Engineered tRNA to overcome nonsense mutation for the rescue of Kir7.1 physiological function

Abstract

Purpose : A nonsense mutation is a point mutation in the codon that introduces a premature termination codon (PTC), resulting in a truncated non-functional protein. It accounts for about 15% of congenital anomalies. Our lab reported a pathogenic nonsense mutation (c.158G>A, W53X) in the KCNJ13 gene that caused autosomal recessive Leber congenital amaurosis (LCA16) due to non-functional inwardly rectifying potassium channel (Kir7.1) protein. We aimed to use anti-codon engineered tRNA (ACE-tRNA) as a novel therapy, with an iso-codon recognizing the above specific nonsense mutation, and validating normal protein translation and functional Kir7.1 ion channel.

Methods : As in vitro disease models, we used HEK293 cells for exogenous expression of W53X Kir7.1 protein through plasmid transfection and patient human induced pluripotent stem cells (hiPSC)-RPE cells harboring W53X mutations. Cultured HEK293 cells were co-transfected with W53X mutant and Trp^UAG tRNA plasmid using PolyJet^TM (Signa Gen). The patient iPSC-RPE confluent cells in transwell culture were co-transduced with helper-dependent adeno (HdAd) viruses carrying a GFP (TAG) reporter and Trp^UAG tRNA. We performed protein expression, imaging, and single-cell whole-cell electrophysiological assays 48 hrs after transfection for HEK293 cells and 7 days post-transduction for hiPSC-RPE cells.

Results : We observed GFP fluorescence in the membrane of Trp^UAG tRNA-treated cells compared to cytoplasmic localization of GFP in the non-treated HEK293 cells, indicating membrane localization of full-length Kir7.1 protein product. Electrophysiological recordings indicated biophysical Kir7.1 channel properties rescue, such as potassium current, membrane potential, and ion-selectivity for Trp^UAG tRNA-treated cells compared to non-treated cells. Similarly, hiPSC-RPE expressing GFP reporter read-through by Trp^UAG tRNA treatment showed functional Kir7.1 channel current and protein expression.

Conclusions : ACE-tRNA provides a high-fidelity protein translation with a safe therapeutic outcome as a trans-gene but nonsense mutation-specific therapy as demonstrated in our functional Kir7.1 ion channel study. We focus on the dose and in vivo delivery optimization approaches towards clinical translation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.