Project 466532

Chimeric antigen receptor (CAR)-T cell therapy has led to improved survival for some patients with relapsed/refractory acute lymphoblastic leukemia (ALL). This therapy involves engineering T cells, a white blood cell, with a new gene, the CAR, so that it can recognize and kill the cancer cells. However, long-term survival remains short for many patients, with poor outcomes associated with a loss of the CAR-T cells. One possible reason for the loss of CAR-T cells is senescence, or aging, of the T cells, resulting in inactivity and cell death. Senescence can occur via telomere-dependent or independent pathways. Telomeres are repeats found on the ends of chromosomes that protect the cellular DNA from damage, and critical shortening of these leads to senescence. Telomere independent senescence is mediated by many different pathways. We propose to examine the pathways of senescence in CAR-T cells and how they differ compared to normal T cells, potentially leading to ways to alter these pathways to improve outcomes for patients by increasing persistence. We will explore telomere-dependent senescence by overexpressing telomerase, the enzyme that lengthens telomeres, in CAR-T using an inducible system so we can turn on and off telomerase activity during repeated activation of the CAR-T cells. We will also examine telomerase activity at various time points from patients who have received CAR-T on a clinical trial. Telomere-independent senescence will be explored using a CRISPR screen. CRISPR is a novel technique that allows for targeted breaks in the DNA, which when left alone, results in loss of that gene, or a knockout. We will use this technique on senescent CAR-T, to identify gene knockouts that revert this, and identify targetable pathways to improve CAR-T persistence.