Just as in battle, the timely delivery of a coded message can change the course of a disease. Few drug classes have garnered more recent attention than mRNA therapies. By harnessing a cell’s translational machinery to produce therapeutic or antigenic proteins, these molecules promise to greatly accelerate the development and deployment of new drugs and vaccines. As an example, consider the current COVID-19 pandemic, where two of the most advanced vaccine candidates—namely those from Moderna and Pfizer/BioNTech—are based on mRNA technology, with several more are in earlier stages. Meanwhile, other companies are teeing up mRNA-based therapeutics as a means for treating genetic diseases, fighting cancer, and targeting a host of other clinical indications.
While mRNA therapies show promise, and some have even been approved, the development of an entirely new drug class is not without challenges. mRNA therapies can be difficult to deliver and dose effectively. Manufacturing a product that can produce a robust clinical benefit without triggering an unwanted immune response or other adverse outcome is not easy either.
In recent years, researchers have figured out how to overcome many of these obstacles. Those advances are speeding research and trials, and have made the pursuit of mRNA medicines that much easier. Provided the momentum continues, the result could be a new era in accelerated drug development.
Stability is the solution
Many of the most powerful medicines available today—and the majority of vaccines—rely on synthetically-manufactured proteins to control cell functions or mimic viral and bacterial antigens. But developing those drugs is typically time-consuming and expensive. Protein synthesis typically occurs in cell culture, and requires carefully controlled environments and extensive purification procedures.
In principle, mRNA therapies sidestep a number of those challenges. They utilize a cell’s translational machinery to code production of the desired protein in vivo, saving considerable time. Yizhou Dong, a pharmaceutical chemistry researcher at Ohio State University, says this is a major advantage when fighting emerging threats like SARS-CoV-2. “Once we understand its genome sequence, within a very short time we can produce mRNA at large scale and test it in cell and animal models, and eventually clinical trials,” he says.
The actual mRNA production is relatively straightforward. Researchers combine a DNA template that encodes a protein of interest with nucleotides and the enzymes needed for a transcription reaction. But delivering an mRNA therapeutic into a cell at a specific dose presents significant challenges.
One of the greatest is stability. Typically, nuclease enzymes degrade mRNA within minutes. As a solution, many researchers have learned to alter certain chemical features of the mRNA transcript to make it less attractive to nucleases. For example, Juozas Šiurkus, senior R&D manager at Thermo Fisher Scientific, notes that his company offers reagents that allow researchers to modulate the length of the polyadenine tail found at the 3’ end of every mRNA transcript. “These [changes] can define the half-life of the mRNA,” Šiurkus notes.
Alternatively, researchers can also modify the chemical structure of the mRNA cap found at the molecule’s 5’ end. The naturally-occurring cap structure typical in mammalian cells has a tendency to be improperly incorporated into mRNAs synthesized in vitro, rendering them less effective. Synthetic ‘anti-reverse cap analogs’, or ARCAs – such as those offered by Thermo Fisher Scientific – can prevent this misincorporation, which results in more stable mRNA with improved translational efficiency.
A carefully modulated response
Even if researchers can enhance mRNA stability, they still have to contend with its immunogenicity. Because many viruses, including SARS-CoV-2, rely on RNA rather than DNA as their genomic material, the human immune system frequently perceives foreign RNA as a threat. “Different RNA sequences can trigger different levels of activation,” explains Dong. “Maybe there's cytokine release or recruitment of different immune cells, but it can be quite a severe response.”
In recent years, researchers have learned that the strategic substitution of certain nucleotides at the mRNA synthesis stage can dampen this effect. For example, cytosine and uracil nucleotides can be respectively swapped with chemically-modified alternatives like 5-methylcytosine or pseudouridine. Such substitutions can mute the immune response while also bolstering the stability of the mRNA and the efficiency of translation. Thermo Fisher Scientific offers a variety of such modified nucleotides as part of its TheraPure product family.
While such modifications can reduce adverse events from mRNA administration, Dong notes that there are some instances where immunogenicity is an advantage. For example, his group is developing a cancer therapy based on mRNA that encodes an immunity-stimulating cytokine. He has found that the innate immune response to the mRNA itself can actually deliver an efficacy boost. “That's very helpful to remove the primary tumour and also establish whole-body anti-tumour immunity,” Dong says. A similar effect could be beneficial for vaccines, where the mRNA molecule itself primes the immune system to respond more potently against the antigenic protein that it encodes.
The last mile of delivery
mRNA can only exert its therapeutic effects if it safely reaches the cytosol of living cells, where it can be processed into protein by the ribosomal translation machinery. A well-chosen delivery vehicle can also protect its mRNA cargo against degradation or immune recognition without the need for extensive chemical modification. “The field has moved forward towards protecting large RNAs by encapsulating them in nanoparticle formulations, so that they’re basically inside the core and well-protected from any nucleases,” explains Gaurav Sahay, a pharmaceutical scientist at Oregon State University.
Most of the clinically-oriented programs in this space have focused on the use of lipid-based nanoparticles. These particles are inherently well-suited for fusion with the lipid membrane surrounding cells, and have a good track record of safety and efficacy in humans. Indeed, the first RNA-based drug to win US Food & Drug Administration approval, Alnylam’s Onpattro, is formulated via encapsulation in lipid nanoparticles, which enable efficient delivery to the liver. Thermo Fisher Scientific also offers its own lipid nanoparticle-based reagent system, Invivofectamine Rx, for the encapsulation and delivery of therapeutic mRNAs.
Sahay, who works extensively with lipid nanoparticles, notes that these are typically formulated with a mixture of four different lipids. These collectively facilitate the delivery of the RNA-laden particles to cell membranes and the subsequent uptake into cells via endocytosis. As a final step, the RNA cargo must be released from the endosomes responsible for nanoparticle uptake.
This last step remains a significant bottleneck in the mRNA delivery process, but researchers are making steady gains in delivery efficiency. For example, Sahay says that the latest generation of lipid nanoparticles can release up to 25% of their payload into the cytosol. This still leaves behind a lot of unused mRNA, potentially necessitating larger or more frequent doses, although even a small quantity of mRNA can have a potent effect. “Just a few mRNA reads can then create a lot of proteins,” says Sahay, noting that he has obtained promising preclinical data from mRNA-based therapies for chronic diseases like cystic fibrosis.
Once the first wave of mRNA therapies has demonstrated the technology’s mettle, Sahay says he can envision a ‘plug and play’ future. Here, each new mRNA would be individually optimized for a given clinical application, and then produced and formulated for delivery by a more or less standard procedure. In such a way, mRNA could become a generalizable platform for accelerated drug and vaccine development.
As a commercial manufacturer of many critical tools and reagents for the full process of mRNA synthesis, optimization, and delivery, Šiurkus is enthusiastic about the role that Thermo Fisher Scientific could play in that future. “We are producing the bread and butter for the people who are making those compounds,” he says.
In research, new developments in tools and methods frequently presage even larger breakthroughs. The easier and faster it becomes to work with and deliver mRNA in vivo the more advances will appear on the horizon. “Messenger RNA can be standardized; it can be reprogrammed easily; and it is much easier to produce than an antibody,” Šiurkus says. “I believe that the era of messenger RNA is just arriving.”
To learn more about mRNA therapeutics reagents and systems, visit Thermo Fisher.