Restoration of tumor suppressor protein with enhanced activity using engineered tRNAs to induce tumor regression

Cancer develops through a gradual accumulation of mutations including nonsense mutations in tumor suppressor genes (TSGs), which drive tumor proliferation and progression due to the impairment or complete loss of tumor suppressor protein function ^1–3 . The ability to restore functional tumor suppressor protein from mutant driver TSGs is a longstanding aspiration in cancer therapy, however, current strategies face formidable challenges in recovering proteins harboring heterogeneous mutations and circumventing dominant-negative effects exerted by endogenous mutant proteins. Therefore, the development of therapeutic interventions that are effective and generalizable across diverse nonsense mutations in cancer, to rescue endogenous mutant tumor suppressor proteins remains an urgent yet unmet need. Here we describe a “Double Suppressor Strategy” that engineered suppressor transfer RNA (sup-tRNA) could overcome nonsense mutations in key driver tumor suppressor genes, such as TP53 , enabling the restoration of full-length p53 with its original or enhanced transcriptional activity through the precise incorporation of desired amino acids. Moreover, Optimized sup-tRNAs, designed guided by a computational model, could effectively suppress tumor cell proliferation by targeted readthrough of nonsense mutations in different tumor suppressor genes. In two xenograft models harboring nonsense mutant TP53 , we observed up to a 60% reduction in tumor weight after sup-tRNA treatment, along with robust recovery of p53 expression and function, and no hematological or histological abnormalities detected. Our findings demonstrate the potential of sup-tRNA as a feasible and effective strategy to rescue and enhance tumor suppressor protein function, thereby preventing cancer development. As a novel therapeutic RNA, sup-tRNA provides a flexible strategy for targeting driver mutations in cancer, thereby expanding the potential of tRNA-based therapy in precision oncology.