‘PAC-MAN’ to the Rescue: CRISPR-Based Tech on Cutting Edge of COVID-19 Therapeutics

BERKELEY, Calif. — Great partnerships depend on a rare combination of shared vision, mutual trust, and complementary skillsets. Scientists from Stanford University and Berkeley Lab’s Molecular Foundry may have found just that in a newly formed collaboration. Since March, research teams from these two institutions have been developing a new therapeutic that uses CRISPR technology in combination with a novel lipitoid delivery system to fight COVID-19 and other RNA viruses.

Last year, Stanley Qi, an assistant professor in the departments of bioengineering, and chemical and systems biology at Stanford, along with his team pioneered a new technology called prophylactic antiviral CRISPR in human cells, or PAC-MAN. Their efforts were initially focused on developing PAC-MAN as a strategy for targeting and destroying influenza in humans. When the COVID-19 pandemic hit in January, they abruptly pivoted their research to adapt the technology for SARS-CoV-2.

Lipitoids, which self-assemble with DNA and RNA, could serve as cellular delivery systems for antiviral therapies that prevent COVID-19 and other coronavirus infections. (Image courtesy R.N. Zuckermann)

PAC-MAN is based on the popular gene-editing tool, CRISPR, which has revolutionized modern medicine. The technology allows for unprecedented control over gene expression in many species, including humans. At its core, CRISPR is a technique for finding a specific sequence of DNA inside a cell and then altering or deleting it.

The process involves two components: an enzyme (Cas protein) that cuts through genetic material; and a piece of RNA that is used as a guide to direct Cas protein to the targeted gene sequence. PAC-MAN uses a specific version of the Cas protein, called Cas13, which slices through RNA such as that which makes up the genomes of viruses like influenza and SARS-CoV-2.

‘Molecular treasure’ emerges as key to fighting COVID-19

In a paper published in the journal Cell, Qi and his team demonstrate that their PAC-MAN strategy can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus in human lung cells. Notably, the authors perform bioinformatics analyses that suggested a group of only six guide RNAs can target more than 90% of all coronaviruses, giving PAC-MAN the potential to become a pan-coronavirus inhibition strategy.

There was one problem. For PAC-MAN to be effective in patients, particularly as an anti-COVID-19 therapy, it needs to be able to be delivered into lung cells. “But my lab doesn’t work on delivery methods,” Qi explains in a media release.

Enter Michael Connolly, a principal scientific engineer associate in the Biological Nanostructures Facility at the Molecular Foundry. His work on synthetic molecules called lipitoids provides a possible solution to the delivery issue with PAC-MAN. Lipitoids were first developed 20 years ago by Connolly’s mentor, Ron Zuckermann, and have since been widely studied for potential therapeutic applications. They are nontoxic to humans and can facilitate entry of RNA or DNA into cells by encapsulating them into tiny nanoparticles, a seemingly perfect delivery system for the PAC-MAN technology.

“Berkeley Lab’s Molecular Foundry has provided us with a molecular treasure that transformed our research,” Qi says.

PAC-MAN’s most recent adventure

In late April, the PAC-MAN/lipitoid system had its first test run in a sample of human lung cells. According to Qi, this approach reduced the amount of synthetic SARS-CoV-2 by greater than 90%. As an immediate next step to validate their technique, the team is collaborating with researchers at New York University and Karolinska Institute in Sweden. There, researchers are conducting animal trials with live SARS-CoV-2 infection. If successful, they hope to further develop the PAC-MAN/lipitoid system as a therapy against other viruses.

“An effective lipitoid delivery, coupled with CRISPR targeting, could enable a very powerful strategy for fighting viral disease not only against COVID-19, but possibly against newly viral strains with pandemic potential,” says Connolly.

One of the silver linings to emerge from the COVID-19 pandemic is how the scientific community has banded together to focus urgently on one topic. The joint effort of researchers from Stanford University and Berkeley Lab’s Molecular Foundry is an archetype of that collaborative spirit. As Qi notes, “It’s very rewarding to combine expertise and test new ideas across institutions in these difficult times.”

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