NEW YORK—A team of scientists at the New York Genome Center, New York University, and the Icahn School of Medicine at Mount Sinai have identified new potential therapeutic targets in the fight against SARS-CoV-2. The researchers performed a genome-scale, loss-of-function CRISPR screen to systematically knock out all genes in the human genome. The team examined which genetic modifications made human lung cells more resistant to SARS-CoV-2 infection. Their findings revealed individual genes and gene regulatory networks that are required by SARS-CoV-2—and that, when suppressed, offer resistance to viral infection.
The study, published in Cell, describes a wide array of genes that weren’t previously considered therapeutic targets for SARS-CoV-2.
“Seeing the tragic impact of COVID-19 here in New York and across the world, we felt that we could use the high-throughput CRISPR gene-editing tools that we have applied to other diseases to understand what are the key human genes required by the SARS-CoV-2 virus,” said the study’s co-senior author Dr. Neville Sanjana, core faculty member at the New York Genome Center, assistant professor of biology at New York University and assistant professor of neuroscience and physiology at the NYU Grossman School of Medicine.
“In March 2020, I had a conversation with Dr. Tom Maniatis, who leads the New York Genome Center, and told him that we were eager to apply our CRISPR-based tools to help fight the pandemic but did not know anyone who could handle the virus in their lab,” Sanjana tells DDN. “He connected me with Dr. Ben tenOever (a former member of Dr. Maniatis’ lab) at Mt. Sinai, and the rest is history.”
The team discovered that the genes were substantially clustered into a handful of protein complexes, including vacuolar ATPases, Retromer, Commander, Arp2/3 and PI3K.
“We were very pleased to see multiple genes within the same family as top-ranked hits in our genome-wide screen,” pointed out Dr. Zharko Daniloski, a postdoctoral fellow in the Sanjana Lab and co-first author of the study. “This gave us a high degree of confidence that these protein families were crucial to the virus lifecycle, either for getting into human cells or successful viral replication.”
Among the top-ranked genes, only ACE2—the receptor responsible for binding the SARS-CoV-2 viral protein Spike—showed tissue-specific expression. The rest of the top gene hits were broadly expressed across many tissues, so these mechanisms may function independently of cell or tissue type.
After completing their primary screen, the researchers used several techniques to validate the role of many of the top-ranked genes. Using human cell lines derived from the lung and other organs susceptible to SARS-CoV-2, the team measured viral infection after gene knockout by CRISPR, gene suppression using RNA interference or drug inhibition.
“For several of the top-ranked gene hits from the CRISPR screen, we wondered whether loss of these genes confers resistance to SARS-CoV-2 by completely distinct mechanisms, or whether some of them might work through a shared mechanism or common pathway,” Sanjana states.
Using recently developed ECCITE-seq technology, the team identified that loss of several top-ranked genes results in upregulation of cholesterol biosynthesis pathways and an increase in cellular cholesterol. Next, they studied the effects of amlodipine—a drug that alters cholesterol levels.
According to Sanjana, “ECCITE-seq lets us get at these different mechanistic hypotheses. With it, we can measure the transcriptome of each CRISPR-targeted cell. We found that for six of the top-ranked genes, CRISPR-driven gene loss resulted in a similar ‘cell state’—namely, increased cholesterol biosynthesis.”
“To test this, we used an established drug, amlodipine, and showed that it also increased cholesterol in the cells. Next, we tested whether cells treated with amlodipine were protected against the virus and indeed they were,” he continues. “This is very promising news because amlodipine has a long-established safety record (FDA approved ~30 years ago) and many people take it for years.”
“Since recent clinical studies have also suggested that patients taking calcium-channel blockers have a reduced COVID-19 case fatality rate, an important future research direction will be to further illuminate the relationship between cholesterol synthesis pathways and SARS-CoV-2,” added Dr. Tristan Jordan, a postdoctoral fellow in the tenOever Lab and co-first author of the study.
The team built on previous research and explored whether loss of some genes might confer resistance to the coronavirus by lowering ACE2 levels. They identified one gene in particular—RAB7A—that has a large impact on ACE2 trafficking to the cell membrane, and showed that RAB7A loss prevents viral entry by sequestering ACE2 receptors inside cells.
“This makes for a clear mechanism: less ACE2 means that it will be harder for Spike to bind and enter cells. So, as we show in Fig 6B, we tested several of the top-ranked hits and measured any potential modulation of ACE2 expression. We found that the gene RAB7A was a very strong modulator of ACE2 on the cell surface. If you remove RAB7A, then ACE2 is greatly reduced,” explains Sanjana. “This suggests that therapeutic inhibition of RAB7A could also be useful for blocking SARS-CoV-2 infection.”
“We are especially excited by how many of the top-ranked genes are therapeutically targetable,” he adds. “We have shown that this ‘genetics-guided’ approach is very useful for finding new therapies, and we are now working to test these in preclinical models.”
“We are looking for drug development partners to help us translate the findings from the genome-wide CRISPR screen into therapies that can help treat COVID-19 patients,” Sanjana concludes. “One of the most promising aspects of our work is that this human genetics-guided approach has led us to discover multiple promising drug targets.
“A key future research direction will be understanding whether different inhibitors—targeting different genes—can be combined together to provide a more robust therapeutic protocol that can attack the virus from several different angles.”
