CAR T-Cell Immunotherapy
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Researchers at the Gladstone Institutes and the University of California San Francisco (UCSF) have used a recent variation of CRISPR technology called CRISPRa to develop a tool that enabled comprehensive functional mapping of gene networks that can modulate cytokine production. The tool will give researchers a faster, more comprehensive way to uncover the gens that play a role in immune cell biology.

The twist of the new CRISPR tool is that instead of using it to edit or otherwise alter genes, it forcibly activates the genes in human cells. The tool allowed the researchers to test nearly 20,000 genes in parallel to learn which are most responsible for particular cell functions, and potentially paving the way for the identification of new, more effective immunotherapies.

“This is an exciting breakthrough that will accelerate immunotherapy research,” said Alex Marson, director of the Gladstone-UCSF Institute of Genomic Immunology and senior author of the new study published last week in Science. “These CRISPRa experiments create a Rosetta Stone for understanding which genes are important for every function of immune cells. In turn, this will give us new insight into how to genetically alter immune cells so they can become treatments for cancer and autoimmune diseases.”

The research is the first to successfully use CRISPRa at a large scale in primary human cells, which are cells isolated directly from a person.

Marson and his team have been using CRISPR-Cas9 technology over the past few years to study immunity by knocking out genes from various types of human immune cells, including regulatory T cells and monocytes. The scientists have made headway using this approach to better understand how to better engineer cells to be more effective at fighting infections.

But this work only served to show how eliminating a gene’s effects could influence cell function. It couldn’t provide the opposite information: how making a gene more active might also have an effect.

To perturb genes into a more active state Marson and his team used CRISPR activation—called CRISPRa. This form of CRISPR allows scientists to attach an activator to Cas9, so that when it binds to a gene, it activates it. Alternatively, they can attach a repressor—an “off” switch—to Cas9 to turn genes off, achieving a result similar to a typical knockout approach (called CRISPRi for CRISPR interference).

T cells are critical in their ability to manage immune response. Not only do they target pathogens, but they also direct other immune cells to increase, or decrease, their response to intruding pathogens or cancer cells. These function are achieved through the production of cytokines, with different types T cells producing different types of cytokines, which have differing effects on immune response.

As Marson noted, having the ability to control T cell cytokines represents a hug opportunity to reshape immune response across range of different diseases. The hope is that this new technology can illuminate which genes control which cytokines.

The research team used CRISPRa or CRISPRi to activate or inactivate nearly 20,000 genes in human T cells isolated directly from multiple healthy volunteers. They screened the resulting cells for changes to cytokine production and homed in on hundreds of genes that serve as key cytokine regulators, including some never-before identified in knock-out screens.

“Our work demonstrates the precision and scalability of this technology in human T cells,” said Ralf Schmidt, co-first author. “And we quickly learned the rules of which genes you could turn on to dial the levels of certain cytokines.”

As the researchers see it, better understanding of cytokine production could be a valuable tool to make existing CAR T therapies even more effective.

“Our new data give us this incredibly rich instruction manual for T cells,” said Marson. “Now we have a basic molecular language we can use to engineer a T cell to have very precise properties.”

Marson’s lab is now studying some of the individual genes identified in their screen, as well as working to further leverage CRISPRa and CRISPRi to discover genes that control other critical traits in human immune cells.

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