Engineered tRNA reduces vision loss in a mouse model of Leber congenital amaurosis

Premature termination codons (PTCs) are a major class of pathogenic variants that underlie rare inherited disorders, including forms of childhood blindness. Therapeutic suppression of these “nonsense mutations” offers a gene- and position-agnostic strategy to restore protein function. Our previous work established that the W53X PTC in the KCNJ13 gene causes Leber congenital amaurosis type 16 (LCA16) by disrupting the inwardly rectifying potassium channel Kir7.1, leading to retinal pigment epithelium (RPE) dysfunction. Here, we present a proof-of-concept approach using anticodon-engineered transfer RNA (ACE-tRNA) to promote targeted translational readthrough. We engineered a suppressor tRNA encoding tryptophan (ACE-tRNA Trp.UAG ) to selectively recognize the UAG stop codon at the W53X site, enabling incorporation of the correct amino acid and restoration of full-length Kir7.1 protein. Delivery of ACE-tRNA via helper-dependent adenovirus (HDAd) resulted in robust rescue of channel function in heterologous systems expressing mutant KCNJ13 and in patient-derived human induced pluripotent stem cell (hiPSC)-RPE cells. Functional recovery was confirmed by electrophysiological assays demonstrating restored inwardly rectifying currents and membrane potential. Importantly, subretinal delivery of HDAd-ACE-tRNA Trp.UAG in a W53X mouse model led to partial restoration of RPE physiology, as measured by electroretinography, without evidence of retinal toxicity. Together, these findings establish ACE-tRNA-mediated suppression as a viable therapeutic strategy for nonsense mutations in multimeric ion channels and provide a translational framework for precision treatment of inherited retinal diseases.