The CRISPR gene editing system has offered scientists a powerful tool for studying the effects of altering specific genes. Now, researchers at the Gladstone-UCSF Institute of Genomic Immunology have devised a CRISPR platform that allowed them to discover new roles of genes in human T cells, which they say could aid the design of better cellular immunotherapies.
By performing genomewide CRISPR screens in primary human T cells like never before, the Gladstone-UCSF team identified gene networks that control the production of interleukin 2 (IL-2) and interferon-gamma (IFN-gamma), both of which are cytokines critical for immune responses.
The study, published in Science, showed how T-cell activity might be tuned—as characterized by cytokine expression profiles—for different therapeutic purposes, the researchers said. The new CRISPR system could help identify targets for designing next-generation cell therapies to treat various immune disorders including autoimmune diseases and cancer, they said.
CRISPR is already widely used in biomedical research. But most studies use it to cut—and therefore silence—genes in cultured cell lines that, like tumors, can grow indefinitely. The Gladstone-UCSF team deployed CRISPR at scale in primary cells that were isolated directly from humans. And they dialed up or repressed gene expression without cutting the DNA, which can reveal important new insights into biological pathways, the team said.
But the process has been challenging.
In the current study, the researchers dialed up the CRISPR-dCas9 system with a lentiviral vector that could more efficiently transfer genetic materials to cells. The system was either attached with an activator to increase target gene expression or used in a typical inhibition manner to turn genes off.
The team used those approaches to screen nearly 19,000 protein-coding genes in human T cells to understand the regulatory pathways behind IL-2 and IFN-gamma. IL-2 has been tested in low doses for autoimmune diseases, and Clinigen’s Proleukin is an IL-2 drug approved for certain cancers. IFN-gamma promotes immune response against viruses and is linked with enhanced response to cancer immunotherapy.
Hundreds of genes turned up that seem to affect cytokine production, including some new regulators that weren’t captured in previous CRISPR screens.
With the CRISPR activation screen, the researchers identified proximal T-cell receptor signaling pathway genes, suggesting dialing up expression of these components could overcome immune signaling “bottlenecks.”
The CRISPRa screen also pinpointed cytokine regulators that were expressed at low levels. For example, while CRISPR interference identified an IFN-gamma signaling circuit involving the NF-kB pathway, the activation screen detected several protein receptors in the TNF family that also signal through NF-kB. Although the receptors weren’t individually required for signaling—therefore missed in the interference screen—they could promote IFN-gamma when overexpressed.
Better understanding of T-cell activation regulators could prove useful in various therapeutic settings. “These CRISPRa experiments create a Rosetta Stone for understanding which genes are important for every function of immune cells,” Alex Marson, M.D., Ph.D., senior author of the study, said in a statement. “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.”
One application the team envisions? Developing more powerful CAR-T cell therapies, in which a patient’s own T cells are modified to target cancer cells. Altering cytokine production through signaling networks identified via CRISPR screens could potentially boost the cancer-fighting skills of engineered T cells.
Among the many factors handicapping CAR-T therapy in solid tumors is cytokine expression in the tumor microenvironment. An unfavorable cytokine expression profile there can inhibit cancer-killing T cells and promote accumulation of suppressive immune cells.
Considerable efforts are going into modulating cytokines for better cancer immunotherapies. Last year, scientists at France’s Pasteur Institute showed an interplay between IFN-gamma and IL-12 was critical in sustaining cancer-killing by CAR-T cells. From the industry side, numerous companies like AffyImmune Therapeutics and Asher Biotherapeutics are among budding biotechs working on early CAR-T products with unique anti-tumor cytokine profiles or novel cytokine therapies to activate T cells.
The Gladstone-UCSF scientists suggest their CRISPR screen platform could help identify targets for those immunotherapy efforts.
“Our new data give us this incredibly rich instruction manual for T cells,” Marson said. “Our team now hopes to use our new instruction manual to create synthetic gene programs that can be CRISPR-engineered into the next generation cellular immunotherapies to treat a wide-range of diseases.”