Abstract
Purpose :
Nonsense mutations in eukaryotic biology cause diverse diseases due to the creation of premature termination codon that results in truncated protein products. These diseases are often rare and ultra-rare, with an unmet medical need for therapeutic development. We have reported a KCNJ13 gene homozygous nonsense mutation c.158G>A, p.Trp53*.in a patient, and a c.496C>T, p.Arg166* nonsense mutation was reported earlier that caused Leber Congenital Amaurosis. Inwardly rectifying potassium channel (Kir7.1) is present in retinal pigment epithelial cells encoded by the KCNJ13 gene. This study aims to use engineered tRNA for the nonsense suppressing readthrough to rescue channel function.
Methods :
HEK293 cells were co-transfected with a control plasmid (a Mo2RD reporter plasmid with either TAG or TGA codon) and a GFP tagged anticodon-engineered tRNA plasmid (4X Trp TAG 2-1 or 4X Arg TGA 3-1). The cells were imaged using a confocal microscope. The functionality of these plasmids to restore Kir7.1 current was assessed in a heterologous system expressing two GFP tagged nonfunctional nonsense mutation constructs, W53* or R166*. To study this, HEK293 cells were co-transfected with either R166*-eGFP-Kir7.1 plasmid along with 4x-Arg TGA, or W53*-eGFP-Kir7.1 plasmid along with 4x-Trp TAG. 72 hours post-transfection, cells were assayed for Kir7.1 current by whole-cell patch-clamp.
Results :
All transfected cells showed red fluorescence indicating PTC correction for either TAG or TGA nonsense mutation with 80% transfection efficiency. Negative control experiments using a scrambled tRNA did not demonstrate any red fluorescence. HEK293 cells expressing W53* plasmid, when co-expressed 4X Trp TAG 2-1, we found expression Kir7.1 protein on the membrane compared to cytoplasmic expressions in the non treated cells. Biophysical properties of the Kir7.1 current like current-voltage plot, blocker sensitivity, and activation by Rb+ persisted in cells treated with 4X Trp TAG 2-1. Evaluation of the functional restoration of R166* mutation is underway.
Conclusions :
Expression of red fluorescence after treating with respective tRNA treatment for Mo2 RD carrying nonsense mutation codons indicate normal protein translation by incorporating a wildtype amino acid. The restoration of Kir7.1 protein expression through membrane localization and functional restoration further assures the therapeutic use of engineered tRNA to correct nonsense mutation errors.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.