As he addressed an audience of virologists from China, Australia, and Singapore at October’s Pandemic Research Alliance Symposium, Wei Zhao introduced an eye-catching idea.
The gene-editing technology Crispr is best known for delivering groundbreaking new therapies for rare diseases, tweaking or knocking out rogue genes in conditions ranging from sickle cell disease to hemophilia. But Zhao and his colleagues at Melbourne’s Peter Doherty Institute for Infection and Immunity have envisioned a new application.
They believe Crispr could be tailored to create a next-generation treatment for influenza, whether that’s the seasonal strains which plague both the northern and southern hemispheres on an annual basis, or the worrisome new variants in birds and other wildlife that might trigger the next pandemic.
Crispr can edit the genetic code—the biological instruction book that makes life possible—within the cells of every living being. That means it can take different forms. The best-known version is mediated by the Cas9 enzyme; this can fix errors or mutations within genes by cutting strands of DNA. But virologists like Zhao are more interested in Cas9’s less famous cousin, the Cas13 enzyme, which can do the same to RNA. In human cells, RNA molecules carry instructions from DNA to make proteins, but the genetic code of influenza viruses is composed entirely of RNA strands—a vulnerability that Cas13 can exploit.
“Cas13 can target these RNA viruses and inactivate them,” Zhao explained.
Human cells do not naturally make either Cas9 or Cas13; both of these enzymes are found in the immune systems of bacteria and microscopic organisms called archaea., where Cas13 enables them to disable invading viruses called phages. Zhao and a wider team of scientists are devising an innovative system to confer the same benefits to humans.
Initially pioneered in the lab as a novel Covid antiviral, their idea is to develop a nasal spray or an injection that uses lipid nanoparticles to deliver molecular instructions to flu-infected cells in the respiratory tract. It’s a two-stage process. The first molecule would be an mRNA that instructs the cells to make Cas13, with the second being a so-called guide RNA that directs Cas13 to a specific part of the influenza virus’s RNA code.
“Cas13 then cuts the viral RNA, disrupting the virus’s ability to replicate and effectively stopping the infection at the genetic level,” says Sharon Lewin, an infectious diseases physician at the Peter Doherty Institute who is leading the project.
While the main aim would be to use the technology as a way of curbing short-term infections, Zhao also envisions the spray being used to prevent infections, for example during a particularly virulent flu season. “You’d basically be preparing the cells in your respiratory tract to produce this Cas13, as a first layer of defense,” he says. “It’s like the army—you’d have those soldiers armed and ready to meet their enemy.”
The main attraction of this idea is that Cas13 can be engineered, via the guide RNA, to target so-called ‘conserved regions’ of influenza’s genetic code. Those are known segments of RNA that are found in virtually all flu strains and are crucial to the virus’s survival. Conventional antivirals such as Tamiflu only target particular strains of flu, which swiftly acquire resistance.
Crispr-Cas13 is one of the leading innovations among so-called pan-fluenza antivirals, but there are others too. Monoclonal antibodies are also designed to target conserved regions of influenza’s genetic code, while other drugs aim to ramp up the production of interferons, the body’s inbuilt alarm system that signals immune cells to attack an invading pathogen.
With the influenza A strain alone killing 12,000 to 52,000 Americans every year, depending on the severity of the flu season, the need for better alternatives is clear. But as Nicholas Heaton, professor of molecular genetics and microbiology at Duke University points out, numerous hurdles still need to be overcome before any Crispr-Cas13 nasal sprays or jabs could be rolled out.
“I like the idea of it, but it’s [still] putting a foreign protein from a bacteria into someone’s body,” he says. “So will the body make an immune response against it?” Heaton also cautions against “off-target effects,” the chance that a Crispr treatment will inadvertently go after your body’s own RNA as well as an invading virus.
One early safety assessment has already been carried out at Harvard University’s Wyss Institute for Biologically Inspired Engineering, where scientists have used human lung and blood vessel cells to create a “lung-on-a-chip.” In the case of severe flu infections, influenza invades and replicates within microscopic air sacs called alveoli, making this a useful model to examine whether training these cells to produce the Cas13 enzyme can help fight off severe flu.
According to Donald Ingber, the institute’s founding director who pioneered the lung-on-a-chip models, studies have shown that the Cas13-powered cells could fight off various strains of flu—from the H1N1 strain responsible for the 2009 swine flu pandemic, to H3N2, which has been responsible for a particular virulent seasonal flu outbreak this winter. Not only that, but there seemed to be no unwanted consequences. “We didn’t see any off-target effects, which was amazing,” says Ingber. “We suppressed viral replication, but also the molecules that mediate inflammation which are secreted when your tissues are infected.”
Still, scientists are understandably cautious. Ingber says that figuring out a way to deliver a lipid nanoparticle containing the instructions to make Cas13—directly to alveoli cells deep within the lungs—is no easy task. Heaton also points out that any antiviral that targets a virus directly may help encourage the pathogen to further mutate, even if it is targeting seemingly integral parts of its genetic code. “Typically, what we find is that nature has a way,” he says. “It’s like with the old Jurassic Park movie.”
Heaton is also working on alternative ways of harnessing the power of Crispr to target flu. Another idea may be to play defense, using the Cas9 enzyme to adjust our own genetic code to make us more resistant to flu. “We all have genes that are being expressed that are allowing the virus to get into your cells and replicate,” he says. “But what if we could find one of these key genetic factors which the virus really needs, and just turn it down a little. Is there something in our biology which we can do with less of, that influenza can’t?”
To explore this, scientists have been carrying out a painstaking series of experiments. Research groups, including Heaton’s lab, take human cells, use Cas9 to remove genes one at a time, and then see whether the influenza virus can still kill them. This has already yielded a key discovery: flu relies upon a gene called SLC35A1, which ensures that certain sugars are present on the outside of our cells. According to Heaton, this gene is the flu’s very own Achilles heel.
“Flu uses those sugars as a receptor,” he says. “Theoretically, if you could make an inhibitor of that gene and have somebody inhale it, that would essentially stop all flu.”
Of course, given that mammals and influenza have been in a biological arms race for millions of years, evolution would likely have already eliminated SLC35A1 if it were possible for humans to survive without it. However, Heaton is not ruling out a more nuanced approach.
“What if we don’t completely eliminate this gene?’ he says. “What if we only eliminate it or reduce it transiently in a specific part of our body. Would that be tolerated? It’s still very early stages with these technologies, but I like this idea of finding genes that can restrict the virus’s capabilities, and then trying to see whether that would be safe.”
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