Abstract
Purpose :
The KCNJ13 gene encodes the inwardly rectifying potassium channel Kir7.1. Nonsense mutations in this gene are known to cause Leber Congenital Amaurosis 16 (LCA 16), leading to early-onset vision loss. This form of autosomal recessive blindness is characterized by severe vision impairment, nystagmus, and photophobia in the pediatric population. We previously demonstrated a readthrough of the Kir7.1 restored the channel function. In this study, we tested the effect of treating the KCNJ13 nonsense mutation W53X with the novel eukaryotic ribosome selective glycoside compounds ELX-01 and ELX-03.
Methods :
Human Embryonic Kidney (HEK293T) cells were plated on a 35mm culture dish until 60-70% confluence. Cells were then transfected with a plasmid carrying KCNJ13 nonsense mutation at 53rd amino acid (c.158G>A, W53X) and fused with Green Fluorescent Protein (GFP). After 24 hours of transfection, the cells were incubated with readthrough compounds ELX-01 and ELX-03 at 1mM for additional 24 hours. The cells were transferred to glass coverslips for whole-cell patch-clamp electrophysiology. Ringer’s solution was continuously perfused as an external solution. Rubidium (Rb+) -Ringer’s solution was used to test the Kir7.1 channel function.
Results :
Detection of GFP fluorescence in GFP tagged W53X transfected HEK293T cell membranes confirmed Kir7.1 protein expression. Previously demonstrated, W53X transfection resulted in non-measurable Kir7.1 current because of the truncated protein product. After we treated these cells with ELX-01 and ELX-03 at 1 mM concentration, the current-voltage curve showed inward rectifying Kir7.1 current. The maximum inward current measured at -160 mV was -90.63 ± 8.23 pA and -68.66 ± 30.56 pA, respectively, compared to only -18.61 ± 7.23 pA without the drug treatment. Upon substitution with Rb-Ringer, the maximum current amplitude did not change in non-treated cell measuring -18.92 ± 5.18 pA but increased to -502.93 ± 50.65 pA, a ~5.64-fold increase with ELX-01 treatment. Similarly, ELX-03 increased the maximum current to -768.02 ± 137.84 pA, representing a ~7.1-fold increase.
Conclusions :
Both ELX-01 and ELX-03 are able to restore the translation of full-length and functional Kir7.1 channels in HEK cells. Further evaluation of these compounds will ensure readthrough effects on LCA16 patient-derived iPSC-RPE and mouse models for preclinical validation.
This is a 2021 ARVO Annual Meeting abstract.