Precision therapeutic tRNA rescue of nonsense mutation R166X in KCNJ13 to restore K⁺ channel function

The KCNJ13 gene encodes the Kir7.1 protein, and mutations cause Leber's Congenital Amaurosis (LCA) and Snowflake Vitreoretinal Degeneration (SVD), leading to early-onset vision loss. One such nonsense mutation, R166X, results in nystagmus, poor night vision, and visual impairment. We examined therapeutic options for the R166X nonsense point mutation. Unlike missense genetic mutations, which can be rectified by gene augmentation and genome editing, nonsense mutations provide an opportunity to test additional readthrough therapies. Attempts to produce human induced pluripotent stem cells (hiPSCs) with the R166X mutation (CGA to TGA) were unsuccessful. This may be due to challenges in chromatin structure and folding that hinder access to the targeted loci. Therefore, we constructed an open reading frame (ORF) stably integrated HEK293T line by inserting the wild type (WT) or R166X-KCNJ13 gene using the FLP-FRT recombinase technique. While genome editing strategies did not repair the R166X mutation efficiently in this cell line, an arginine anticodon-engineered tRNA (ACE-tRNA^Arg.UGA) restored K⁺ channel expression and function. This study provides an example in precision medicine where translational readthrough strategies can rescue channel function at a mutation that is difficult to correct via genome editing.