mRNA Therapeutics: A Promise On The Horizon
Despite the undoubting progress in humanity's battle against diseases, there remain maladies, which still pose much trouble and worries. Gratefully, in the past 30 years, a new type of medicine has emerged, bringing hope to many. Messenger RNA (mRNA) is a messenger that carries genetic instructions from the DNA to the protein-making machinery. In 1990, Wolff et al. found that mRNA could be directly delivered to adult mice without a drug carrier. Admittedly, the results were poor; nonetheless, this experiment paved the way for future advances in this field (Wolff et al., 1990). mRNA's popularity skyrocketed during the COVID-19 pandemic due to its role in the race to establish a vaccine against SARS-CoV-2 (Chanda et al., 2021). An mRNA-based vaccine possesses a unique feature: it simultaneously stimulates innate immunity by binding to pattern recognition receptors and adaptive immunity by providing antigens (Pardi et al., 2018). Still, that only represents one of the many possibilities of the therapeutic use of mRNA, which this article explores.
What is mRNA, and what is its structure?
In eukaryotes, messenger RNA (mRNA) is a single-stranded RNA molecule created during the transcription process from DNA. Its primary purpose is to bear crucial information concerning protein structure. The mRNA molecule consists of a sequence of nucleotides known as codons, which correspond to specific genes or proteins. This sequence encompasses four distinct nucleotides: adenine (A), cytosine (C), guanine (G), and uracil (U) (uracil replaces thymine (T) found in DNA). mRNA is responsible for transporting the genetic instructions encoded in DNA from the nucleus to the ribosomes, the cellular factories where protein synthesis occurs. mRNA is a temporary copy of a particular gene or set of genes, providing the necessary information for ribosomes to synthesise the corresponding protein molecules. By facilitating the transmission of the genetic code, mRNA plays a pivotal role in determining the structure and functionality of proteins (Alberts et al., 2002). The naturally occurring mature mRNA comprises the five fundamental subsequent elements: the 5′ cap, the 5′-UTR, the coding region, the 3′-UTR and the poly(A) tail (Figure 1) (Weissman, 2014).
Eukaryotes possess a molecule at the 5′ ends of RNA, followed by a bridge binding it to the first nucleotide. That structure is called a 5′ cap. Its purpose is to regulate pre-mRNA splicing, initiate translation, protect mRNA from cleavage and recognition of self-RNA by the receptors of the innate immune system. It is a crucial component that partakes in efficient mRNA translation (Weissman, 2014).
At the 3′ ends of human mRNA, a long chain of around 250 nucleotides (nt) is situated. That polyadenylated region bears the name of poly(A) tail. It influences mRNA's stability and translational efficiency with the 5′ cap (To & Cho, 2021).
The coding region, also called the coding sequence (CDS) in mRNA, is encompassed by the 5′ cap and the poly(A) tail at the 3′ ends. It is the part of mRNA that the ribosome reads during translation. The coding region begins with a start codon, typically AUG (adenine-uracil-guanine), which initiates translation. It is followed by a series of codons, each consisting of three nucleotides, which encode the protein's amino acid sequence. The coding region ends with a stop codon, such as UAA, UAG, or UGA, which terminates translation and marks the completion of the protein chain (Weng et al., 2020).
UTRs —untranslated regions— are separate sections on each coding region's side. They contain specific nucleotide sequences crucial in regulating post-translational gene expression, mRNA degradation rate and stability. Unlike the regulatory elements in DNA, UTRs are built on a combination of primary and secondary structures on which they base their biological activity. The 5′-UTR contains the Kozak sequence - an essential nucleic acid motif with the start codon (methionine) (Qin et al., 2022).
Why mRNA?
The first attempt to use mRNA for an in vivo protein expression was made approximately three decades ago. At first, due to the instability of mRNA and inept distribution in vivo, researchers were hesitant about using it and focused their work on DNA-based and protein-based therapeutic approaches. However, especially in recent years, it has been made apparent that mRNA holds the potential to revolutionise vaccination, protein replacement therapies and genetic disease treatment (Kauffman et al., 2016).
One of the essential advantages of mRNA-based treatment is its safety. Unlike DNA, mRNA does not need to cross the nuclear membrane to become functional, eliminating the risk of insertional mutagenesis (Wei et al., 2023). It can be challenging to create small molecules that can boost the function of a faulty enzyme in a different way. It can also be tough to produce a properly structured and modified protein to replace the missing enzyme. In contrast, generating mRNA that carries the instructions for making the normal enzyme is relatively easy. When this mRNA is given to the correct cells, it can replace the lacking enzyme and help restore normal function (Magadum et al., 2019).
Figure 2 summarises the pros and cons of using mRNA in medical approaches.
mRNA manufacturing is flexible, cost-effective, and commonly relies on in vitro transcription processes (IVT). IVTmRNA generates higher circulating protein levels in vivo compared to DNA plasmids and viral vectors. One major difficulty in delivering proteins directly into the bloodstream is maintaining their intricate three-dimensional structures, essential for biological functionality. However, this drawback can be overcome by directly providing mRNA to target cells (Magadum et al., 2019). Another advantage of mRNA-based therapy is a reduced risk of anaphylactic reactions, which can occur with traditional protein-based enzyme replacement therapy (Prieve et al., 2018).
Naturally, mRNA-based therapy is not free of impediments. These include the short half-life of mRNA and mRNA-mediated protein, low rate of in vitro transcription, and activation of the innate immunity resulting from mRNA transfection. This process also carries risks, as it is difficult to predict the precise penetration of mRNA into individual cells and to affirm whether every mRNA molecule has been released from the liposome that encapsulated it (Zarghampoor et al., 2019). Ideally, therapeutic mRNA exhibits low immunogenicity, prolonged stability and robust translation. One of the most challenging aspects of therapeutic mRNA use is successfully delivering the molecule to the target cell without provoking an immune response and traversing the cell barriers (Wei et al., 2023).
Possible Applications
mRNA-based therapeutics are expected to become a powerful therapy for various diseases, such as infectious diseases, genetic disorders, cancer, and cardiovascular conditions. As aforementioned, mRNA was the key player in developing the COVID-19 vaccine. Instead of using weakened or inactive virus particles, mRNA vaccines deliver a portion of the virus's genetic material to the cells. This genetic material contains instructions for producing the spike protein normally found on the virus's surface. When the mRNA enters the cells, it acts as a set of instructions, guiding them to create the spike protein. These spike proteins are harmless and stimulate the immune system, initiating a protective response against them upon encounter. Prior to COVID-19, considerable efforts were made to develop mRNA vaccines for other viruses like influenza, Ebola, Zika, rabies, and HIV, with some progressing to clinical trials. However, further validation is needed to explore the application of mRNA vaccines against pathogens other than SARS-CoV-2 (Wei et al., 2023). Figure 3 demonstrates potential applications for mRNA-based therapeutics.
Attempts at using mRNA-based therapeutics may be limited to three distinct approaches: immunotherapy, transcript replacement therapy and reprogramming. The principal idea behind each of them is to stimulate immune responses against target cells (mostly viral-infected or cancerous), treat monogenic diseases arising from mutations in the genetic code, and prompt stem cell-like pluripotency to generate a desired type of cells, respectively (Granot & Peer, 2017).
Immunotherapy is a treatment that harnesses the immune system's power to fight diseases, including cancer. mRNA-based immunotherapy takes advantage of modified mRNA to boost and strengthen the immune response against specific targets. In 2015, Kübler et al. conducted clinical trials in which they administered mRNA to patients with prostate cancer. Their research proved fruitful and described the technology as able "to generate immune responses against virtually any protein antigen" (Kübler et al., 2015).
In replacement therapy, the preeminent idea is to use the patient’s cells as factories, i.e., instead of directly administrating the drug, the functional protein is manufactured and expressed by the recipient of the therapeutic mRNA. The main advantage of such an approach relies on the individual post-translational modifications, which results in high-quality production whilst simultaneously negating any inducement of defence mechanisms in the host. Cellular reprogramming includes prodding fully differentiated adult cells back into pluripotent cells with the help of Yamanaka factors [Oct4, Sox2, Klf4 and cMyc] (Kwon et al., 2018).
The mechanism of action of mRNA therapy for monogenic disorders is to restore the deficient or defective enzyme/protein by utilising mRNA. Martini et al. investigated the efficacy and safety of mRNA therapy in treating three distinct monogenic disorders: methylmalonic acidemia (MMA), acute intermittent porphyria (AIP), and Fabry disease. The studies demonstrated promising results in animal models, showcasing the effectiveness and tolerability of mRNA therapy following intravenous administration, both as single and repeat doses. However, while preclinical data is encouraging, it requires evaluation in a clinical setting (Martini & Guey, 2019). Figure 4. depicts simplified mechanisms of action of mRNA-based therapeutics.
Chanda et al. investigated using mRNA encoding human telomerase (TERT) as a therapeutic for senescence, that is, the irreversible growth arrest and functional decline of cells. Cellular ageing is influenced by telomere erosion, where telomeres (protective caps on chromosomes) become shorter with each cell division. When cells reach their limit for dividing, the telomeres become critically short, which triggers a response that leads to cell cycle arrest and damage. Telomerase is a protein that can lengthen telomeres and reverse this process. It is found in stem cells and some adult stem cells, which explains why they can divide more. Most other cells do not have telomerase, but it can be activated in rapidly dividing immune cells. A concern with telomerase therapy is the potential risk of cancer. In approximately 85% of cancers, human telomerase becomes active again. However, it is implausible that the transient expression of human telomerase using mRNA will increase the cancer risk (Chanda et al., 2021).
Optimisation is crucial
Unfortunately, typical mRNA is unstable, which disqualifies it from clinical applications. In their ground-breaking studies, Karikó et al. modified the structure of mRNA by changing specific components, which made it more stable (Karikó et al., 2008). Optimised mRNA promises an efficacious treatment for copious chronic genetic diseases. Incorporating stabilising elements into particular sections of mRNA, like the 3' untranslated regions, enhances its stability. For example, when using the 3' UTRs of α- and β-globin, the efficiency of mRNA translation is significantly improved. Moreover, including specific segments from viruses or human proteins can further enhance translation efficiency.
The composition of the coding region greatly influences translational efficiency. For example, changing a given codon with one that is synonymous but occurs more frequently in a given cell improves the elongation rate. The downside of optimising codons is that some proteins demand a slow translation rate to ensure correct folding. The phenomenon where there is a difference in the frequency of usage of synonymous codons is called codon usage bias and is the result of the degenerative nature of the genetic code (Qin et al., 2022).
mRNA capping is a crucial process in mRNA maturation that influences mRNA stability, splicing, translation initiation, export, and immune recognition. The cap structure serves multiple roles in ensuring the proper functionality and destiny of mRNA molecules, thereby influencing the efficiency and precision of protein synthesis. As a result, extensive research has been conducted to explore various modifications, with particular attention given to adding methyl groups (Zarghampoor et al., 2019).
Package delivery
A successful therapeutic approach hinges on the correct choice of a vector. The most commonly used non-viral delivery system for mRNA is lipid-based nanoparticles (LNPs) (Figure 5). These LNPs contain different lipid and polymeric materials, allowing them to enter cells through endocytosis. Researchers like Wang et al. have studied lipid-protamine particles to encapsulate mRNA, showing improved protein expression and mRNA stability (Wang et al., 2013). In a study by Rybakova et al., LNPs were used to deliver a cancer drug called trastuzumab, targeting a specific protein overexpressed in breast cancer. This delivery method effectively slowed tumour growth without causing harmful side effects (Rybakova et al., 2019).
The clearance and biodegradability of the delivery system also are a matter of concern. When administered intravenously, most drugs and nanoparticles tend to accumulate in the liver due to the structure of the circulatory system. However, targeting other tissues can be challenging. LNPs, with their size of around 100nm, face difficulty entering the bloodstream, preventing effective filtration by the kidneys and resulting in their accumulation in organs (Kowalski et al., 2019).
Conclusion
mRNA-based therapeutics represent a paradigm shift in medicine, offering new avenues for effective treatments. Continued exploration and investment in this field will undoubtedly lead to further breakthroughs and advancements in mRNA-based therapies, addressing unmet medical needs and improving the lives of countless individuals.
