A magazine for friends of the Warren Alpert Medical School of Brown University.

The Rest of the Story


We thought DNA would explain it all. Turns out RNA has something to say about life and human disease.

Last October, the two scientists whose breakthrough discoveries made possible the SARS-CoV-2 vaccines received the Nobel Prize in Physiology or Medicine. Their finding, in 2005, was deceptively small: a modification to a messenger RNA molecule, which let it elude the body’s immune system, enter the cell, and prompt it to produce antibodies. That tweak proved the viability of mRNA vaccines, and 15 years later saved millions of lives worldwide.

Prior to the COVID-19 pandemic, mRNA vaccines were developed for rabies and several cancers; clinical trials are still underway. But RNA, many experts believe, is poised to do so much more.

“COVID was just the tip of the iceberg. Literally, the tip of the iceberg,” says Mukesh K. Jain, MD, the dean of medicine and biological sciences at Brown.

Jain is referring not just to the diseases we know are related in some way to RNA—several cancers, flu, Huntington’s, spinal muscular atrophy, Alzheimer’s, ALS, to name just a few—but to the ones we don’t. And that number is vast: due to shortcomings in available technology, we know the function of, at most, 5 percent of all RNAs. The rest, says Professor of Molecular Biology, Cell Biology, and Biochemistry Juan Alfonzo, PhD, is “dark matter.” Alfonzo, a world-renowned RNA biologist, sums up the fundamental quandary confronting his field: “What the heck is going on?”

He’s determined to get to the bottom of the mystery, as the director of the brand-new Brown RNA Center and a leader in a global effort to identify all of our RNA, in all of its forms—a project that Jain says would be “one of the largest scientific endeavors since the Human Genome Project.”

If the endeavor succeeds, Alfonzo adds, the outcome will be no less than “a new understanding of medicine and biology.”

To grasp the potential gains of fully sequencing our RNA, you have to appreciate just how little scientists know about it now. Until relatively recently, “we used to think of RNA as this boring thing between DNA … and protein. Nothing happens. It’s a direct copy of DNA,” says Vivian Cheung, MD, another internationally acclaimed RNA researcher—and close friend of both Alfonzo and Jain—at the University of Michigan.

You may remember from biology class RNA’s four nucleotides—guanine, adenine, cytosine, and uracil—and that DNA encodes mRNA, which carries the instructions to make protein.

About three-quarters of our DNA is transcribed into RNA, Alfonzo says. But only 1 percent makes mRNA. The other 74 percent of the genome, he says, “used to be called junk DNA, because nobody knew what it was doing. But now you start sequencing RNA and you’re like, wow, all of this is making RNA.” These are the non-coding RNAs: transfer RNA and ribosomal RNA—which are also involved in protein synthesis—but also microRNA, and circular RNA, and small interfering RNA, and piwi-interacting RNA … and the list goes on. Most if not all seem to regulate gene expression. But how, exactly? “Who knows?” Alfonzo says.

“Out of that 74 percent [of the genome that makes non-coding RNA], it’s calculated that we know the function of 4 percent to 5 percent of it,” he continues. “So the remaining 70 percent, we have no clue what it’s doing. And even when we said we know the function of 4 percent to 5 percent, we know a function.”

But wait, there’s more: RNAs are composed not only of the four primary, or canonical, nucleotides; there are more than 185 modifications—and counting—to those nucleotides that are critical to RNA function. This is the understanding that won biochemist Katalin Karikó and immunologist Drew Weissman the Nobel Prize last year: the mRNA had to be modified to generate the right immune response.

“Back then, people really thought, who cares about putting a decoration on RNA? It’s completely academic,” says Cheung, who was on the University of Pennsylvania faculty with Karikó and Weissman when they made their discovery. “And it turns out to save how many people’s lives, and now win a Nobel Prize.”

But what the precise RNA modifications are that make the SARS-CoV-2 vaccine possible, Alfonzo says, is unknown. The technology to accurately sequence single molecules of RNA—and identify the locations of the modifications on each molecule—does not exist. “So if they wanted to improve [the COVID vaccine], they have to do it empirically. Because currently, they cannot even tell you what is the prevalence at each position,” he says. It’s like trying to read a book without the full alphabet, or any punctuation.

In RNA research circles, a certain quote by former Secretary of Defense Donald Rumsfeld comes up a lot, about known knowns, and known unknowns, and unknown unknowns. Alfonzo gets frustrated when colleagues declare anything to be “known” in relation to RNA modifications.

“If you don’t even know what is out there, how can you know you know it?” he says.

Cheung likens today’s RNA sequencing technology to a backyard telescope, while what we need is the James Webb Space Telescope. In just two years since it launched, “our knowledge about the universe has completely changed,” she says. “And I think that the technology for RNA has that potential.”

In 2020 Cheung emailed Alfonzo, who was then at Ohio State University, and asked him, “Are you as bothered as I am by the state of technology to sequence RNA directly?” Alfonzo—who studies tRNA editing and modifications and how they contribute to mitochondrial function—says he replied, “‘Bothered, Vivian?’ And I sent her like five commentaries that I’ve written through the years, making the point precisely: that we’re missing a huge chunk of information in the cell.”

For years, scientists studying RNA had to content themselves with complementary DNA (cDNA) sequencing, which uses reverse transcription to convert RNA back into DNA, to then determine the RNA sequence. But DNA has no complements for RNA’s modifications—so that critical information is lost in the process.

Now researchers have two direct sequencing methods at their disposal, but both have shortcomings. Mass spectrometry, Alfonzo says, is “super accurate,” and able to identify and quantify modifications, but it can only handle short strands of RNA and requires large amounts of sample. Nanopore sequencing, on the other hand, can analyze long, single molecules, and does so very accurately with DNA—“but for RNA, the error rate is just ridiculous,” he says.

A year after that 2020 email exchange, Alfonzo, Cheung, and four coauthors from around the country published a paper in Nature Genetics decrying the state of RNA sequencing technology and calling for the development of better machines that can sequence full-length RNAs and identify all modifications. Noting that the Human Genome Project had not yielded the full genetic picture that humanity had hoped for, they wrote that a complete sequence of our RNA and its modifications—the RNome—“should advance understanding of gene regulation and lead to new frontiers in health and medicine.”

Not long after Jain read that article, he was in a position to answer their call to action. “I like to say they’re the brains, and I’m the brawn,” Jain says of Alfonzo and Cheung. Before he arrived at Brown as dean in March 2022, he worked with them on a proposal, to the National Academies of Science, Engineering, and Medicine, that it assemble an international committee of experts to draw up a plan to develop new technology and sequence the RNome. When NASEM agreed, Jain then went to The Warren Alpert Foundation—which named the Medical School he would soon lead—and secured funding for the committee.

“I said, you have a once-in-a-lifetime opportunity to support the National Academy’s efforts to develop a roadmap to sequence RNA, which, if it gains federal funding support, could catalyze one of the greatest scientific efforts since the Human Genome Project,” Jain says he told the foundation.

The NASEM workshop, which Alfonzo helped plan, took place last March; the group continued to meet throughout the year to put their report together. In addition, an RNome working group, led by Cheung, met in Providence in January to develop an action plan. The scientists hope these efforts will inspire public and private entities around the world to unite to sequence the RNome, just as they did for DNA.

“If it is funded, it is my hope that our community may be able to successfully compete and become an anchor site advancing RNA science, RNA therapeutics, and RNA diagnostics in Little Rhody,” Jain says. “At least that’s my pie-in-the-sky goal.” “Rhode Island has a long history of being a manufacturing hub,” Cheung adds. “It would be exciting to think of Rhode Island becoming an industry hub of RNA technology.”

Jain came to Brown determined to establish a center for RNA research. He said as much to President Christina H. Paxson during his recruitment interviews, and to Kimberly Mowry, PhD, the chair of the Department of Molecular Biology, Cell Biology, and Biochemistry, the first time he met her.

“I was very excited,” says Mowry, who studies the localization of mRNA molecules within oocytes. “We’ve had a lot of people at Brown over the years who are very interested in RNA, but we haven’t had the critical mass of a bunch of different labs working in this area.”

When the University approved the Brown RNA Center last fall, a number of existing faculty were ready to affiliate, bringing expertise in biology and genetics and chemistry and engineering. Alfonzo will deepen the bench with several new recruits, one of whom started in January; eventually most of their labs will be housed together, on one floor of the planned Integrated Life Sciences Building, which Brown estimates will be built in three to four years.

Having a group of RNA scientists in one place who are thinking about different approaches to similar problems “is going to really help us make breakthroughs,” says Mowry, the Robin Chemers Neustein Professor of Biomedicine. “The types of science and the types of discoveries that can be made are pretty much limitless with a focus on RNA,” she adds; the Brown RNA Center will help “accelerate progress and discoveries in RNA science, and that will be to the benefit of everybody, in terms of understanding how cells work, in terms of therapeutics, and in terms of disease.”

Indeed, that is Alfonzo’s vision for the new center: “to get people who like to collaborate.” He says that while there is value in focusing on a particular research area—say, RNA modifications—he adds, “when you are too focused, you miss a lot. … To me, the most important thing is to get people who are really, really, really, really good at whatever they do related to RNA, and if on top of that they have a collaborative mind, then things will happen.”

Mowry endorses Alfonzo’s goal for the center, adding with a laugh, “I’m excited about the high emphasis on bringing in collaborative faculty, who also think RNA is as cool as I think it is.”

In addition to the emphasis on collaboration, Jain says that he and Alfonzo also discussed how to organize the center: “The vision is that you have a core group that does hard-core science and new discovery; then you have a group that helps develop technologies; and then you have a translational group that thinks about the new science and the technologies that you’re developing, and how it can impact human health.”

Jain adds: “Whatever they decide scientifically, it’s going to be important. Otherwise, why the heck would anybody do it?”

One group of scientists was pivotal to the success of the COVID vaccines, though their contribution wasn’t recognized by the Nobel committee: the developers of lipid nanoparticles, tiny vesicles that can deliver small molecules inside cells. “It’s not just the RNA biologists” who made the vaccine possible, Cheung notes. “You need the engineers to figure out how to wrap the RNA into lipids and to be able to give it as a shot to someone. There are so many complications from basic science to engineering to production.”

Alfonzo acknowledged this need with his first recruit to the Brown RNA Center: Theresa Raimondo ’11, PhD, a biomedical engineer who creates lipid nanoparticles to deliver RNA therapeutics. For her postdoc at MIT, she focused on delivering immunotherapies to ovarian cancer—work she’s continuing in her new lab in Providence.

“Delivering RNA to a specific organ and a specific cell type is really a challenge, and that’s limiting what we can do therapeutically,” Raimondo says. “I’m excited about better understanding how to control the delivery of these particles, and then engineering and optimizing a system that is going to be really
targeted, safe, efficient, and therapeutic.”

As a PhD student at Harvard, Raimondo worked on protein delivery for tissue regeneration and muscular dystrophy. But receptors for proteins and antibodies are on the outside of the cell, while RNA therapies act inside of the cell. “Protein-based [therapies]… and small molecules [are]limited to what they can bind,” she says. “But with RNA … if we can get it inside cells, which we can with our lipid nanoparticles, we can target essentially any part of the genome—so we can even treat things like genetic disorders.”

While Raimondo’s chief research focus is cancer immunotherapy—she was talking to potential collaborators at Brown’s Legorreta Cancer Center even before she arrived on campus last month—she’s also interested in chronic inflammatory diseases and degenerative diseases, “and in using RNA-based strategies for gene editing, CRISPR editing,” she says.

RNA’s potential in disease prevention and treatment is seemingly endless. In addition to rabies and several cancers, the long list of mRNA vaccine targets under development includes RSV, malaria, HIV, herpes, flu (including the elusive universal flu vaccine), tuberculosis, even acne. A number of RNA-based therapies are already in use, many for rare diseases, like spinal muscular atrophy. Cheung, a pediatric neurologist, says that drug, nusinersen, is “almost like a miracle.”

“Every baby born 10 years ago with SMA, 100 percent of them, died,” Cheung says. “Today, with this drug, kids are going to kindergarten, to grade school, walking, running.”

Cheung’s lab at Michigan recently figured out how the APOE gene, the highest risk factor for Alzheimer’s, is controlled by the folding and modifying of a particular non-coding RNA. Scientists have to know how a gene is regulated before they can devise a therapy, she points out. Alfonzo adds that about half of the mutations associated with mitochondrial diseases occur in non-coding RNAs, but we don’t know how those defects make people sick.

As technology to sequence RNA improves, and scientists come closer to understanding the mechanisms underlying these challenging diseases, not only will more treatments be possible, but experts believe RNA technology should make them easier and cheaper. Protein-based therapeutics, such as antibodies, enzymes, and hormones like insulin, take a long time to develop, require massive infrastructure, “and they’re so amazingly expensive,” Cheung says.

On the other hand, “making RNA drugs is cheap if we know how,” she continues. “There’s no design. It’s literally spelling a word. If we know how to spell the word, we can put it together into a medicine.” This is one reason the COVID vaccine could be developed, and then updated, so quickly. “The cancer vaccine is made with exactly the same basic ingredients as the COVID vaccine. Just the spelling is different,” Cheung says. “It’s a completely different way of thinking about drug manufacturing.”

Developing the technology to sequence RNA and map its modifications will have benefits beyond human health, Alfonzo says: industry and agriculture stand to make big gains as well. Modified RNA has enabled crops like rice and potatoes to dramatically increase their yields. Researchers have found an mRNA modification that affects barley’s cadmium absorption and the salt tolerance of sweet sorghum. A Massachusetts start-up is developing RNA sprays to replace chemical pesticides and fungicides.

“Food insecurity is a huge problem,” Jain says. “I see an opportunity where Brown … can do
things at the intersection of RNA and plants while we’re doing the intersection of RNA and human biology.”

Synthetic biologists are creating RNA fluorescent biosensors that can detect environmental pollutants in the air and water, Alfonzo adds, and “even engineering new enzymes into organisms for bioremediation.” And RNA plays a starring role in the CRISPR gene editing process, which has applications across biology—from gene therapy to plant breeding to engineered materials like silk fibers.

Much of this research, Alfonzo says, “is still in diapers … but the proof of principle is there.” He says he’s attended many talks and realized that the presenters’ lack of knowledge of RNA modifications is stymying their work. “I sit there thinking, I know what’s killing them,” he says. “Everybody has an RNA problem and a modification problem. They just don’t know it.”

When the Human Genome Project was first proposed, no one thought it could be done—among other challenges, the technology to accurately sequence DNA didn’t yet exist. But with the backing of governments and private companies around the world, the project succeeded. Given RNA’s complexities, Alfonzo says the RNome project will likely be five times bigger. But we have to try, he adds, because DNA doesn’t tell the whole story; “the full story happens at the RNA level.”


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