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Pawan K Shahi, Dalton Hermans, Nathaniel York, Simran Brar, Katarzyna Borys, Elizabeth E Capowski, Ellen Welch, De-Ann M Pillers, David M Gamm, Bikash R Pattnaik; Read-through of LCA16 nonsense mutation therapy using iPS-RPE cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1103.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations of KCNJ13 gene, retinal pigment epithelium (RPE) inwardly potassium channel (Kir7.1) protein, cause Leber congenital amaurosis (LCA-16). We recently discovered a nonsense mutation in KCNJ13 gene (c.158G>A: p.W53X) in a young boy. Nonsense mutations are target of read through drugs that permit translational read-through to generate full length protein product. We hypothesized that read-through drugs will suppress W53X non-sense mutation and produce a functional Kir7.1 protein using patient derived iPS-RPE cells.
iPSC-RPE were differentiated from LCA16 patient (LCA16 iPSC-RPE) and a healthy individual (ctrl iPSC-RPE) skin fibroblast using the controlled and defined methods. Molecular and biochemical assays were performed on differentiated cells to verify RPE cell identity and maturity. We used isolated cells to perform whole-cell electrophysiology and compare Kir7.1 current recordings between drug (Gentamicin, novel small molecules RTC-14 and NB-84) treated for 36 hours vs. untreated and control cells.
Monolayer of hexagonal cells with pigmented appearance was seen after 6 weeks of culture in transwell. Both PCR and immunostaining confirmed the expression of RPE cell markers RPE65, bestrophin-1, and ZO-1. Kir7.1 was detected by PCR in both ctrl iPSC-RPE and LCA16 iPSC-RPE cells. Only LCA16 iPSC-RPE had the mutation 158G>A. Kir7.1 protein product in LCA16 iPSC-RPE was also truncated. Electrophysiology revealed Kir7.1 inwardly rectifying current (IKir7.1) measuring 144pA @ -160 mV with a zero current potential (Vm) of -54mV. Rb+, which is highly permeable through the Kir7.1 channel increased inward current by 10 fold. Compared to ctrl iPSC-RPE cells, LCA16 iPS-RPE cells measured IKir7.1 of 98 pA, Vm of -28mV and no effect of Rb+. Gentamicin (500µM) had no measurable change in LCA16 iPS-RPE cell whole cell parameters. On the other hand, both small molecules RTC-14 (5µM), and NB-84 (500 µM) treatment rescued Vm completely with variable recovery of current.
LCA16 iPSC-RPE expressed all the RPE markers and can be used as a model for the LCA16. Read-through drugs RTC-14 and NB-84, completely rescued Kir7.1 current in patient iPSC-RPE cells but not gentamicin. Hence, we plan to develop NB-84 as a drug for inherited retinopathy such as LCA16 through a personalized medicine approach.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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