 The use of modified nucleotides, careful purification of the final mRNA product using affinity and column chromatography, or co-administration of immune modulating transcripts can help to mitigate the effects of these unintended components within RNA-based therapeutics.5,18
The future of RNA medicine
The current momentum from the rapid development and deployment of mRNA vaccines for Covid-19 has made RNA-based drugs a reality. The success of these formulations has pushed the promise of RNA therapeutics forward and led to an increased number of mRNA medicine start-up companies and academic collaborations focused on further developing circRNA and saRNA innovations. While targeted delivery to specific organs remains a challenge, researchers have the unique opportunity to leverage recent discoveries and advances in nucleic acid functionality, delivery strategies and stability modifications to refine therapeutics and treatments and optimise production of these flexible and novel medicines. While not discussed here, manufacturing, storage, and distribution challenges will also need to be addressed to ensure manufacturers are able to meet capacity demands, scale production, and provide access to treatment in a cost-effective manner.
Through a renewed enthusiasm for the clinical potential of RNA-based therapies and a continued focus on refining drug safety and efficacy profiles, these treatments are positioned to transform clinical care and improve patient outcomes for a range of conditions long after the Covid-19 pandemic comes to an end.
About the author
Dr Pirkko Muhonen is a Senior Field Application Scientist, Nucleic Acid Therapeutics, at Thermo Fisher Scientific. She has expertise in mRNA production workflow end-to-end solutions and is specialised in mRNA in vitro transcription raw materials.
- WHO Coronavirus (COVID-29) Dashboard. World Health Organization. https://covid19.who.int/. Updated November 9, 2021. Accessed November 9, 2021.
- COVID-19 Vaccines Work. Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/vaccines/effectiveness/work.html. Updated November 9, 2021. Accessed November 9, 2021.
- Roberts TC, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev Drug Discov. 2020;19(10):673-694. doi:10.1038/s41573-020-0075-7.
- Kowalzik F, Schreiner D, Jensen C, Teschner D, Gehring S, Zepp F. mRNA-Based Vaccines. Vaccines (Basel). 2021;9(4):390. Published 2021 Apr 15. doi:10.3390/vaccines9040390.
- Bloom K, van den Berg F, Arbuthnot P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021;28(3-4):117-129. doi:10.1038/s41434-020-00204-y.
- Blakney AK, Ip S, Geall AJ. An Update on Self-Amplifying mRNA Vaccine Development. Vaccines (Basel). 2021;9(2):97. Published 2021 Jan 28. doi:10.3390/vaccines9020097.
- Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern. World Health Organization. https://www.who.int/news/item/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern. Published November 26, 2021. Accessed December 1, 2021.
- Omicron-Specific Vaccines Can Be Ready in 100 Days, Pfizer and Moderna CEOs Say. Observer. https://observer.com/2021/11/pfizer-moderna-ceo-predict-vaccine-protection-omicron-variant-covid/. Published November 30, 2021. Accessed December 9, 2021.
- More than half of U.S. adults have gotten a COVID vaccine. https://fortune.com/2021/04/21/covid-vaccinations-state-tracker/. Published April 21, 2021. Accessed 11/24/21.
- COVID-19 Vaccine Booster Shots. Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/vaccines/booster-shot.html. Updated 11/19/21. Accessed 11/24/21.
- He AT, Liu J, Li F, Yang BB. Targeting circular RNAs as a therapeutic approach: current strategies and challenges. Signal Transduct Target Ther. 2021;6(1):185. Published 2021 May 21. doi:10.1038/s41392-021-00569-5.
- Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018;9(1):2629. Published 2018 Jul 6. doi:10.1038/s41467-018-05096-6.
- Elkhalifa D, Rayan M, Negmeldin AT, et al. Cemically Modified mRNA Beyond COVID-19: Potential Preventive and Therapeutic Applications for Targeting Chronic Diseases. Biomedicine & Pharmacotherapy. Available online 28 October 2021.
- Dolgin E. COVID Vaccine Flop Spotlights mRNA design challenges. Nature. 2021; 594(483).
- Gebre MS, Rauch S, Roth N, et al. Optimization of Non-Coding Regions for a Non-Modified mRNA COVID-19 Vaccine [published online ahead of print, 2021 Nov 18]. Nature. 2021;10.1038/s41586-021-04231-6. doi:10.1038/s41586-021-04231-6.
- Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery [published online ahead of print, 2021 Aug 10]. Nat Rev Mater. 2021;1-17. doi:10.1038/s41578-021-00358-0.
- Let’s talk about lipid nanoparticles. Nat Rev Mater6, 99;2021. https://doi.org/10.1038/s41578-021-00281-4
- Granados-Riveron JT, Aquino-Jarquin G. Engineering of the current nucleoside-modified mRNA-LNP vaccines against SARS-CoV-2. Biomed Pharmacother. 2021;142:111953. doi:10.1016/j.biopha.2021.111953.