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

MECP2 nonsense mutations account for a large fraction of Rett syndrome (RTT) cases, and these patients may benefit from translational readthrough approaches aimed at promoting ribosomal bypass of premature termination codons (PTCs). Several readthrough approaches have already been considered, including small drugs like gentamicin and Ataluren. However, chemical readthrough agents suffer from limited specificity and carry the risk of introducing amino acid substitutions, an important limitation given the extreme sensitivity of MeCP2 to even subtle sequence alterations. These considerations highlight the need for strategies capable of restoring full-length MeCP2 with complete amino acid fidelity. Anticodon-engineered tRNAs (ACE-tRNAs) represents a promising alternative. Generated by modifying the anticodon of natural tRNAs to recognize a specific stop codon, ACE-tRNAs are aminoacylated by endogenous tRNA-synthetases and can support the synthesis of wild-type, full-length proteins under physiological regulation of expression. Using cellular models expressing common RTT-associated nonsense variants, we investigated the ability of ACE-tRNAs to selectively suppress PTCs and rescue full-length MeCP2. We show that ACE-tRNAs promote efficient and selective readthrough, restoring the synthesis of full-length MeCP2. Functional assays demonstrated 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. We are now optimizing ACE-tRNA delivery in human and mouse RTT neurons to evaluate whether they can promote sufficient readthrough to surpass the therapeutic threshold. These studies aim to provide proof of principle that ACE-tRNAs represents a promising and economically sustainable therapeutic option for a substantial subset of RTT patients.