New RNA editing tool could improve cancer treatment

Cancer cell therapies can be potentially improved with a CRISPR RNA-editing platform, according to a new study published February 21 in Cell.

The new platform, Multiplexed Effector Guide Arrays, or MEGA, can modify the cells’ RNA, which has allowed Stanford University researchers to regulate the metabolism of immune cells in a way that enhances the cells’ ability to target tumors.

Lead author and Stanford graduate student Victor Tieu wanted to improve chimeric antigen receptor (CAR) T-cell therapy. In this cancer treatment, T cells, a type of white blood cell, are engineered with the CAR protein, a receptor that allows cells to better track cancer cells. Although CAR T therapy has successfully treated blood cancers, including lymphomas and multiple myeloma, the engineered immune cells have not performed well against solid cancers such as pancreatic and lung cancers.

Indeed, solid tumors have a larger structure into which immune cells can penetrate: the cells become exhausted before they can progress in destroying the tumors. T cells have evolved to fire quickly and attack viruses, meaning they often burn through their energy stores too early when fighting cancer.

“We were really interested in how we could improve these cells to improve clinical outcomes,” Tieu said. “Most of the tools we have now are just not very good.”

The researchers tested their tool on CAR T cells in laboratory cultures containing tumor cells and in mice with cancer. “Our finding is that it works 10 times better, in terms of reducing tumor growth and maintaining T cell proliferation over the long term,” said lead author Stanley Qi, associate professor of bioengineering at Stanford and researcher at the Sarafan ChEM-H institute.

Stop cellular exhaustion

Previous research efforts to improve CAR T cell therapy have used CRISPR-Cas9 to modify the cells’ DNA. However, this gene-editing platform carries risks because it permanently removes pieces of DNA, which can have unintended consequences and even make the T cells themselves cancerous.

So the Stanford team took a different route, exploring whether CRISPR-Cas13d, which uses a molecular scissor that cuts RNA not DNA, could enable reversible changes in gene expression in T cells. Unlike Cas9, Cas13d can easily target multiple genes at the same time. In the paper, the researchers demonstrated that they could make 10 changes at once to human T cells.

“RNA is the next layer after DNA, so we’re not actually touching any genetic code,” Tieu said. “But we are still able to achieve large changes in gene expression, capable of altering the behavior of the cell.”

To see if this tool could successfully improve CAR T cell function, they identified 24 genes that might be involved in T cell exhaustion. They then tested 6,400 paired gene combinations in culture, with different genes turned down. using the MEGA tool, and identified new pairs of genes that worked particularly well together to enhance anti-tumor function.

Turning T cells into marathon runners

In another experiment, the team tuned a set of metabolic genes in T cells to switch the cells from sprinters to marathon runners, giving them the stamina needed to eliminate tumors. They compared these MEGA CAR T cells to unmodified T cells and CAR T cells, both in laboratory cultures containing tumor cells and in mice with cancer. After three weeks, they tested the extent of the tumors as well as how well the T cells survived.

Initially, MEGA cells lagged in their anticancer activity. “At first I was like, ‘Oh, these cells are worse,'” Tieu said. But, after a while, these cells persevered against the tumor cells while the CAR T cells and regular T cells wore out, leading to a 10-fold improvement in reduced tumor growth and T cell proliferation. .

The secret was changing the way cells spent their sugar, moving from a fast-burning process of glycolysis to oxidative phosphorylation.

“We were able to use this technology to engineer the mRNAs in this sugar-utilization pathway inside T cells that regulate their choice of which sugar molecule to use,” Qi said. As a result, “we were able to really maintain the persistence of these T cells, so that the T cells could live longer in the tumor site and also exert much better performance.”

Not only did the MEGA platform fine-tune the genes regulating T cell metabolism, but this fine-tuning could also be regulated using a drug. When an antibiotic called trimethoprim was present, it activated the RNA changes, curbing the cells’ glycolysis metabolism and turning them into endurance athletes in their attack on tumor cells. When the drug wore off, the cells returned to their original gene expression.

This drug-based control mechanism “allows you to create a safety switch” for immunotherapy treatments, said co-author Crystal Mackall, the Ernest and Amelia Gallo Family Professor and professor of pediatrics and medicine at Stanford.

Although the platform is still in its early stages, researchers hope it may eventually prove useful in clinical settings.

Tieu plans to continue developing the platform for this purpose. “It would be really cool to try to turn this into a real clinical product,” he said. “I think there’s great potential to really improve CAR T cell therapy in ways that people couldn’t have done before.”