What can it be used for?
Recently, mRNA has been making headlines. The main reason is for its potential to fight Covid-19. However, the more we’re beginning to investigate its possible uses, mRNA might be the answer we’re looking for to treat a variety of conditions. What exactly is mRNA? How can it be used in the fight against diseases?
Not long ago, mRNA was an obscure term. However, mRNA technology is not new. If you do a simple Google search for “mRNA,” you’ll get access to close to 80 million links. This boom of interest results from the coronavirus pandemic and the development of two of the most important vaccines in history: the mRNA-based Covid-19 vaccines. Despite the speed at which the vaccines were created, the path was far from short. In truth, the vaccines drew on the work of hundreds of researchers over more than 30 years.
To understand mRNA, we first need to talk about DNA. DNA contains our genetic code and is located in the nucleus of our cells. It is a double-stranded molecule made up of a sugar-phosphate backbone and four nucleotide bases (adenine (A), guanine (G), cytosine (C), and (T), thymine). These nucleotide bases contain our genes, which are the blueprints for making the thousands of proteins that we need to survive. However, proteins cannot be produced directly from DNA. Instead, an intermediate is required to carry instructions from DNA in the nucleus to the cytoplasm, where proteins are made. However, proteins cannot be produced directly from DNA. Instead, an intermediate is required to carry instructions from DNA in the nucleus out to the cytoplasm, where the protein synthesis machinery is. Messenger RNA, or mRNA, is the link between the two.
RNA is structured similar to DNA in that it is made up of a sugar-phosphate backbone (although it does have one extra oxygen atom) and four nucleotide bases (ACGU instead of ACGT, where the U stands for uracil). mRNA also contains a polyadenylated tail (poly-A tail) and 5’ cap structure. Through transcription, a pre-mRNA strand is produced by RNA polymerase from a DNA template in the nucleus. Capping, polyadenylation, and splicing in the nucleus convert the pre-mRNA into fully processed mRNA. This mRNA transcript is a copy of the genetic code required to synthesize the specific protein of interest. Three-base units in the mRNA, called codons, specify which amino acids the final protein will be composed of.
Next, the mRNA is exported from the nucleus and travels to the cytoplasm, where it engages with ribosomes. Ribosomes act like tiny factories manufacturing proteins because they take the mRNA code, translate it and produce the coded proteins. The poly-A tail is shortened during the process, and eventually, the mRNA is de-capped and destroyed. Synthetic mRNA uses the same ribosome machinery as mRNA produced in the cell, enabling it to be translated into the protein of interest.
For synthetic mRNA to successfully be translated, it must evade detection by the innate immune system. The innate immune system operates by identifying common signals of damage or infection. There are two types: Danger-Associated Molecular Patterns (DAMPs) and Pathogen-Associated Molecular Patterns (PAMPs), depending on if they are found in the host environment or in pathogens. Pattern Recognition Receptors (PRRs) bind to DAMPs/PAMPs and trigger a downstream immune response. Synthetic mRNA can function as a DAMP, causing activation of the innate immune system leading to inflammation, inhibition of translation, and mRNA degradation. To avoid detection, mRNA can be sequence-optimized, chemically modified, and capped (synthetic mRNA isn’t capped by default, so capping must be built into the manufacturing process). One tactic is removing uridine triphosphate from the final mRNA sequence. Modified nucleoside triphosphates (NTPs) can be substituted for the uridine triphosphate base. Sequence optimization takes advantage of synonymous codons, which are different codons that encode the same amino acid. Many mRNA therapeutics use both synonymous codons for uridine depletion and modified NTPs.
Researchers have been delving into the mysteries of mRNA. It was first identified in 1961. Also discovered in the 1960s were liposomes, which are fatty membrane structures. In 1978, scientists used liposomes to transport mRNA into mouse and human cells to induce protein expression because they protect the mRNA and fuse with cell membranes to deliver the genetic material into cells. In 1984, a Harvard University team used an RNA-synthesis enzyme (taken from a virus) and other tools to create biologically active mRNA in the lab (this method remains in use today) and injected it into frog eggs. They showed that it worked just like the real thing. Since RNA had a reputation for being unstable, the Harvard team decided not to patent the approach. Instead, they gave their components to Promega Corporation, which made the RNA-synthesis tools available to researchers.
In late 1987, Robert Malone performed a landmark experiment following the Harvard team’s tactics to synthesize mRNA for his experiments. However, he added a new kind of liposome that carried a positive charge, which improved the material’s ability to engage with the negatively charged backbone of mRNA. Next, he mixed strands of mRNA with droplets of fat and then bathed human cells in the mixture. The cells absorbed the mRNA and began producing proteins from it. Realizing that this discovery might have far-reaching potential, he wrote on January 11, 1988, it might be possible to “treat RNA as a drug.” Later that year, Malone’s experiments showed that frog embryos also absorbed such mRNA. This marked the first time anyone had used fatty droplets to facilitate mRNA’s passage into a living organism.
After Melton found that the mRNA could be used both to activate and prevent protein production, he helped form a company called Oligogen (later renamed Gilead Sciences) to explore ways to use synthetic RNA to block the expression of target genes. The goal was to see if treating disease was possible. For many years after Malone’s experiments, mRNA was seen as too unstable and expensive to be used as a drug or vaccine. Dozens of academic labs and companies worked on the idea, struggling with finding the right formula of fats and nucleic acids.
In 1989, Malone left graduate studies early to work at Vical, where he and the team showed that the lipid–mRNA complexes could spur protein production in mice. In 1991, Vical entered into a multimillion-dollar collaboration with Merck, one of the world’s largest vaccine developers. Merck scientists evaluated the mRNA technology with the hope of creating an influenza vaccine but eventually abandoned the approach.
Another exciting advance came in 1992 when scientists at the Scripps Research Institute used mRNA to replace a deficient protein in rats to treat a metabolic disorder. However, it would be another two decades before independent labs reported similar success. Then, in 1993, researchers at a small biotech firm, Transgène, showed that an mRNA in a liposome could elicit a specific antiviral immune response in mice. The researchers patented their invention and continued to work on mRNA vaccines. However, the patent lapsed after Transgène’s parent firm decided to stop paying the fees needed to keep it active.
During the 1990s and most of the 2000s, nearly every vaccine company that considered working on mRNA opted to invest its resources elsewhere because mRNA was thought to be too prone to degradation and production too expensive. Instead, the idea of using mRNA started to take hold in oncology circles as a therapeutic agent. It was thought that by taking immune cells from the blood and encouraging them to take up synthetic mRNA that encoded tumor proteins and then injecting the cells back into the body, they could train the immune system to attack lurking tumors. While researchers had some success in mice, when they approached companies about commercialization opportunities, they were told there wasn’t any economic potential in the technology. However, the work had a significant consequence. It inspired the founders of CureVac and BioNTech to begin work on mRNA vaccines. CureVac was the first to achieve success. Their strategy involves altering the genetic sequence of the mRNA to minimize the amount of uridine. They reported in 2000 that direct injections could elicit an immune response in mice. Within a few years, human testing began.
In 2007, RNARx (based out of the University of Pennsylvania) made a key finding: altering part of the mRNA code helps synthetic mRNA slip past the cell’s innate immune defenses. At the time, few scientists realized the therapeutic value of these modified nucleotides. In September 2010, that changed when a Boston Children’s Hospital team described how modified mRNAs could be used to transform skin cells into embryonic-like stem cells and then into contracting muscle tissue. As a result, some team members co-founded Moderna, which tried to license the patents for modified mRNA. After Moderna and RNARx failed to reach a licensing agreement, UPenn granted exclusive patent rights to a small lab-reagents supplier, Cellscript. The company only paid $300,000 in the deal but would make hundreds of millions of dollars in sublicensing fees from Moderna and BioNTech.
By the late 2000s, several big pharmaceutical companies were entering the mRNA field. In 2008, both Novartis and Shire established mRNA research units, with the former focusing on vaccines, the latter on therapeutics. BioNTech also launched that year. Boosted by a 2012 decision by the US Defense Advanced Research Projects Agency to start funding industry researchers to study RNA vaccines and drugs, other start-ups soon got into the mix. By 2015, Moderna had raised more than $1 billion on the promise of harnessing mRNA to induce cells in the body to make their own medicines. When that plan faltered, Moderna prioritized a less ambitious target: making vaccines.
By the beginning of 2020, Moderna had advanced nine mRNA vaccine candidates for infectious diseases into people for testing. However, only one had progressed to a larger-phase trial. So, when Covid-19 struck, Moderna was quick off the mark, creating a prototype vaccine within days of the virus’s genome sequence becoming available online. The company collaborated with the US National Institute of Allergy and Infectious Diseases (NIAID) to conduct mouse studies and launch human trials. All of this was done in less than 10 weeks. BioNTech also was quick to respond. In March 2020, it partnered with Pfizer. Their product went from first-in-human testing to emergency approval in less than eight months.
Importance of Transport Carriers
Many experts highlight another innovation that was crucial for mRNA vaccines. It’s the tiny fat bubbles, lipid nanoparticles (LNPs), that protect the mRNA and shuttle it into cells. Beginning in the late 1990s, the laboratory of Pieter Cullis pioneered LNPs for delivering strands of nucleic acids that silence gene activity. The nanoparticles are comprised of a mixture of four fatty molecules. Three contribute to structure and stability, whereas the fourth (ionizable lipid) is a substance that is positively charged under laboratory conditions. While it offers similar advantages to the liposomes developed and tested in the late 1980s, these ionizable lipids convert to a neutral charge under physiological conditions such as those in the bloodstream. This limits the toxic effects on the body. In addition, the four-lipid cocktail allows the product to maintain its stability inside the body and be stored longer on the shelf. By the mid-2000s, a new way to manufacture these nanoparticles had been invented. The process uses a ‘T-connector’ apparatus, which combines fats (dissolved in alcohol) with nucleic acids (dissolved in an acidic buffer). When the two solutions are merged, the components spontaneously form densely packed LNPs. Since this technique proved to be more reliable than other ways of making mRNA-based medicines, almost every company now uses some variation of this LNP manufacturing system.
mRNA Potential Uses
As a result of all the recent advancements, mRNA has emerged as a promising way to create new classes of medications. One focus is on preventive vaccines for emerging infectious diseases. Another area being explored is using the technology as therapeutic vaccines for cancer, cystic fibrosis, heart disease, rare genetic conditions, and other chronic diseases. Due to its versatility, mRNA can be used for therapeutic protein replacement, gene editing, cell therapy, and many more uses. Sequence engineering and chemical modifications offer precise control mechanisms, and rapid development timelines with scalable manufacturing make mRNA an attractive solution for numerous therapeutic applications. It’s almost limitless in what it can do.
Current mRNA therapeutics function either via direct (in vivo) introduction of mRNA into the patient or by ex vivo use of mRNA to modify cells which are then injected. With in vivo, a delivery vessel (LNPs) is used to facilitate its successful uptake into host cells. A massive advantage of mRNA therapy is that the mRNA never enters the nucleus of the cell, which means it doesn’t edit the person’s DNA. The downside of this is that mRNA therapy isn’t a one-off treatment. So, the person would need to receive mRNA injections/infusions at specific intervals. Another plus is that mRNA is degraded in the body by normal cellular processes, reducing the risks of metabolite toxicity.
Infectious Diseases: Given the success of the mRNA-based COVID vaccines, an obvious next step is to expand the use of mRNA vaccines for other infectious diseases. Vaccines mimic an infection by exposing the body to antigens present in the target infectious agent. This means that an effective vaccine will cause an immune response that leads to antigen-specific B and T cells being created without triggering a dangerous reaction from the body. mRNA vaccines are very simple to make because all you need to know is what your pathogen is and the target for the immune system. Then, you sequence it and make the necessary piece of mRNA, and then you can inject it into someone. How fast can this process go? Well, it took Moderna just two days from the release of the virus sequence to finalize the sequence for its Covid vaccine. It then took only 25 days to manufacture the first clinical batch. Considering that typical vaccine development takes years, this is an incredible feat. Generally, the delivery system stays the same, and the only thing that changes between different mRNA therapies is the coding region of the mRNA. Self-amplifying mRNA (SAM) vaccines are a variant of the current mRNA vaccine technology. They utilize alphavirus genetic replication machinery to amplify the mRNA message within the cell. The result is lower dosing is required to produce the same expression level. Some areas being looked at for vaccine development include influenza, cytomegalovirus (CMV), Zika virus, respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), rabies, yellow fever, rotavirus, and malaria.
Genetic Diseases: The potential of mRNA as a protein replacement therapy to treat certain rare genetic diseases has caused quite a stir. It’s easy to see why since many rare genetic diseases are caused by defects in or deficits of proteins. The theory is that mRNA could be designed to trigger the expression of those missing proteins or healthy versions of the defective proteins. Traditional protein therapy is used to replace secreted proteins. With mRNA therapy, the ability to replace intracellular and transmembrane proteins could be possible, broadening the spectrum of diseases that can be treated. BioNTech recently released a new study showing that mRNA might combat multiple sclerosis. Other research is looking at treating inherited metabolic disorders, rare liver diseases, and cystic fibrosis.
Cancer Immunotherapy: Immunotherapy entails utilizing the immune system to target cancer cells like the immune system targets infection. There are many types of mRNA immunotherapy currently being researched. One approach involves the development of personalized cancer vaccines by using mRNA to express tumor-associated epitopes in host cells. Once presented by the cell, the patient’s immune system will recognize the antigen and mount a response against the cancer. Clinical trials are currently underway using this technology to treat various types of cancer, including lymphoma, skin cancer, ovarian cancer, prostate cancer, breast cancer, non-small cell lung cancers, gastrointestinal cancer, and pancreatic cancer.
Regenerative Therapeutics: mRNA therapy is being tested to promote the growth of new tissues. One area being studied is the creation of new blood vessels to help patients who have myocardial ischemia (a type of heart disease which occurs when blood flow to the heart is reduced, preventing the heart muscle from receiving enough oxygen).
Other Areas: Research is also ongoing in using mRNA vaccines and therapies to prevent and treat HIV, tuberculosis, and various autoimmune conditions. Traditionally, antibody therapy requires the infusion of monoclonal antibodies produced by hybridomas. Unfortunately, the high costs and supply chain challenges have impeded access. Given the relatively low expense of mRNA manufacturing, mRNA antibody therapy is an attractive alternative. Another area of exploration is employing mRNA to introduce non-native proteins into cells, such as tools for gene editing like the CRISPR-Cas family of nucleases.
Who’s Leading the Way?
Moderna is a big player in the mRNA arena, with more than two dozen prospective mRNA therapies and vaccines in the pipeline. They’ve also partnered with Merck to use a personalized mRNA cancer vaccine along with the immunotherapy drug Keytruda for patients with colorectal and head and neck cancers. Early results were promising. BioNTech has a similar number of new mRNA studies and research projects in the works. CureVac has launched studies into a half-dozen other potential medical uses. AstraZeneca is testing a new mRNA-based treatment for heart failure. Translate Bio Inc. is studying mRNA for cystic fibrosis. CRISPR editing company Intellia Therapeutics is evaluating an mRNA-based therapy for the rare inherited disease transthyretin amyloidosis.
Challenges to Overcome
Many scientific and regulatory obstacles lie ahead for these new mRNA-based therapies. It’s important to note that not all the recent news on mRNA has been positive. Targeting mRNA to organs while minimizing side effects still presents challenges. Few have made it to clinical trials, and those that have aren’t always successful. For instance, CureVac reported disappointing results in a clinical trial of its new mRNA-based drug for prostate cancer. The CV9014 drug didn’t boost the survival rates or halt the disease’s progression, the two primary goals of the study. The setback is a reminder that mRNA faces significant hurdles as a therapeutic, particularly in cancer treatment because each type has its own set of challenges.
A significant difference between traditional vaccines and mRNA therapeutics is that vaccines require just one or a few doses. Once the immune system is trained, the protein produced from mRNA degrades and doesn’t need replenishment. With mRNA, repeated doses are required to resupply a protein over a lifetime, which means potential side effects, such as the buildup of lipid nanoparticles in the body or an inflammatory response to foreign RNA, are very possible. To make this less likely to occur, companies are designing mRNA to look as natural to the body as possible, delivering it in biodegradable nanoparticles, and improving the amount of protein the body makes from a single dose of mRNA to reduce the frequency and size of doses required. It’s vital to remember that none of the mRNA therapies under study have been conclusively validated in clinical trials for safety and effectiveness, nor has the Food and Drug Administration (FDA) greenlighted any of these treatments.
No question, the Pfizer and Moderna COVID-19 vaccines have put mRNA technology on the map! They provided real-world proof this developing branch of medicine offers a practical way to save lives. The reason mRNA holds such promise is that it combats disease in an entirely different way meaning the opportunities for using mRNA to prevent or treat diseases look to be vast. If mRNA therapeutics prove to be as successful, they’ll transform the drug industry!