Development of single fluorophore biosensors for multiplex imaging of CAR T Signaling - Project Summary The complexity and dynamics of protein kinases and epigenetic modifications in cellular processes necessitate the development of precise biosensors for live imaging. This proposal aims to develop single-FP-based, high- performance, ultrasensitive biosensors through directed evolution in mammalian cells. These biosensors will be used in multiplexed imaging and dynamic visualization of signaling activities in situ, with a specific focus on improving chimeric antigen receptor T cells (CAR-T) for cancer immunotherapy. Addressing the limitations of CAR-T therapy, particularly T cell exhaustion in solid tumors, requires a better understanding of the molecular mechanisms involved. Although kinases and epigenetic markers, particularly H3K27me3, play key roles in T cell regulation, our understanding of their spatiotemporal dynamics during cancer-immune interactions and through the course of CAR-T cell rejuvenation remains limited due to the absence of appropriate investigative tools. Thus, parallel examination of these key regulators in cancer-immune interacting environments should reveal new insights into the systematic behaviors and identify essential links for therapeutic manipulation. My hypothesis is that 1) the reversal of CAR-T cell exhaustion involves a rejuvenation of ZAP70 and Lck kinase’s function and a reprogramming of the H3K27me3, transitioning from an exhausted state to a naïve T cell-like state; and 2) transient knockdown of exhaustion-related genes could prevent and reverse exhaustion of CAR-T cells, which can be reflected by the kinase and epigenetics coordinated response patterns. Leveraging the combined expertise of my mentors, I have demonstrated the feasibility of engineering single-FP biosensors for tyrosine kinases, directed evolution of single-FP biosensors in mammalian cells and established a transient gene knockdown system that can be remotely controlled by focused ultrasound (FUS). Building on these achievements, three distinct aims have been further proposed: Aim 1 focuses on engineering novel single-FP prototype biosensors for monitoring tyrosine kinases or epigenetics in CAR-T cells of different phenotypes during cancer cell engagement and establishing a multiplexed imaging platform. Aim 2 involves developing ultrasensitive single-FP biosensors through directed evolution, high-throughput screening, and next- generation sequencing. Aim 3 focuses on the application of these biosensors for multiplexed imaging and manipulation of kinase-epigenome signaling in CAR-T cells using a FUS controllable gene knockdown system. This biosensor engineering platform can potentially be extended to develop any other kinase and epigenetic biosensors for live cell imaging. Similarly, the novel FUS-controllable gene knockdown system could be generalized for broader manipulations of various cellular processes. Successful execution of this project could revolutionize biosensor engineering and kinase imaging, profoundly impacting our understanding and treatment of cancer and other diseases.
