Overcoming premature termination codons in neurodevelopmental diseases with Anticodon Engineered tRNAs

A wide range of clinical conditions and neurodevelopmental diseases are caused by nonsense mutations that generate premature termination codons (PTCs), resulting in non-functional protein products. Several approaches to promote PTC readthrough, such as the use of small molecules, have shown limited efficacy. These compounds often incorporate the wrong amino acid, leading to missense mutations and raising safety concerns. Anticodon-engineered tRNAs (ACE-tRNAs), which can recognize stop codons (UGA, UAA, or UAG) and facilitate the incorporation of amino acids at PTC sites, offer an alternative strategy to restore the production of full-length, functional proteins. My research has focused on PTCs in the CDKL5 and MECP2 genes, mutations of which cause CDKL5 deficiency disorder (CDD) and Rett syndrome (RTT), respectively. Roughly 15% of CDD patients and 35% of RTT patients carry nonsense mutations, leading to truncated CDKL5 and MECP2 proteins. Thus, I have investigated ACE-tRNAs as potential therapeutics to restore full-length and functional protein synthesis. In this study, the efficacy of tRNA therapy was tested in transfected HEK293T cells expressing different CDKL5 or MECP2 PTCs in the presence or absence of UGA-tRNAArg. Obtained results demonstrated a significant restoration of full-length protein synthesis for both CDKL5 and MECP2. For CDKL5, we also observed a recovery of its catalytic activity, that was never obtained with readthrough molecules. Importantly, we proved that ACE-tRNA didn’t induce readthrogh at natural termination codons (NTCs). These results provided the proof of principle, supporting the translation of this research into a genomic context. We will investigate the efficacy of ACE-tRNA in restoring the synthesis of full-length, functional proteins in primary mouse neurons expressing different PTCs, human neurons derived from CDD and RTT patients’ IPSCs and mouse models of these diseases.