SPECIFIC: A systematic framework for engineering cell state‐responsive synthetic promoters reveals key regulators of T cell exhaustion

Cell state-specific synthetic promoters are essential tools for studying and manipulating cellular function, yet their design remains challenging, particularly for complex states such as T cell exhaustion. Here we present SPECIFIC (Synthetic Promoter Engineering for Cellular State Identification and Functional Analysis), an integrated framework that leverages chromatin accessibility profiling and machine learning to systematically identify and validate cell state-specific synthetic promoters. By comparing exhausted T cells from both mouse OT-I and human CAR-T models, we identified 56 conserved transcription factor binding motifs associated with T cell exhaustion. From these motifs, we engineered a subset of the most promising candidates into synthetic promoters driving an exhaustion-responsive gene circuit that senses and responds to T cell dysfunction. Several synthetic promoters, particularly those containing NFATc2 or MEF2C binding sites, demonstrated remarkable specificity in recognizing the exhausted state and effectively attenuated T cell dysfunction by reducing both CAR expression and exhaustion markers. This study establishes a generalizable approach for designing cell state-specific regulatory elements and provides new strategies for improving CAR-T cell therapy through programmed control of gene expression.