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CHOP Scientists Enlist CRISPR to Learn How to Disrupt SARS CoV-2’s Entry Routes
limjr [at] email.chop.edu (By Jillian Rose Lim)
As we learn more about the COVID-19 symptoms that patients present on the outside, scientists are piecing together how the virus takes hold on the inside. One crucial puzzle piece to COVID-19’s complex biology involves the study of genes: What role do specific genes and the proteins they encode play in the spread of infection throughout the body?
At Children’s Hospital of Philadelphia, Stephanie Sansbury, a trainee in the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, typically uses CRISPR-based genetic screens to better understand neurodegenerative diseases. In recent weeks, however, Sansbury also devotes much of her time to conducting such screens for SARS CoV-2, the coronavirus that causes COVID-19. Working in the lab of Ophir Shalem, PhD, assistant professor of Genetics at CHOP, Sansbury is leveraging her expertise in developing and using advanced genomics tools to contribute to our understanding of the pathology of COVID-19.
“Generally speaking, what we’re trying to do is adapt the kind of genetic screen we do all the time to a different readout that’s related to coronavirus,” said Sansbury, who is also a PhD student at the University of Pennsylvania’s Perelman School of Medicine. “Usually, we’re interested in diseases that have to do with nerve degeneration, so we try to find genes that affect protein clumping, which is a pathological hallmark of many neurodegenerative diseases. But as [Dr. Shalem] and I were advising another lab group on CRISPR, we realized we could use the same approach to ask coronavirus-related questions.”
Genetic screens are crucial for understanding the function of specific genes in the body. Using a high-throughput CRISPR-based approach, scientists can determine which genes influence certain physiological traits. These traits can include anything from why cancers might become resistant to certain drugs, to why a virus is able to enter and infect so many cells in the body.
When it comes to COVID-19, existing evidence suggests that SARS COV-2 grasps onto two particular proteins to enter a cell: the ACE2 receptor and the TMPRSS2 enzyme, both of which sit on the cell surface. But in order to reduce the number of these proteins, scientists must first identify the genes required to maintain their expression.
Blocking the Entryways of SARS COV-2
Think of a cell as a tiny, carefully-guarded unit, complete with doors that protect it from its outside environment. Then, imagine protein receptors as the handles of those doors, spread all throughout the cell’s surface.
When SARS COV-2 enters the body, it latches onto the cellular door handles it recognizes as ACE2. In order to latch on to the ACE2 handle, the proteins on the surface of the virus first need to be activated (or “primed”) by TMPRSS2. Together, ACE2 and TMPRSS2 provide the entryway the virus needs to infect the body. By identifying which genes are required to maintain production of these proteins, scientists could potentially lay the framework for targeted treatments.
“If we temporarily reduce surface expression of these two proteins, that might mean the cell would be less susceptible to infection,” Sansbury said. “So, if we can find a collection of genes that affect the expression of these two proteins on the cell’s surface, then those genes might be good therapeutic targets.”
The ideal outcome for this research appears as a list of genes ranked in order of their importance for production of ACE2 and TMPRSS2 proteins.
“What we hope to find are genes whose function are necessary in order for these two proteins to be expressed,” Sansbury said. “We would then compare that list to a database of existing FDA-approved drugs that target the proteins these genes encode. The hope is that our data would bolster the work of other groups who are already screening drugs by pointing out ‘these are the genes that we need to focus on disrupting to effectively prevent viral infection, or to reduce viral spread throughout the infected tissue.’”
Why ACE2 and TMPRSS2?
Though the current research project is very much experimental, strong evidence points to the importance of ACE2 and TMPRSS2, and suggests that reducing their expression is likely to help.
“An important paper showed that if you express these proteins in cells that are infected by other types of viruses, they become susceptible to SARS-CoV-2,” Sansbury said. Furthermore, other viruses in the coronavirus family have used these proteins to gain entry into cells, and scientists know that ACE2 is regulated as a result of inflammatory processes.
“One thing that could be happening – and again, this is still just a theory – is that the abundance of ACE2 changes after some initial infection because of the local response to viral infection,” Sansbury said. “And then that leads to a cascade where, as the virus grows in your cells, it's more likely to infect neighboring cells or instigate tissue damage.”
Sansbury emphasizes the investigational aspect of the work, since the scientific community still has a number of unanswered questions. One of those is whether outside the lab setting (where research is conducted on cell lines grown in a petri dish) and in a real human lung, ACE2 and TMPRSS2 are indeed the most essential proteins.
“They seem to be the most promising targets for this experiment,” Sansbury said. “But, if new information comes out suggesting that a different receptor might play a larger role, then we switch gears and study that. In other words, I think like the rest of the community, we're working with the best available knowledge, and of course, it’s not the full story yet.”
Furthermore, because the lab’s expertise lies in genomics technology like sophisticated genetic screens rather than in virology, Sansbury views their genetic screening efforts as one piece of a larger response to generate data that experts in coronavirus biology and infectious diseases can use to fight COVID-19.
“Our goal is to do this experiment to the best of our ability and put the data out there so that people who know much more than we do about these viruses, and about whatever proteins we find, can take the information and generate and pursue their own hypotheses about the role of these proteins in infection and how to disrupt infectious or damaging processes,” Sansbury said. “The deliverables would be a recommendation for which genes to target with drugs, and a better understanding of how these two surface proteins are regulated in a lung cell model.”
A Scientific Community Comes Together
Sansbury is still in the middle of these large-scale experiments – generating millions of mutant cells to test for ACE2 and TMPRSS2 expression levels – so future findings are forthcoming. But even so, her research has already highlighted a positive aspect to research and science that Sansbury says makes her proud to be part of the thriving scientific community at CHOP and Penn.
“The whole scientific community has been incredible: really open about sharing data, supportive, and rigorous,” Sansbury said. “To see this group of creative, resourceful, capable people come together and pause what they've been working on for decades to focus on this problem – it's inspiring and comforting. Knowing that thousands of researchers around the world are doing everything they can during this pandemic has been a source of hope in an otherwise dark time.”
Learn more about the Shalem Lab at CHOP.