T-FITNESS began by exploring the molecular mechanisms driving T cell exhaustion. By generating and analyzing small RNA-sequencing data from both in vivo and in vitro models, the consortium identified and validated a set of microRNAs consistently associated with exhaustion. These findings enabled the development of synthetic gene circuits that respond to intracellular exhaustion signals. Simultaneously, the project prioritized key transcription factors involved in the regulation of exhausted T cells through integrative bioinformatic analysis of public and in-house datasets. A user-friendly tool for single-cell regulatory network analysis was also developed and is now in its final stages. These insights informed the design of miRNA-responsive synthetic circuits, which demonstrated robust and tunable control of gene expression in primary human T cells. To advance toward clinical application, T-FITNESS developed optimized genome editing protocols, achieving high-efficiency knock-ins with minimal impact on cell viability. Strategies included donor template optimization, repair pathway modulation, and the use of high-fidelity editing tools, all of which enhanced editing precision and performance. Functional studies confirmed that circuit-equipped T cells exhibited reduced exhaustion, improved proliferation, and sustained effector function under chronic stimulation in vitro. To advance T-FITNESS technology to the clinic, GMP-compliant manufacturing workflows were established using automated platforms. These workflows achieved high editing efficiency, scalability, and product quality, making them suitable for therapeutic development. With support from the Hop-On facility grant, the project also initiated the development of protein-based inhibitors targeting exhaustion-related transcription factors. In parallel, additional miRNA-responsive systems were created, offering new strategies for context-sensitive gene regulation. Finally, a collaboration with project XPAND was established to co-develop enhanced genome editing platforms for T cells and hematopoietic cells, with activities set to begin in 2025.
