The tumour challenge
One of the major challenges when developing cancer immunotherapies is that tumours exist in an environment that suppresses T cells and other immune cells, allowing tumour cells to form and grow. Therapeutic T cells, engineered from a patient’s own T cells to recognise and kill tumour cells, can get exhausted or dysfunction as they battle this environment, becoming unable to take down the cancer cells.
“Genome editing methods allow you to reprogramme the immune response to target cancer cells,” explained Shy. “Thus far, this has primarily been accomplished by inserting a new surface receptor via semi-random integration with viruses. This approach makes it difficult to target specific locations in the genome and leads to manufacturing bottlenecks due to the cost and complexity of viral production.”
In response to the challenge presented by tumours and previous attempts at editing T cells, the researchers instead used a set of CRISPR screens that allowed them to turn off each gene in the genome, one at a time, in a pool of human T cells. By knocking out one particular gene, they created cells that are not just potent tumour cell killers but also more persistent killers over a long period of time.
“In comparison to semi-random integration with viruses, here we used CRISPR-based targeted transgene insertions, which allow us to directly correct or reprogramme specific locations in the genome,” Shy said. “This allows you to be far more precise with the changes you are introducing. We can disrupt specific genes, correct pathogenic mutations or make use of native regulatory elements to improve the function of cells. There are also potential safety benefits because we know the exact location we are modifying in the genome and can avoid any spots we think might be risky. In addition, the fully non-viral approach we optimise here allows us to bypass issues associated with sourcing virus.”
A new target
“One challenge with non-viral insertion has been the toxicity of the large DNA templates we use to introduce a new gene. To get efficient ‘knock-in’, we need to deliver high concentrations of these templates into cells. We were able to reduce the toxicity of these templates by switching from double-stranded to single-stranded DNA and enhance the delivery of these templates by including Cas9 binding sites on each end,” Shy explained. “Cas9 binds directly to the templates and drags them all the way to the nucleus through nuclear localisation signals present on the protein.”
From their CRISPR screen, the team found a handful of candidates that could render the T cells resistant to key aspects of the immune‑suppressive microenvironment often found in tumours. They found one gene of interest named RASA2, which had never been associated with immune cell function before.
The team focused on RASA2 to find out whether controlling expression of the gene in human T cells might make them more sensitive immunotherapy agents. Leveraging models, the team created T cells with the RASA2 gene knocked out. They then subjected these T cells to various “stress tests” by exposing them repeatedly to cancer cells as well as to models of the tumour microenvironment.
They compared the performance of these cells to that of the original therapeutic T cells that still contained a functioning RASA2 gene. Long after the original cells had lost their cancer-fighting abilities, the cells with RASA2 knocked out remained remarkably tireless.
The results were consistent in tests on different types of engineered T cells in which the team had blocked RASA2 and across cells from many different human donors and in models of both liquid and solid cancer. They found that the knockout cells continued to kill the cancer cells.
“To broadly validate the approach, we showed the system worked with lots of different templates, target sites and types of human blood cells,” commented Shy.
Improving existing therapies
“One important demonstration from my perspective was showing that we could use this system to manufacture clinically-relevant doses of engineered T cells with all the same equipment and reagents we would need to use for patients,” concluded Shy.
The research group is now investigating this in pre-clinical models to streamline its effectiveness and assess its safety, ensuring that the T cells only recognise and attack cancer cells, rather than healthy cells.
In collaboration with other labs, the researchers are laying the groundwork for a clinical trial by combining multiple novel technologies with RASA2 deletion, aiming to improve a T-cell therapy already in use.
“We are actively moving this technology toward the clinic at UCSF,” said Shy. “We hope these types of approaches will improve the safety and function of cancer immunotherapies, help reduce costs and expand patient access, and provide a platform that can be rapidly extended toward other indications. There are endless opportunities for continued improvements to target cancer, infectious diseases and many inherited disorders.”
Dr Brian Shy is a Clinical Instructor in UCSF Department of Laboratory Medicine, Member of the Gladstone-UCSF Institute of Genomic Immunology, and Medical Director for the UCSF Human Islet and Cellular Transplantation GMP facility. His research focuses on the development and therapeutic application of cellular engineering tools.
- Shy BR, Vykunta VS, Ha A, et al. High-yield genome engineering in primary cells using a hybrid ssdna repair template and small-molecule cocktails. Nature Biotechnology. 2022.