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Rabid-Seq Method Offers NGS-Based View of Cellular Interactions in the Brain

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NEW YORK – A new next-generation sequencing-based method offers insight into cellular interactions in the brain and can even be used to discover new drug candidates for neurological diseases.

The method, recently published by researchers from the Broad Institute and Harvard Medical School, combines cell-cell interaction tracing using modified rabies virus with a barcoding scheme that is read out with single-cell sequencing. The modified virus tags each cell it encounters with the mCherry fluorescent marker, which enables their isolation with cell sorters.

"At the end of the day, what you get is a dataset of single-cell analyses," said Francisco Quintana, the study's corresponding author and a professor of neurology at Harvard Medical School. "It could be RNA-seq, or you could go for single-cell ATAC-seq [assay for transposase accessible chromatin by sequencing]. But the important point is that it's annotated with barcodes to track cell-cell interactions."

Quintana and his team described the method, rabies barcode interaction detection followed by sequencing, or Rabid-seq, last month in a paper in Science. In this study, they relied on transgenic mice engineered to have particular cells express the target protein of the virus using a Cre-lox genetic engineering system. That allowed them to focus on astrocytes, a type of glial cell in the brain that are known to play a role in neural signaling. In principle, the cell type of interest can be tunable by encoding receptors for the virus in different cells.

Already, the researchers were able to apply the method to identify a drug target and candidate molecule in an experimental model of multiple sclerosis.

"For years we’ve been trying to understand how inflammation in the CNS [central nervous system] is regulated," Quintana said. In particular, his lab has focused on CNS "resident" cells, including glial cells. Cell interactions are key activities of these cells in neurological diseases, he said, and Rabid-seq is an attempt to study them in a more unbiased manner.

Using modified rabies virus to study neuronal interactions in the brain is not new, but previous methods required microscopy. These methods were "not high throughput, with not much depth of information," Quintana said.

In the study, the researchers used the Smart-seq protocol for single-cell transcriptomics but have now transitioned to using 10x Genomics' Chromium platform.

In a proof-of-concept study described in the paper, the researchers used Rabid-seq to identify a drug target, screen for drugs against that target, and identify the mechanism of action for the compound.

In mice with experimental autoimmune encephalomyelitis, or EAE, an inflammatory condition of the CNS used to model multiple sclerosis, the researchers looked at astrocyte subsets associated with disease pathology and their interaction with microglia cells. Using transcriptomic data generated with Rabid-seq, they zeroed in on a set of signaling proteins that mediated interactions between the cells and promoted EAE pathology.

One of those proteins, Ephrin-B3, is a receptor that harbors a tyrosine kinase domain that could be interfered with by a small molecule kinase inhibitor that penetrates the CNS and ameliorated EAE in their disease model.

Using Rabid-seq on cells treated with the small molecule showed that it worked by regulating mitochondrial function. "The use of Rabid-seq to not only identify cell-cell interaction mechanisms and targets for therapeutic intervention but also understand the mechanisms of action of drugs highlights its potential," Quintana said.

The method can also be paired with epigenetic assays to see how cell interaction networks influence epigenetic regulatory patterns. It is also not necessarily limited to use in the brain but could be applied to CNS cells in other parts of the body, like the gut, or to study inflammation in other organs outside the CNS, Quintana said.

"We are actively looking into both licensing [IP] and starting a company" around Rabid-seq, he said, noting that the researchers have applied for a provisional patent for their barcoding scheme. Quintana has founded a startup before: AnTolRx, a Boston-area therapeutics company.

Meantime, his lab is working to integrate Rabid-seq with in situ transcriptomics methods, particularly multiplex error-robust fluorescence in situ hybridization, or MERFISH, developed by Xiaowei Zhuang's group at Harvard.

They are also working on a version of the method that doesn't require transgenic animals, so that it could be used with wild-type mice, human tissue samples, and nonhuman primate model systems.

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