By David Ricketts, DPhil, Bernard Sagaert, Stefaan De Koker, PhD, and Nevin Witman, PhD
In addition to COVID-19, viral diseases that are driving the development of mRNA-based vaccines range from influenza to genital herpes. These vaccines are forthcoming because mRNA development platforms are versatile. They help shorten the timelines and reduce the costs of vaccine development. Moreover, they are readily adapted to new targets—not just additional vaccine targets, but also therapeutic targets. Now that mRNA technology is of proven versatility, the industry is moving swiftly to exploit its potential in a vast range of therapeutic applications, ranging from autoimmune diseases to protein replacement therapies.
Long before COVID-19, mRNA vaccines were developed with oncology in mind, and it seems inevitable that the next major clinical breakthrough will come in the cancer sector. Companies such as Moderna, BioNTech, and CureVac are currently running clinical trials in diseases in advanced melanoma and other malignancies such as ovarian, colorectal, and pancreatic cancers. It is entirely conceivable that an mRNA cancer vaccine could reach the market by the end of the decade.
Cancer vaccines, however, will have to overcome stubborn challenges. For example, vaccine development for solid tumors must be personalized to target patient-specific neoantigens and thereby prevent or delay relapse following the surgical removal of cancerous tissue. Although manufacturers are developing increasingly innovative ways of manufacturing mRNA—for example, CureVac has struck a partnership with Tesla to explore whether bioprinters could automate the production process—the costs are likely to be high.
Nonetheless, mRNA technology could help overcome the manufacturing challenges encountered with CAR T-cell therapies such as Kymriah and Yescarta. These therapies have shown great promise against hematological cancers and lymphoma, but they rely on manufacturing processes that are time consuming, difficult to scale, and costly. Encouragingly, new evidence has emerged demonstrating that a vaccine-like injection of mRNA can induce CAR T cells in situ. Last year, as reported in Science, a team from the University of Pennsylvania together with Acuitas Therapeutics utilized mRNA to engineer regular T cells into functional CAR T cells inside the body. These cells reduced fibrosis and restored cardiac function in a mouse model of heart failure.
Another application that stands to benefit from mRNA technology is gene editing. For example, CRISPR-Cas9 mRNA and synthetic single guide RNA ribonucleoproteins make the nonviral delivery of gene editing components an attractive alternative to adeno-associated viruses. Although multiple clinical trials have shown that adeno-associated viruses have long-term efficacy, safety concerns remain. These include the potential for insertional mutagenesis and even carcinogenesis. The lower immunogenicity of nonviral approaches allows for redosing, while the greater control offered reduces off-target effects and toxicity.
Optimizing payload and delivery
All this serves to illustrate the many opportunities that lie ahead for RNA vaccines and therapeutics. But while we have seen many advances in the generation, purification, and cellular delivery of RNA, challenges remain, including optimizing the RNA payload, reducing toxicity, and improving targeting efficiency.
Many companies wish to explore the possibility of using an mRNA payload in a customized lipid nanoparticle (cLNP), a possibility that has already been realized in COVID-19 vaccines. etherna has dedicated a great deal of research to improving LNP technologies as well as optimizing the mRNA payload. Particularly, when it comes to mRNA construct design, there are many factors to consider beyond merely the coding sequence of the target. The capping of mRNA is crucial to minimize degradation and reduce the innate immune response, while well-designed and carefully paired UnTranslated Regions (UTRs) can significantly increase the expression of mRNA. Optimizing the final component of the construct, the poly A tail, is important to ensure the stability and integrity of the product.
mRNA and cLNP components also need to be carefully synergized for each individual application as both have biological activity and aggregate physiochemical properties. An integrated approach is required that involves not only the optimization of the mRNA payload, but also the screening of a library of ionizable lipids to identify the optimal formulation regarding both lipid chemistry and the mRNA-to-lipid ratio. For example, a more immune-stimulatory cLNP is applicable for vaccines where you want to build an antibody titer; alternatively, immune-silent LNPs might be more applicable for gene editing or treating an autoimmune disease.
Another key issue relates to establishing methods of improving the targeting efficiency of mRNA cLNPs, as cLNP biodistribution is generally broad and mainly directed to the liver. Therapeutics need to be targeted at the organ or even the cell type of interest while delivering enough of the payload deep into the tissue to elicit the desired response.
Broadly speaking, two different approaches try to address this problem. The first is called “active targeting.” Here, a chaperone molecule is chemically conjugated to the cLNP to help guide it toward the desired location. The second is known as “passive targeting.” Here, the cLNP can be designed or manufactured to have a predisposition for certain tissues by modifying the lipid composition and particle size. At etherna, we have shown that by carefully fine-tuning these two parameters, we can preferentially target spleen or liver following intravenous administration of cLNPs/mRNAs.
Researchers working in the gene editing field are particularly interested in finding better ways of targeting mRNA-LNPs toward the heart, lung, brain, kidney, and other tissues. Improved targeting would make it easier to design therapeutics for diseases that afflict these organs, including rare diseases such as enzyme replacement deficiencies and metabolic disorders. The Holy Grail is to find a way of engineering mRNA-cLNPs capable of crossing the blood-brain barrier and unlocking enormous treatment opportunities ranging from brain tumors to neurodegenerative diseases such as multiple sclerosis.
Eliminating the cold chain
Although the rollout of COVID-19 vaccines has been a remarkable success, it was estimated in early 2023 that 2.3 billion people remain unvaccinated against the SARS-CoV-2 virus, with 89% of those living in the developing world. These discouraging figures are due, in part, to the cold chain requirements for mRNA-LNP COVID-19 vaccines. The need to store these vaccines between −50°C and −15°C hinders vaccination distribution, particularly in poorer nations with limited infrastructure. As a result, there has been considerable research over the last three years into lyophilization or freeze-drying. This commonly used pharmaceutical industry technique removes water from drug formulations to increase the stability and shelf-life of products. If technologies can be developed to lyophilize mRNA-cLNP vaccines, it would be possible to ship the vaccines worldwide without the need for cooling or freezing.
However, this process is far from straightforward and requires careful selection of lyophilization buffers, cycle process parameters, and temperatures. When the product is recovered, it is vital to demonstrate that the characteristics and traits of the mRNA and cLNP components have not been altered to a point where they are either unsafe or inefficient. Key physicochemical parameters such as particle size, proper payload encapsulation, and stability of the lipid components are critical to biological performance and must be retained during both lyophilization and subsequent storage.
Several research groups have successfully demonstrated the ability to lyophilize cLNPs containing either small interfering RNAs (siRNAs) or mRNA. However, retaining efficacy after reconstitution with water and during weeks or months of storage has proven challenging. Although siRNA-LNPs were successfully lyophilized by researchers at Carnegie Mellon University, they showed much lower efficacy in vitro following recovery. Another relevant lyophilization study was conducted by scientists at EyeGene, a Korean company. They successfully lyophilized mRNA-cLNP COVID-19 vaccines and showed that the reconstituted products could still induce strong immune responses in mice, but they did not examine whether the products remained stable after time in storage.
Almost every RNA company is attempting to develop new techniques to overcome these hurdles, and there are promising signs. Tekmira Pharmaceuticals has lyophilized an siRNA-cLNP for treating Zaire Ebola virus infection and shown that the reformulated version has equivalent efficacy in a Phase I trial.
The goal is to produce lyophilized vaccines that are thermostable and have a shelf life of more than six months. Such vaccines would be in huge demand. More important, they would be suitable for transport to nations around the world where the cold chain has traditionally proven cumbersome. No one has achieved this goal yet, but it is attracting significant resources.
The patent landscape
Previously undruggable pathways can be targeted by mRNA therapeutics. Accordingly, mRNA therapeutics represent a disruptive technology, one that is expected to change the standard of care for many diseases. However, innovations in RNA therapeutics may face nontechnological challenges. One such challenge concerns third-party intellectual property (IP), an important factor from the mRNA and cLNP perspective.
For example, several companies hold potentially restrictive patents pertaining to core components of mRNA technology. There are patents held on the most prevalent capping technologies, and much IP has locked up the use of modified nucleoside triphosphates, the building blocks of RNA. Other companies are in control of IP relating to lipid formulations and specific lipids used in cLNPs. The IP landscape is complex and dynamic. A number of summary articles provide an overview, including one written by partners at Neal Gerber Eisenberg.
This complex IP landscape could prove to be rate-limiting for the field because RNA startups will have to determine if they can afford the commercial licenses for these different technologies. When companies select modified nucleoside triphosphates, they already find that they must consider licensing issues in addition to what might be biologically optimal.
These are all hurdles that this nascent field will have to overcome. However, given the potential of mRNA platforms to tackle so many unmet clinical needs and the enormous amount of investment being directed toward mRNA, we are certain that the future is bright.
David Ricketts is director, business development, Bernard Sagaert works as interim CEO, Stefaan De Koker, serves as vice president, discovery, and Nevin Witman acts as a consultant at etherna.