Abstract
Purpose :
The KCNJ13 gene encodes the weak inwardly rectifying potassium channel Kir7.1, which is located in the retinal pigment epithelium (RPE). Several mutations in the KCNJ13 gene cause early onset Lebers Congenital Amaurosis (LCA16) and Snowflake Vitreoretinal Degeneration (SVD). In this study, we treated a LCA16 Kir7.1 nonsense mutation (W53X) using small-molecule read-through drugs G418 and Elox03, the nonsense mediated mRNA decay (NMD) inhibitor caffeine, NMD knockdown, and suppressor tRNA.
Methods :
HEK 293 cells were transfected with N-terminal GFP-fused W53X mutant plasmid. Cells were co-transfected with human SMG-8 siRNA or tryptophan-carrying anticodon engineered (ACE)-tRNA for 48 hours, treated with 150 µM G418 or Elox03, or co-treated with 150 µM G418 and 500 µM of caffeine. All treated cells were incubated with drugs for 48 hours. Immunocytochemistry (ICC) and whole-cell patch clamp was performed on treated cells. Function of Kir7.1 channel was measured in the presence of K+ to determine current, Ba+ to block current, and Rb+ to enhance current. The students T-test was used and significance was determined at the P < 0.05 cutoff level.
Results :
W53X transfection resulted in non-measurable Kir7.1 current as compared to the wild-type Kir7.1 channel. Upon treatment of cells with G418 we detected membrane expression of Kir7.1 and recorded a 100-fold increase in inward Rb+ current amplitude (P < 0.01), in addition to a six-fold increase in the inward Rb+/K+ current amplitude ratio (P < 0.01). When compared to the treatment of cells with G418 alone, co-administration of G418 with caffeine resulted in a two-fold increase in the inward Rb+/K+ current amplitude ratio (P < 0.01), and interestingly, a 29 mV hyperpolarizing shift in resting membrane potential (Vm) (P < 0.001). Similarly, when compared to G418 treatment alone, G418 treated SMG-8 knockdown cells led to a two-fold increase in inward Rb+ current amplitude (P < 0.001) and inward Rb+/K+ current amplitude ratio (P < 0.01), with a 32 mV hyperpolarizing shift in Vm (P < 0.001). Suppressor tRNA led to membrane expression of Kir7.1 and a two-fold increase in inward K+ current amplitude (P < 0.01).
Conclusions :
Functional assays using our disease mutant model clearly demonstrated the potential of small-molecule read-through drugs, NMD inhibition, and suppressor tRNAs for therapeutic usage of PTC-causing mutations.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.