Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness

Abstract

Pathogenic variants of the KCNJ13 gene are known to cause Leber congenital amaurosis (LCA16), an inherited pediatric blindness. KCNJ13 encodes the Kir7.1 subunit that acts as a tetrameric, inwardly rectifying potassium ion channel in the retinal pigment epithelium (RPE) to maintain ionic homeostasis and allow photoreceptors to encode visual information. We sought to determine whether genetic approaches might be effective in treating blindness arising from pathogenic variants in KCNJ13. We derived human induced pluripotent stem cell (hiPSC)-RPE cells from an individual carrying a homozygous c.158G>A (p.Trp53*) pathogenic variant of KCNJ13. We performed biochemical and electrophysiology assays to confirm Kir7.1 function. We tested both small-molecule readthrough drug and gene-therapy approaches for this ''disease-in-a-dish'' approach. We found that the LCA16 hiPSC-RPE cells had normal morphology but did not express a functional Kir7.1 channel and were unable to demonstrate normal physiology. After readthrough drug treatment, the LCA16 hiPSC cells were hyperpolarized by 30 mV, and the Kir7.1 current was restored. Similarly, we rescued Kir7.1 channel function after lentiviral gene delivery to the hiPSC-RPE cells. In both approaches, Kir7.1 was expressed normally, and there was restoration of membrane potential and the Kir7.1 current. Loss-of-function variants of Kir7.1 are one cause of LCA. Using either readthrough therapy or gene augmentation, we rescued Kir7.1 channel function in iPSC-RPE cells derived from an affected individual. This supports the development of precision-medicine approaches for the treatment of clinical LCA16.

KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells

“…To further see the structure of protruded RPE cells, we observed cultured hiPSC-RPE cells of WT and KCNJ13-KO by transmission electron microscopy. As previously reported, 22 the WT hiPSC-RPE cells had elongated apical microvilli, apically distributed melanosomes, and basal membrane infoldings (Supplementary Fig. S4A and not shown).…”

“…Samples for electron microscopy were prepared with the modified method reported by Shahi et al 22 Briefly, hiPSC-RPE on Transwells were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1-M cacodylate buffer (CB) (pH 7.4), for 1 hour at 4°C. The samples were rinsed in 0.1-M CB and then post-fixed in 2% osmium tetroxide (OsO 4 ) in CB for 1.5 hours.…”

Sensing through Non-Sensing Ocular Ion Channels

“…The treatment also led to the restoration of the CNGA1 subunit, co-localized with CNGB1in rod of mice who performed better in vision-guided behavior tests than the untreated mice [250]. Gene therapy has shown success in the iPSC-RPE recessive disease model for the restoration of Kir7.1 and Best1 channel functions [242,244]. Mostly, recessive loss-of-function mutations are the prime target for gene therapy, but it is also capable of rescuing dominant loss-of-function mutations (BEST1; A10T, R218H, D302A, L234P, A243T, and Q293K) with similar efficacy in an iPSC-RPE model without the inactivation or disruption of the mutant allele [257].…”

“…), Ataluren (PTC124), and its oxadiazole analogs which act as a nonsense suppressor [291][292][293][294][295][296][297]. These drugs are effective in in vitro and in vivo disease models of eye diseases like LCA16 and retinitis pigmentosa [242,298]. Nonsense mutations reflect the stop codon, UAG, UGA, and UAA in the mRNA transcript much earlier than the usual stop codon followed by PolyA.…”

“…Several drugs like caffeine and SMG1 kinase inhibitors attenuate the NMD pathway [289,[303][304][305]. To date, there is only one report available for RT suppression of nonsense mutation leading to channel dysfunction in which NB84 treatment of an LCA16 patient (KCNJ13; W53X) derived hiPSC-RPE rescued the Kir7.1 channel functions by incorporating a near-cognate amino acid at PTC site [242].…”