Anticodon-edited tRNAs: a novel strategy to suppress premature stop codons in Rett syndrome

Mutations that either reduce or increase the function of the X-linked MECP2 (methyl-CpG-binding protein 2) gene are linked to a group of typically severe neurological disorders. Specifically, MECP2 deficiency is primarily associated with Rett syndrome (RTT), while MECP2 gene duplication is the main cause of the MECP2 duplication syndrome (MDS). Currently, there is no cure for these disorders. Although several studies have demonstrated that reintroducing the wild-type MECP2 gene can restore normal function in Mecp2-deficient mouse models, gene replacement strategies for RTT carry significant risks, particularly due to the potential for overexpression, which can lead to symptoms associated with MDS. As a result, it is critically important to regulate MeCP2 protein levels tightly. In this context, a promising therapeutic approach—specifically for nonsense mutations, which account for approximately 35% of RTT cases—is the use of translational read-through (TR). In particular, the tRNA suppressor strategy involves anticodon-edited tRNAs (ACE-tRNAs) that recognize premature stop codons and insert the corresponding canonical amino acid, enabling the synthesis of the full-length protein. Here we demonstrate that ACE-tRNAs for arginine (ACE-tRNAargUGA) are restoring full-length MeCP2 levels in HEK cells co-transfected with constructs expressing the R168X/R255X or R270X MeCP2 pathogenic derivatives and ACE-tRNAargUGA. Notably, no readthrough of canonical STOP codons was observed. We are now investigating the therapeutic efficacy of this strategy in a genomic context using patient-derived iPSCs. First results will be reported.