Anticodon-engineered tRNAs restore full-length MeCP2 expression and function in Rett syndrome nonsense mutations

Background: Rett syndrome (RTT) is a severe neurodevelopmental disorder most commonly caused by loss-of-function mutations in the MECP2 gene, including a substantial fraction of nonsense variants. Despite intense research efforts, no curative therapy is currently available for RTT, although several therapeutic strategies are under investigation. Among these, translational readthrough approaches, aimed at promoting ribosomal bypass of premature termination codons (PTCs) using small molecules, have attracted considerable attention in recent years. However, chemical readthrough agents raise concerns due to their limited specificity and the high sensitivity of MeCP2 to amino acid substitutions, underscoring the need for strategies capable of restoring full-length MeCP2 while preserving its amino acidic and functional integrity. Methods: Using cellular models expressing common RTT-associated nonsense variants of MECP2, we investigated the potential of anticodon-edited transfer RNAs (ACE-tRNAs) to selectively suppress MECP2 nonsense mutations and restore full-length, functional MeCP2 protein. Results: We show that ACE-tRNAs promote efficient and dose-dependent readthrough, leading to the production of full-length MeCP2. Readthrough efficiency differed among nonsense mutations, with only specific variants benefitting from ACE-tRNA level optimization. Functional analyses revealed that ACE-tRNA–rescued MeCP2 correctly localizes to heterochromatic foci and regains the ability to recruit the transcriptional corepressor TBL1, two hallmark features of MeCP2 activity. In contrast, scrambled control tRNAs failed to restore nuclear localization or protein–protein interactions. Conclusion: Together, these findings demonstrate that ACE-tRNAs enable efficient and functionally relevant readthrough of MECP2 nonsense mutations, highlighting mutation-specific requirements and supporting the feasibility of a precision readthrough strategy for RTT. Although further in vivo validation and delivery optimization is required, ACE-tRNA-based readthrough represents a promising and complementary strategy for the treatment of nonsense mutations associated with RTT.