On February 19, Tim Abbott, a PhD candidate at Stanford University’s Bioengineering Department, checked the results of an experiment that he was running as a part of a team using the gene-manipulating Crispr technology to fight coronavirus. Abbott was working out of the lab of Stanley Qi, a pioneer developing Crispr tools that can mess with cancer cells and the like to fight diseases. Using an approach the lab called PAC-MAN (Prophylactic Antiviral Crispr in huMAN cells), the idea was to attack the coronavirus by directing a Crispr torpedo at it, attacking the virus’s genetic makeup that allows it to penetrate human cells and then use the cell’s machinery to self-replicate. In this particular experiment, he had introduced the lab’s Crispr-based system for finding and destroying SARS-Cov 2 (what scientists call the new coronavirus) into a solution containing an inert synthesized fragment of that virus.
Like all Crispr systems, this one was composed of two parts: an enzyme and a strand of so-called "guide RNA. " The RNA directs the enzyme, in this case, Cas-13d, to latch onto specific spots in the coronavirus's genome where it then makes a series of cuts. You can think of it like a pair of scissors programmed to scan a cookbook and chop up only the page containing the recipe for SARS-Cov-2. After Abbott analyzed the data, he called over Marie La Russa, a research scientist managing the project, to verify what he’d seen.
The coronavirus-targeted Crispr had reduced the amount of virus in the solution by 90 percent. If effectively delivered, this kill rate, they theorized, might be enough to stop the disease in a human. That result, along with others included in a paper released last weekend—in preprint form and not yet peer-reviewed—suggests that we may be entering an era of developing new Crispr-based weapons against deadly viruses, from flus to coronaviruses. “The PAC-MAN approach,” the authors wrote, “is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.
” But before you spring from your isolation-in-place for a cheer, underline “potentially. ” As the Stanford team readily admits, their paper is more a blueprint, or proof of concept, than an actual medical treatment ready for testing in animals or humans. The project has some serious X-factors, including the fact that they weren’t able to test PAC-MAN on the actual coronavirus. They still haven’t developed a system to get it into human cells.
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And, as Fyodor Urnov, a professor in UC Berkeley’s Department of Molecular and Cell Biology, points out, even if it works, there’s still a long horizon between preprint and clinical testing. “There is, frankly, zero chance that this approach can be tested in humans in the next four to six months,” says Urnov. “By analogy, if we were trying to go to the moon and come back safely, what this work shows is one can build a rocket that achieves escape velocity. ” No, it’s not a quick fix, but working on moon shots isn’t a bad idea.
“We are at point in human history where every thoughtful idea should be pursued, well beyond the tools we have, which were developed in the 13th century (quarantine), the 17th century (medicines) and the 18th century (inoculation/vaccination),” says Laurie Zoloth, senior advisor to the provost at the University of Chicago for programs on social ethics. “Crispr is very new, very unproven in human disease, but it is logical that it should work. ” The gene-editing power of Crispr technology has been increasingly directed at fighting diseases, originally against genetic ones. But more recently, it’s been harnessed to fight infectious diseases, including, now, the new coronavirus.
For instance, multiple teams inside and outside of academia are working on using Crispr for more effective tests. Mammoth Biosciences, a private company, claims to have developed a test for Covid-19 that cuts the result time from several hours to under 30 minutes. Sherlock Biosciences has produced a protocol that could possibly enable something that would work like a pregnancy test, giving a positive signal on a test strip. Efforts using Crispr to actually prevent or fight coronavirus are also emerging from existing projects designed to fight influenza and other infectious viruses.
In 2018, Darpa began a four-year program called Prepare. According to its call for proposals, the idea was to use genetic approaches to “generate new medical countermeasures for future use in humans. ” Qi’s lab at Stanford was one of several grant recipients. In April 2019, they began working on a Crispr-based means of fighting influenza.
Naturally, as the coronavirus spread earlier this year, the team took notice, and in late January they switched their focus to the virus that’s now changed the way we live. Tackling this particular virus was a challenge. The coronavirus, says Qi, has 30,000 nucleotides, and the Crispr-powered guide RNA can only target regions of 22 nucleotides to cut. It took a lot of bioinformatics computation and experimentation to locate the best spots to attack.
The attack itself, says Qi, is a double-barreled genetic assault, affecting the target. “One effect is to decrease the concentration of the virus genome inside the human cells,” he says. “The second is to block the production of the viral proteins” that it would otherwise use to create copies of itself and overwhelm the body’s defenses.
“The nature of the attack inspired its arcade moniker.”
”I like videogames,” says Qi. “The Pac-Man tries to eat cookies, and it is chased by a ghost. But when it encounters a specific kind of cookie called the power cookie—in our case will be a Crispr Cas13 design—suddenly it turns itself to be so powerful. It can start eating the ghost and start cleaning up the whole battlefield.
” But before PAC-Man can try to clean up anything, the Stanford group has a lot of work to do to prove that their concept will work in actual human bodies. The biggest caveat so far is that they didn’t use the actual SARS-Cov-2 coronavirus in the experiment. Since they could not get sufficient samples of the coronavirus—and did not have the authorization to handle the dangerous virus—Qi’s group created what they called a synthesized, non-replicating version that they felt expressed the relevant genetic characteristics to serve as a stand-in for the actual virus. The Stanford group contends that their result matters, even without the live virus.
“Using fragments of the coronavirus show that there are regions that we can target and interfere with,” says Abbott. “This won't end at just a concept—this will eventually be brought into something that's rapidly deployable. ” But other researchers say that in order to prove you can neutralize the virus, you have to use the real thing. “These viruses replicate rapidly,” says Philip Santangelo, a professor at Georgia Tech University who is part of a team (that also received one of the Darpa Prepare grants) working on a similar Crispr-based approach to fighting influenza-like outbreaks, and is now studying the coronavirus.
“Their study doesn’t deal with those kinetics. ” Read all of our coronavirus coverage here. Another problem: There’s not yet a proven delivery system for a Crispr-based treatment for viruses. One of the continuing problems of Crispr medicine is a way to get the treatment to the right cells.
The lung, where the coronavirus battle must be fought, is a particularly tricky battlefield—relatively inaccessible and full of mucus that could interfere with targeting. Qi says that while there are a number of potential options, he has yet to find the ideal way to introduce the PAC-MAN RNA to the virus. Perhaps, he suggests, someone else’s solution to this problem is already out there. “That’s one reason why we posted the paper so quickly,” he says.
“Some people may have amazing delivery methods. ” Santangelo’s team at Georgia Tech, which is collaborating with several other universities, believes the answer is a nebulizer, a mist-inhaling device that allows subjects to breathe in the Crispr-based treatment. This week, in fact, they are testing the nebulizer/Crispr combo on a mouse. Finally, the paper’s title describes using Crispr as a “prophylactic strategy” to combat the novel coronavirus.
That means a system designed to keep uninfected people free of the virus. But Crispr is still very novel, and so far there have been only three FDA-approved human trials in the United States using Crispr to treat a disease. While none have found that it caused damage, researchers are cautious; earlier experiments with introducing genetically modified cells into people have led to extreme inflammation that, in some cases, killed the patient, or nearly so. “That's one of the things that would need to be ironed out,” says La Russa.
“If your immune system doesn't like having that protein in yourself, then maybe the risk-reward balance is better to have it be used just as a treatment. ” And finally, like any new treatment, a Crispr prophylaxis for a virus would still need to go through testing in both animals and humans, and then pass the FDA’s rigorous review process. For all of that, the Stanford paper may one day be seen as a milestone. The ultimate promise of a Crispr-based system is that once you identify the genetic targets of a new virus, altering a previous treatment is a simpler process, and can be quickly implemented.
“So maybe down the line in the future, we would have the system that is completely agnostic to which virus that you're [fighting],” says Abbott. “All you would have to do is change one simple part, and then, boom, you’re actually protected against this new novel virus. ” (He concedes that the FDA would still have to sign off on any new use for the therapy. ) That’s probably one reason why why Urnov, the Berkeley professor, calls the Stanford paper “an important, but far from the only, tile in the overall jigsaw puzzle of how to take on emerging viral threats to human health.
” Yes, we could badly use a completed puzzle right now. But this intriguing, while speculative, preprint hints that maybe by the time the next pandemic rolls around, we’ll have more weapons to fight it than our current vaccines and drugs. For now, as we shelter in place, the only Pac-Man we’re going to get will be on video screens. WIRED is providing unlimited free access to stories about the coronavirus pandemic.
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