Advances in mRNA Therapeutics and Vaccines

Medicine is the testament to the virtue of human innovation - a collective desire to reduce the burden of disease and an ever evolving journey to enhance the quality of human health. The origin of medicines can be traced back to ancient times when human began to use the natural resources to treat injuries and illness. Highly developed and documented evidences of medicinal practices comes from the world’s most ancient civilisations such as Egypt, India, China and Greece1. Traditional Chinese medicine2, which dates back more than 2,500 years, used a combination of herbs, acupuncture, and other techniques to treat a wide range of conditions. An ancient Indian system of medication Ayurveda3, dating back to 5000 years, also relies on natural substances such as herbs, minerals, and animal products for treating illnesses. During the middle ages, medicine in Europe was mostly influenced by the works of Greek and Roman physicians such as Hippocrates and Galen4. However, the development of modern medicine is often attributed to the scientific and technological advancements of the 19th and 20th centuries5, which led to the discovery and development of drugs, vaccines, and other medical treatments. Today, medicine is a highly evolving field which encompasses a wide domain of disciplines including but not limited to basic research, public health, clinical practices and policy.

Virtually all conventional drugs currently available in the market are small molecules. Small molecules are low molecular weight (<900 Dalton) organic compounds that target specific biomolecules involved in disease pathways through a variety of mechanism including inhibiting enzyme activity, disrupting protein-protein interactions, or blocking receptor- ligand interaction. Owing to its easy oral bioavailability, versatility and low immunogenicity, small molecules have made significant contributions in treating a wide spectrum of disease. However, small molecules have shown serious limitations such as off-target toxicity, drug resistance, limited druggable target sites and non-optimality when it comes to either tailoring personalised therapy or curing genetic disorders. This necessitates the upsurgence of next generation drug modality including mRNA and protein based therapeutics. mRNA- based therapeutics have advantages over protein-based drugs due to their ability to target intracellular proteins, their high specificity, and the ease and rapidity of their production6. This article gives a wider perspective onto the structural and functional relevance of mRNA in medicine.

Untapped potentials of nucleic acids

As per the central dogma proposed by Francis Crick in 1958, DNA is primarily responsible for storing genetic information which gets transcribed into mRNA and translated into proteins. Proteins exhibits multiple functions in body including enzymatic reactions, signal transduction, and structural support. However, multiple decades of intensive research has yielded numerous secondary applications of nucleic acids (Figure 1). In addition to mRNA and regulatory RNA (siRNA, miRNA and ASO), there are unique classes of RNA as well as DNA called ribozyme and DNAzyme, respectively, which are capable of catalysing chemical reactions such as redox7 and click chemistry8, in turn, mimicking the role of protein enzymes. Further, there are aptamers – single stranded RNA/DNA - which can recognise and bind to its target with high specificity and selectivity9. DNA, specifically, is now being considered as an excellent material for constructing nano-scale devices and objects with promising application in the delivery of mRNA in cells beside its potential application in making DNA nanowire, nano-electronics, and data storage devices10,11. RNA and DNA manifest such unusual functions on the virtue of their 3-dimensional structures. Other than the widely known two-stranded double helical structure, nucleic acids are capable of making non-conventional three-stranded and four-stranded topology such as triplexes12 and G- Quadruplexes13 with major potential functional implications in disease and drug development. Particularly, G-Quadruplexes have been shown to be involved in the life cycle of multiple viruses. Recently, G-Quadruplexes have been recognised as a potential target for antiviral drugs against a variety of viruses including Hepatitis C Virus, Zika Virus, and Ebola Virus14. The next section talks about some of the major biophysical and computational approach that are used to study these DNA/RNA structures and their interactions.

Figure 1. Diverse functions of Nucleic acids

Destination mRNA-therapeutics: tools that helped write the travel guide

The advancement and integration of biochemistry, biophysics and bioinformatics has been instrumental in advancing the field of mRNA therapeutics and developing new therapies for a range of disease including cancer, genetic disorders and infectious diseases. Figure 2 shows a diverse range of techniques that are frequently used to probe and elucidate structural-functional association of nucleic acids. Particularly, NMR and X-ray crystallography15 are used to determine high resolution 3-D structure of biomolecules such as proteins and nucleic acids, including mRNA. This information has been vital in identifying potential targets for mRNA vaccines and to design RNA molecules that are optimized for binding to desired targets. In addition, other biophysical techniques such as single-molecule imaging, CD spectroscopy and cryo-electron microscopy (cryo-EM)16 are used to study the structure and function of mRNA molecules and optimize their design for use in vaccines.

Further, Differential Scanning Fluorimetry (DSF), Fluorescence Resonance Energy Transfer (FRET) and Surface Plasma Resonance (SPR)17 are used to study macromolecule interactions including protein-RNA, protein-protein and RNA-RNA interactions.

Additionally, the advancement in the field of RNA sequencing, bioinformatics and machine learning augmented MD simulations18,19 has been critical in the development of mRNA therapeutics. Researchers have used bioinformatics tools to identify highly conserved regions of viral proteins that can be targeted by mRNA vaccines. This has led to the development of highly effective mRNA vaccines for diseases such as COVID-19.

Computational methods allow researchers not only to design mRNA but also to analyse large databases to scan for potential adverse and off-target interaction of mRNA vaccines and therapeutics.

Figure 2. Biophysical and computational tools to study RNA and DNA

mRNA vaccine and therapeutics

Vaccination is one of the most successful medical technique for disease prevention, control and eradication. Historically, vaccines have been created to defend against disease causing

micro-organism. However, in the future, vaccines could have the ability to act as both preventive and curative measures for a range of ailments including cancer. Conventionally, heat and chemical denaturation were used to produce deactivated vaccines20. Although, the use of killed whole organism-based vaccine had enormous success in eradiating a number of severe human infectious diseases such as polio, smallpox, rubella, mumps, measles21 etc., they have certain drawbacks such as the risk of the pathogen turning virulent, inadequate immune response, and challenges related to production and storage. In contrast, mRNA vaccines22 are a more recent advancement in vaccine technology that offer numerous benefits, such as enhanced safety, design and production flexibility, and rapid response capability for emerging infectious diseases. Further, non-viral nucleic acid-based vaccine manufacturing is a safe and efficient alternative to traditional methods. It eliminates the need for large-scale growth of highly pathogenic organisms and reduces the risks of contamination and release of dangerous pathogens.

There were a lot of key challenges that had to be overcome to use mRNA in therapeutic and vaccination. These included optimizing translation efficiency and mRNA stability, avoiding adverse effects and immune activation, and developing efficient delivery methods to target specific tissues and cells. Additionally, it was important to address the issues of scaling up the production of mRNA and making it cost-effective. To overcome these limitations, the use of chemically modified nucleotide such as N(1)-methyl-pseudouridine (m1Ψ), pseudouridine (Ψ), 5-methylcytidine (5 mC), cap-1 structure and optimized codons have shown promising results23. These modifications help in preventing the recognition of in vitro transcribed (IVT) mRNA by the innate immune system, thereby avoiding any undesirable immune response. They also improve the translation efficiency of the mRNA. Further, HPLC and FPLC purification methods has allowed for the production of highly pure mRNA. This is important as immature contaminant mRNA during in-vitro transcription raises serious issues of translation inhibition and innate immune activation.

Currently, there are two classes of mRNA vaccines: convention mRNA vaccines and self- amplifying mRNA vaccines24. Conventional mRNA vaccines are composed of a single- stranded mRNA encoding the antigen of interest, delivered to the host cells by lipid nanoparticles. Once inside the cells, the mRNA is translated into protein, inducing an immune response. In contrast, self-amplifying mRNA vaccines contain not only the mRNA encoding the antigen but also a viral replicase that can continuously generate more copies of the mRNA, resulting in a higher expression of the antigen and a stronger immune response. While conventional mRNA vaccines require large amounts of mRNA to be injected into the body to elicit an immune response, self-amplifying mRNA vaccines can replicate themselves within cells, resulting in the production of a much larger quantity of antigen.

This leads to a more robust and longer-lasting immune response, potentially requiring fewer doses of the vaccine. Additionally, self-amplifying mRNA vaccines can be designed to target multiple antigens simultaneously, increasing their potential effectiveness against a variety of infectious diseases.

Further, mRNA therapeutics is an emerging area of medicine that employs modified mRNA to treat various diseases. Clinical and preclinical studies have shown its potential in treating various conditions like genetic disorders, cancer, and infectious diseases. For instance, Moderna's mRNA-4157 and BioNTech's BNT111 are mRNA-based cancer immunotherapies

that trigger the immune system to identify and destroy cancer cells25. Additionally, mRNA can be used to address rare genetic disorders, such as cystic fibrosis and phenylketonuria (PKU), by delivering functional mRNA to replace the deficient or absent protein.

mRNA Encapsulation and Delivery

One of the most important aspect of utilizing mRNA for vaccination and therapeutics involves the selection of career module for mRNA delivery inside cells. The administration route and formulation is crucial to evade the stimulation of the innate immune response and to determine the kinetics and magnitude of antigen expression. The delivery of mRNA into cells is challenging due to its large size, negative charge, and susceptibility to degradation by nucleases in the bloodstream. To overcome these challenges, various delivery systems have been developed, such as lipid nanoparticles (LNPs), cationic polymers, and protamine with promising results26. A very unconventional approach of using DNA nanorobots and 3-dimensional DNA cage has also been proposed to safely encapsulate and deliver RNA in cells10. However, detailed mechanistic studies and further improvements will optimize mRNA delivery, including increasing the efficiency of delivery, minimizing off-target effects, and enhancing stability in vivo.

Conclusion

In conclusion, mRNA vaccine and therapeutics have emerged out as a ground-breaking technology with immense potential for the prevention and treatment of various diseases. Numerous preclinical and clinical studies have made significant progress towards the potential use of mRNA vaccines and drugs, indicating that mRNA-based prevention and treatment options can be translated to human applications. The success of mRNA vaccines against COVID-19 has opened up new possibilities for the rapid development and production of vaccines against other infectious diseases. mRNA therapeutics offer a versatile and customizable approach for the treatment of various diseases, including cancer and genetic disorders. Additional investigation is required to enhance the administration and effectiveness of mRNA-based treatments, and to investigate their prospects in other medical fields.

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