Targeting a coronavirus

On the afternoon of March 12, Bruno Miguel da Cruz Godinho, PhD, sent an email to his colleagues in the Khvorova lab with the subject line “RNAi versus coronavirus.” The email didn’t contain much: the lab’s most advanced chemistries at the time and a link to a paper published only a few days earlier in Nature identifying two potential genetic targets that might slow SARS-CoV-2, the coronavirus that causes COVID-19.

Anastasia Khvorova PhD, had a simple, four-word reply for her 25-person lab: “We can do it.”

Developing an RNAi therapeutic for a virus, however, has never been done. It involves sequencing DNA; performing bioinformatics and designing a chemical scaffold for delivering a drug; animal testing; clinical trials; and drug production. To create an RNAi-based treatment for SARS-CoV-2, the Khvorova lab would be stepping outside its normal area of expertise, embarking on a complete bench-to-clinic research trajectory, while trying to do it in record time.

“It’s like trying to fly the plane while you’re building it,” said Dr. Khvorova, the Remondi Family Chair in Biomedical Research and professor of RNA therapeutics and molecular medicine.

Co-discovered by 2006 Nobel Laureate and UMMS Professor of RNA Therapeutics Craig Mello, PhD, RNAi is a natural molecular process inside cells. It uses small RNA molecules to halt the translation of genes into proteins. These molecules, such as small interfering RNAs (siRNA) and antisense oligonucleotides (ASOs), effectively turn the gene off by stopping the protein building machinery inside the cell—a process scientists call silencing.

Scientists, such as Khvorova and Jonathan K. Watts, PhD, associate professor of RNA therapeutics and biochemistry & molecular pharmacology, seek to turn this natural cellular phenomenon into a powerful therapeutic for treating human disease.

“Theoretically, the power of RNAi is that it allows for the quick design and development of drugs,” said Khvorova. “Once you’ve unlocked the chemical scaffold for efficiently delivering therapeutic doses of siRNA or ASOs to the right tissue, all you need is the right genetic sequence to target. It would be the perfect platform for addressing future pandemics because it would be plug-and-play.”

The molecules that drive RNAi, however, can’t simply be delivered to patients. They’re too fragile and unstable. Plus, there’s the challenge of getting these molecules to the tissues where they are needed and nowhere else. If too many siRNAs or ASOs find their way into the wrong cell types, they might cause unintended or off-target complications.

Khvorova and colleagues identify chemical and biological properties that allow effective and efficient delivery, distribution, retention, cellular uptake and biological availability of small RNA molecules to specific tissue cells. Maximizing the efficiency of these properties is crucial to developing successful RNAi therapeutics. By screening a wide range of chemically engineered and naturally occurring bioactive conjugates, they identify novel chemical scaffolds that support delivery of robust quantities of siRNAs and ASOs to the heart, kidneys, muscle, placenta, vasculature and brain—tissues previously not reached by RNAi.

Bruno Miguel da Cruz Godinho, PhD

Last year, Dr. Godinho, a postdoctoral associate in Khvorova's lab, and his colleagues discovered a new class of gene-silencing RNAs he christened Di-siRNA. Godinho et al. developed the chemical scaffold, which consists of two siRNA molecules connected through a neutral linker, which enabled broad distribution throughout the nervous system and correct genetic mutations in neurodegenerative diseases such as Huntington’s, amyotrophic lateral sclerosis (ALS) and Alzheimer’s. This breakthrough earned Godinho, who holds a Milton Safenowitz Postdoctoral Fellowship from the ALS Association, the honor of being named one of STAT’s 2019 Wunderkinds and a Dr. Alan M. Gewirtz Memorial Scholar.

Left to right, Daniel O'Reilly, PhD, Pranathi Meda Krishnamurthy and Minwook Shin, PhD, of the Khorova lab

ROB CARLIN PHOTOGRAPHY

Single gene mutations, such as those that cause ALS or Huntington’s, are the focus of the Khvorova lab. These types of diseases are more clearly defined than illnesses caused by viruses, thus designing a therapeutic is relatively straight forward, explained Daniel O’Reilly, PhD, postdoctoral associate in the lab who works on oligonucleotide chemistry.

“COVID-19, any type of infectious disease really, isn’t in the scope of what we normally do in the lab,” said Dr. O’Reilly. “That’s the beauty of RNAi, though. You can almost always do something with it.”

One of the first steps for the lab was to identify highly conserved areas of SARS-CoV-2. These are regions of the virus DNA that are relatively stable and do not often mutate as it replicates when moving from cell to cell and person to person. The first DNA sequence of SARS-CoV-2 became available on Jan. 12. This was a critical first step to developing an RNAi therapeutic. Without the viral sequences to plug and play into scientists’ vision of an RNAi platform, there could be no drug.

Kathryn Monopoli, a first-year graduate student pursuing her PhD through a joint program with Worcester Polytechnic Institute, wrote a bioinformatics program to scan and analyze the SARS-CoV-2 sequence for these critical conserved areas in a single day. Pre-COVID, such a project would have taken months to complete. Her work, said Khvorova, identified three highly conserved stretches of the virus’ DNA that were ideal targets for an RNAi drug. That same day, members of the team began synthesizing these stretches of sequences so the lab could begin running experiments.

Anastasia Khvorova, PhD

Jonathan K. Watts, PhD

Across the hall from Khvorova in the Albert Sherman Center, the Watts lab works at the interface of synthetic chemistry and biomedicine, studying the modulation of gene expression in cells and organisms. Like Khvorova, he studies how siRNAs and ASOs can be therapeutically delivered to various tissue types, including the lungs.

“The lung has a whole different set of properties than the brain,” said Dr. Watts. “One of the biggest challenges to delivering any therapy to the lung is the immune response. In the brain you don’t have that problem, but the lungs are constantly under assault and are an immune hotspot.”

As luck would have it, the Di-siRNA scaffold that Godinho discovered during his research on Huntington’s disease also proved effective in the lungs, without triggering an immune response.

“We really hit the jackpot there,” said Annabelle Biscans, PhD, a postdoctoral associate in the Khvorova lab.

Working together, the Watts and Khvorova labs designed a new chemical scaffold that can deliver both Di-siRNA and ASOs to a cell at the same time with the same chemistry. This had never been done before. The advantage of this approach, according to Godinho, is that siRNAs mostly silence transcription in the cytoplasm of the cell, while ASOs, besides working in the cytoplasm, also achieve silencing in the nucleus. This dual approach for gene silencing makes it more likely to be effective against SARS-CoV-2 at lower thresholds.

“The beauty of our collaboration with the Watts lab is that we’re able to tap into scientists who are experts in both siRNA and ASO chemistries,” said Dr. Biscans, the project’s lead biochemist who spearheaded the lab work. “The chemistry is so important. If we don’t build the right drug with the right chemistry, then it won’t be strong enough or efficient enough to successfully treat the illness.”

UMMS halted normal research operations on March 13. While lab projects on campus didn’t completely stop, scientists were analyzing data, designing experiments and writing papers from home. A handful of labs, including the Khvorova and Watts labs, were granted permission to continue operating on COVID-19 research.

“When we were first granted access back to the lab, none of us knew how it was going to go,” said Ken Yamada, PhD, postdoctoral associate in the Khvorova lab. “We didn’t know how COVID-19 was going to affect access to the building and our ability to do experiments.”

O’Reilly added, “At first, there were a lot of logistical issues. How would we stand six feet apart? How would sharing equipment work?”

Dr. Yamada and O’Reilly, who both design the chemistry for the siRNAs and ASOs and conduct biochemical experiments on these designs, began working in shifts for safety. Others, such as Pranathi Meda Krishnamurthy, a research associate and biochemist in the Watts lab and mother to a 6-year-old son, began working longer shifts to minimize the number of times she had to travel between the lab and home. “I didn’t want to potentially expose my family any more than I had to,” she said.

The shutdown also had unintended benefits. Scientists who might otherwise be working on their own projects were freed up to work on COVID-19. Reagents were available for experiments and lab instruments that might be shared between projects were now dedicated to COVID-19 because it was the only research going on on campus.

The project was able to move forward quickly because scientists had access to more resources to work on COVID-19, explained Chantal M. Ferguson, MD/PhD student in the Khvorova lab.

Chantal M. Ferguson, MD/PhD student in the Khvorova Lab

ROB CARLIN PHOTOGRAPHY

“We were really able to build momentum,” she said. “We have expertise from chemists, pharmacologists, biologists and bioinformaticians, and each group is invested and interested in the work of the others, making the environment extremely fun, interesting and innovative. Things just started to snowball,” said Ferguson.

The financial support of the Medical School was also crucial to generating progress. Drawing from institutional dollars available for research, the Medical School lent $1 million to the Khvorova and Watts labs so they could begin working immediately. The expectation is that as grant funding for COVID-19 flows to these labs, the funds will be reimbursed to the institution.

“It’s not typical for an investigator to make a phone call to leadership and that same day have $1 million for research,” said Khvorova. “UMass Medical School is unique. This isn’t the type of research you can just do in the garage. The leadership has strategically invested resources, tens of millions of dollars, over the years, building new facilities and purchasing equipment, that have put us in a position to capitalize on this moment.”

The result of this strategic investment in resources is that the Khvorova and Watts labs are ready to try their best chemistries and targets in nonhuman primates. They have three chemical scaffolds poised for preclinical tests against both viral and host cell targets, including the ACE2 receptor identified in the Nature paper that prompted Godinho’s original email. Some of these drugs will be delivered locally to the lungs through a nebulizer while others will be delivered systemically through injection.

This truncated timeline, however, creates additional challenges for scientists. Normally researchers would spend years meticulously working to answer basic biological questions about the virus, host cells and the biochemistry of the drugs. “We have one shot to get this right in order to have a short-term impact on COVID treatment,” said Watts. “We have a good number of cocktails and sequences that work in cells. We’re collecting as much information as we can on these to help make the best decisions about which ones to take into the nonhuman primates.”

Even if their first attempt doesn’t yield the expected results, chemically modified siRNAs are informational drugs—the base sequence determines its target RNA, while the chemical architecture dictates pharmacokinetic behavior— allowing siRNAs or ASOs and chemical structures to be easily reprogrammed to target any gene sequence.

“I think there’s a silent revolution happening,” said Khvorova. “The pandemic has just accelerated the development, but it was going to happen anyway. We are fortunate. We are the right people in the right place at the right time. If you ask me, five years from now, will this research lead to a new platform for responding to pandemics, will we be able to respond quickly and efficiently to the next pandemic using RNAi as a therapeutic, the answer will be 100 percent yes.”

The right people in the right place

Khvorova and Watts are among a growing list of principal investigators at UMass Medical School researching SARS-CoV-2 with the goal of expanding our understanding of the novel coronavirus so new vaccines or treatments can be developed.

Ann M. Moormann PhD, MPH, professor of medicine and an epidemiologist who normally studies malaria in Kenya, is conducting a longitudinal study of health care workers, patients and UMMS employees. Taking serology and venous blood samples from these populations, she is testing not just for antibodies but for the presence of other immune cells such as dendritic cells, T cells and natural killer cells, all of which play important and different roles in protecting the body from viral attacks.

“We’re doing global health at home now,” Dr. Moormann said.

Ann M. Moormann, PhD, MPH

One of the chief questions Moormann hopes to answer is how long SARS-CoV-2 antibodies persist after the infection has been cleared. She is also exploring the extent to which exposure to other pathogens, such as the coronavirus that causes the common cold, does or does not confer protection.

“That’s a real question right now,” said Moormann. “Once you’ve had the virus, we don’t know how long those antibodies will hang around. Will it be for eight months or eight years?”

Another important quest is whether B and T cells, the memory cells of the immune system, will retain information about the SARS-CoV-2 virus after infection. If they do, the body would be able to mount a quicker and more robust antibody response to future infections. This means future outbreaks or infection would be far less severe than what has been seen during the initial pandemic.

In order to analyze the immune system’s response to COVID-19, Moormann is drawing upon a collection of prepandemic serology samples she collected as part of her work on malaria. These COVID-19 naïve cells are being compared to post-pandemic serology samples she is collecting. Moormann and colleagues will also be able to expose these naïve cells to COVID-19 in cell cultures and analyze how they respond to the novel coronavirus.

“Once we understand how the immune system responds to the virus, we can begin building smarter vaccines and treatments,” said Moormann. “Science has to go beyond what the human immune system does. It’s not enough to just trigger an immune response using an attenuated form of the virus. We have to be smarter than both the virus and the immune system.”

Moormann’s work is being funded by The Massachusetts Consortium on Pathogen Readiness (MassCPR), a multi-institutional initiative convened by Harvard Medical School to combat the global COVID-19 pandemic and to prepare for future outbreaks. In March, the consortium awarded $16.5 million in research funding to projects that address the most pressing challenges of the disease. Moormann was one of three UMMS researchers to receive this funding.

For information about additional research related to SARS-CoV-2 being conducted at UMMS, visit umassmed.edu/coronavirus/research. ■

By Jim Fessenden