Coding for protection

Proteins are what make living creatures what they are. They form the building blocks and spark the molecular reactions fundamental to life, including the creation and management of the immune system, which keeps us protected from disease. The production of proteins hinges on single molecular strands of nucleic acids called messenger RNA, or mRNA. Strands of mRNA in cells act as couriers—hence messengers—carrying instructions, a recipe of sorts, to the ribosomes, where proteins are actually made.

What if you could make mRNA instruct ribosomes to make whatever protein you wanted? This is the concept driving a growing number of researchers who are working on mRNA as a potential therapy or vaccine strategy.

Vaccines typically work by introducing a foreign substance, a non-harmful virus or bacteria, into the body to stimulate an immune response against it. Then, due to immunological memory, if that same pathogen is encountered again, the body can mount a quick and powerful immune response against it. If researchers could introduce custom-made mRNA into cells, the hope is they could direct the body to make specific kinds of proteins, thereby prompting an ongoing immune response against the cancer or pathogen of choice.

University of Pennsylvania immunologist Drew Weissman is compelled by the possibilities for mRNA, particularly when it comes to using this approach as a type of production engine for delivering vaccine immunogens. “We’ve got about 15 different pathogens currently under study,” he says. “We think this is going to be the vaccine platform of the future.”

Researchers have been directly injecting mRNA into mice in an attempt to influence protein production since at least 1990, if not earlier. The most active branch of mRNA research started in a hallway in Philadelphia 20 years ago. There, at the University of Pennsylvania, the Hungarian biochemist Katalin Karikó, who was working in neurosurgery, met Weissman at the Xerox machine. She was working with mRNA, while he was working with dendritic cells and their role in stimulating immune responses by presenting antigens. “A couple of months later, we wrote our first proposal to the U.S. National Institutes of Health for immunization with mRNA encoding HIV-specific antigens,” Karikó says.

By 2010 the Harvard stem cell researcher Derrick Rossi and his team, drawing on the techniques developed by Karikó and Weissman, produced something remarkable—a glowing dot on a mouse leg. What was remarkable was that they made this glowing dot by engineering mRNA to code for a protein expressed in fireflies that produces the bioluminescence in the insect’s light-producing abdominal organ. Then they injected the mRNA directly into a mouse. The result, a glowing dot on the mouse’s leg, signaled that the animal was producing the bioluminescent protein as a result of its cells taking up the mRNA code.

The group then published research using modified mRNA to generate human pluripotent stem cells, which was one of Time magazine’s top 10 scientific discoveries in 2010. It led Rossi to co-found the biotechnology company Moderna Therapeutics, where he remained on the scientific advisory board for the company’s first four years. The company is now valued at US$7 billion, with $1.6 billion in capital.

What began with photocopies and fireflies is now a growing field that includes efforts in different stages of development targeting influenza, melanoma, prostate cancer, lung cancer, herpes, Zika, Ebola, respiratory syncytial virus, rabies, Toxoplasma gondii, and HIV, according to a review article by Norbert Pardi, a University of Pennsylvania research associate and medical professor (Nat. Rev. Drug Discov. 17, 261, 2018). It is still a nascent field but different systems are developing for different pathogens, and now researchers are focusing on optimizing techniques to boost safety and consistency. Most mRNA candidates so far are in preclinical development, but data is emerging from a few early-stage clinical safety studies.

The process of introducing engineered mRNA into the body starts with getting it into cells. Pardi, in his review, describes several methods of introducing mRNA solutions into the body, among them are nose drops; intradermal, intramuscular, subcutaneous, and intravenous injection; or injection directly into lymph nodes or the spleen. At the outset, researchers employed injections of naked mRNA. But naked mRNA, Pardi says, gets degraded by extracellular RNases, nucleases outside the cell that chop RNA into smaller components.

While naked mRNA still offers a potential avenue of inquiry, researchers are now much further along in developing what’s called complexed mRNA, which is mRNA encapsulated in nanoparticles that protects it from degradation. Researchers are employing different solutions for different targets: negatively charged nanoemulsion or lipid nanoparticles for flu, hepatitis C, rabies, and HIV; or liposomes in a solution mixed with a protein called protamine for complexing mRNA for cancer therapies, for instance. Although scientists still do not fully understand the molecular mechanics involved, complexing does seem to aid in shielding the mRNA from the body’s defenses and increasing cellular uptake.

Once inside cells, the magic happens. “The cells translate the RNA to protein, and the proteins do what they are supposed to do,” Pardi says. If for instance the mRNA is introduced into dendritic cells, the intermediaries of the body’s innate and adaptive immune systems, the information carried by the mRNAs could facilitate the production of proteins that would act as antigens and prompt an immune response.

Chemical and nanomaterials engineer Omar Khan, a former Massachusetts Institute of Technology researcher and now chief scientist at Tiba Biotech in Boston, sees broad applications for this approach. “It is neither a vector nor an antigen in the classic sense. We’d rather call it a versatile expression platform, useful for expression of all kinds of biomolecules, including antigens,” Khan says. “Vaccines based on mRNA could be fantastically versatile, but correct delivery is an ongoing challenge. Technology that allows for efficient, effective, and safe delivery of the mRNA is what will really open up the field and bring it closer to the clinic.”

Better techniques for creating RNA sequences, which make mRNA more translatable and stable, are helping. Purifying mRNA using techniques like liquid chromatography, which filters out detrimental mRNA strands, can yield a mix significantly more potent for producing protein in dendritic cells. Modifying the molecules that make up the mRNA code inhibits the induction of antiviral inflammatory immune responses, which could cause potential side effects and raise safety concerns.

The most advanced clinical studies involve an mRNA-based vaccine candidate for rabies being developed by CureVac, a biotech based in Tübingen, Germany. But there are many other companies, big and small, also actively pursuing mRNA-based therapies or vaccines. Another German company, Mainz-based BioNTech, is involved in mRNA platform research. Karikó is now a vice president at BioNTech, leading the company’s mRNA-based protein replacement program. BioNTech recently engaged Weissman’s lab to run a preclinical research program for mRNA vaccine candidates against infectious diseases. There are also a handful of others now in the field: Tiba, Translate Bio, eTheRNA, and Moderna Therapeutics.

Moderna is running Phase I safety trials of mRNA-based vaccine candidates against cytomegalovirus, chikungunya virus, Zika, and metapneumovirus. Influenza virus is also drawing interest: the most recent flu season killed 80,000 people in the U.S. alone, the highest death toll in more than 10 years, and researchers are in pursuit of new formulations for a more universal flu vaccine (see A Mean Flu Season Swings a Spotlight on Vaccines, IAVI Report, Vol 22, No. 1). “I’m very optimistic for flu vaccine. I think it’s doable,” Pardi says. Weissman and Pardi are testing a nucleoside-modified mRNA vaccine candidate targeting the hemagglutinin stalk of influenza, a less variable target on the virus. So far they’ve been able to induce immune responses in mice, rabbits, and ferrets (Nat. Comms. 9, 3361, 2018). CureVac and Moderna are both pursuing mRNA-based flu candidates, as is BioNTech.

Larger pharmaceutical companies are also becoming involved in mRNA research, mostly through deals with biotechs. Earlier this year, BioNTech announced a licensing and equity deal potentially worth up to $425 million with Pfizer to partner on the company’s flu vaccine development efforts. Eli Lilly is collaborating with CureVac on its cancer vaccine candidates targeting tumor neoantigens, which are fragments of protein found on cancer cells. The Bill & Melinda Gates Foundation is investing $52 million into CureVac to support construction of a vaccine manufacturing facility and will separately fund the firm’s development efforts for vaccines against infectious diseases.

This enthusiasm, as well as the money and brainpower being invested in this approach stem from mRNA’s attractive qualities. “Since RNA is injected into the host and the host makes the protein, you can be more certain that these proteins will be functional and properly folded,” Pardi says. “That is a really important thing.”

Another quality that makes mRNA advantageous is its persistence. Weissman and Pardi worked on a Zika mRNA candidate, giving a single dose in an experiment with macaque monkeys, and found that the neutralizing antibody levels induced by the candidate remained steady after a year. Other candidates required two or three shots to gain the same level of neutralizing antibodies required to protect against Zika virus, Weissman says. The team first gave mice single-dose intradermal injections of an mRNA-based vaccine candidate encapsulated in lipid nanoparticles. The mRNA carried the code for pre-membrane and envelope glycoproteins from a Zika strain isolated in the 2013 outbreak. The vaccine elicited Zika-specific CD4+ T-helper cells and neutralizing antibody responses. The team followed this experiment in mice with a monkey study using the same candidate (Nature 543, 248, 2017).

The mRNA platforms appear to also offer advantages in terms of safety and speed of production, and these are significant, Weissman and Pardi say. “It’s two reactions to make and purify the RNA and you are done,” Weissman says.

The mRNA platforms also promise rapid optimization and on-demand production, making them well suited for rapid responses to emerging pathogens. “If you are in an epidemic and need to get a vaccine to people rapidly, I think mRNA has a lot of advantages. The fewer steps you have to get to an immune response the better,” says Wayne Koff, who heads the Human Vaccines Project and was formerly chief science officer at IAVI. Moderna is now a partner of the Human Vaccines Project.

Efforts to develop mRNA-based vaccine candidates are further ahead for pathogens such as flu, rabies, and Zika viruses than for HIV, which poses unique challenges. Researchers cite, as they often do, its rapid mutability as one obstacle. Weissman says another is designing an effective and mature antigen. Antibodies that are broadly effective against HIV are often themselves highly mutated in order to bind to and neutralize an ever-mutating, sugar-covered virus. Coding mRNA to replicate this is a complex challenge.

But while it may be challenging, several research groups are pursuing it. The ongoing work ranges from designing potential vaccine candidates to so-called passive injection strategies to experimental cure research.

Xun Sun, formerly at University of Pennsylvania and now a researcher at Sichuan University’s Key Laboratory of Drug Targeting and Drug Delivery Systems, led a team employing cationic micelles—ionized molecular particles formed in a liquid—to complex and deliver mRNA to dendritic cells in mice. Results showed a detectable immune response specific to HIV’s Gag protein (Drug Deliv. 23(7), 2596, 2016). Pardi, Weissman, and colleagues, including Karikó when she was still at University of Pennsylvania, injected mice with naked mRNA that had been engineered in the lab and encoded for the HIV Envelope (Env) protein gp160, along with an adjuvant solution. The team used this as a prime, administered in two doses, and then boosted with an intramuscular injection of Env protein. Even given the potential drawbacks posed by using naked mRNA, the team was able to detect an immune response in antigen-specific CD4+ and CD8+ T cells in the mice (AIDS Research and Human Retroviruses 30, A249, 2014).

Weissman is currently working with stabilized HIV cell surface trimers for use as immunogens in combination with an mRNA-in-nanoparticle solution. His team also works closely with Bart Haynes’ group at the Duke Global Health Institute to develop novel immunogens for testing as experimental HIV vaccines. And within the Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), Weissman’s team works with an adjuvant development discovery team at Beth Israel Deaconess Medical Center to optimize the immunogenicity of its vaccine candidates. Weissman and Pardi did their one-shot Zika experiments in collaboration with CHAVI-ID, and Haynes says there are more possibilities for collaboration.

“Our general experience is that the mRNAs are doing quite well,” Haynes says, with immunogen designs including Env trimers, gp160 Env proteins, Env proteins that might induce non-neutralizing antibodies—similar to what researchers discovered during the RV144 trial—and others. “We’re working hard to learn how best to use mRNA to express [HIV] Envelope,” he says. “We’re moving rapidly to be able to make mRNA in lipid nanoparticles at our facility here.” Haynes wants to be able to quickly assess immunogenicity of the mRNA constructs in Phase I clinical trials.

Weissman also thinks research results point the way for mRNA as a potential platform for so-called “passive” transfer of HIV-specific broadly neutralizing antibodies (bNAbs) as a way to prevent HIV infection. Weissman, Pardi, and the University of Pennsylvania team worked with Acuitas Therapeutics of Vancouver in deploying a lipid nanoparticle solution carrying modified mRNA encoding for VRC01, one of the first bNAbs of many isolated in recent years that are giving researchers high hopes for developing new vaccine candidates or antibody-based prevention products.

Weissman and his group showed that weekly injections of the VRC01-encoded mRNA caused humanized mice to produce levels of antibody maintained at 40 micrograms per milliliter of blood, and that the translated antibody from a single injection can protect the mice from HIV challenge (Nat. Comms. 8, 14630, 2017).

mRNA also has the attention of researchers pursuing an HIV cure. The Belgian eTheRNA is part of a European Commission-funded group, including the AIDS Research Group at the August Pi Sunyer Biomedical Research Institute (IDIBAPS) in Barcelona and the Free University of Brussels, that over the last five years developed and conducted tests of an mRNA-based therapeutic vaccine. Twenty-one HIV-infected volunteers were enrolled in a Phase I safety trial, which indicated the group’s formula was safe and well tolerated (AIDS 32(17), 2533, 2018). The group went on to launch a Phase IIa immunogenicity trial with 34 volunteers, the results of which are now under analysis and should be available soon, says IDIBAPS infectious diseases service team leader and project coordinator Felipe García.

In reviewing the field, Pardi injects a good dose of caution. “When I talk to company people, they are usually very, very optimistic that mRNA is the best and it’s going to work everywhere. I’m much more cautious because it has turned out so many times—DNA vaccines are probably a good example—that they work very well in mice and then they didn’t work well in people,” he says. “I always emphasize that I’m very cautiously optimistic and really want to start as many clinical trials as possible. Then we will judge if mRNA is really so good or needs to be improved. The next few years will answer this question.”

Michael Dumiak, based in Berlin, reports on global science, public health, and technology.