Advancing entirely virus-free CRISPR CAR T cells to clinic- Are they as good as lentiviral CAR?

We have delivered a number of experimental chimeric antigen receptor (CAR) T cell therapies into clinical trials using specialist facilities at Great Ormond Street Hospital. These have helped treat children & adults with certain types of leukaemia who would otherwise have no further options for their disease. These projects were taken from concept and testing in the lab, through to manufacturing in clean rooms and clinical use in patients. All have relied on using disabled viruses to add genes into T cells to reprogram them. Some viral CAR T cells have undergone extra changes using genome editing - molecular scissors to snip out bits of other genes - and this allowed 'universal' T cells to be used without any matching. Now its becoming possible to use similar technology to both cut genes and insert new ones, and this means we can engineer cells without the need for viruses to add the CAR gene. We think the approach could eliminate supply bottlenecks, especially for making virus stocks for each new CAR, a major hold up issue for clinical trials. We have shown in our lab work so far that the efficiency of CAR insertion using CRISPR is almost as high as when we use viruses, and the cells behave and function just as well in short-term dish experiments. We now need to make sure that the process works at a larger scale, in a way that can be used in clean rooms, and that the cells behave no differently when they activated and responding against human leukemia in animals over a longer period.

This application will manufacture and compare CRISPR CAR T cells using existing lentiviral or new virus-free strategies to add the CAR. The tests will be on a scale suitable for future human trials and under conditions that can be transferred to a clean room setting. The cells will be tested in the lab and also in mice carrying human cancers, and analysed in depth to produce maps of which genes are active in the different populations of cells. We will include tests to find out which combinations of edits work best, and include versions where CAR expression is driven by where in the genome it has been inserted.

The project will adopt a new CAR against CD38 which is already being tested in our lab. It was derived from the monoclonal antibody Daratumumab, which is being widely investigated against multiple myeloma and autoimmune disorders and so has an extensively mapped safety and toxicity profile. Plans for an 'off-the-shelf' universal CAR38 therapy to explore deep cell-mediated clearance of disease are being formulated, and this project will help determine which strategies to use to manufacture the CAR T cell banks ahead for a trial. If CAR T cell production can be switched to entirely virus-free platforms, it will save time and money, and in the long run allow many more versions to be advanced to a clinical stage.