Rescue of Kir7.1 function by gene augmentation in LCA16 patient derived iPSC-RPE cells

Abstract

Purpose : The Retinal Pigment Epithelium (RPE) plays a vital role in vision physiology. The KCNJ13 gene is expressed in the RPE cells and encodes an inwardly rectifying potassium channel responsible for maintaining K⁺ homeostasis in the subretinal space and controlling RPE membrane potential. Using patient-derived iPSC-RPE, we have shown that a nonsense mutation in the Leber’s congenital amaurosis-16 (LCA16) gene resulted in a truncated and non-functional Kir7.1 channel. Here, we used LCA16 iPSC-RPE to test gene-augmentation through viral vector mediated expression of wild-type gene as a potential therapy.

Methods : We used non-integrating vector to reprogram LCA16 patient’s fibroblasts to generate iPSC-RPE cells. The cells maintained in Transwell inserts were used for whole-cell patch clamping. We transduced mature cells with lentivirus and Adeno-Associated Virus (AAV2) carrying a GFP fused normal wild-type KCNJ13 gene. Kir7.1 expression was confirmed through confocal microscopy and whole-cell patch clamp electrophysiology of transduced cells. We delivered either lentiviral or AAV2 particles to the mouse subretinal space and indirectly determined Kir7.1 expression through GFP fluorescence.

Results : iPSC-RPE formed a tight monolayer within six weeks of culture. The whole-cell current at -160 mV measured 98 pA with a resting membrane potential (Vm) of ~28 mV (n=8). Only lentivirus (not AAV) carrying the wild-type KCNJ13 gene (pLV-EF1a-GFP-Kir7.1, AAV-EF1a-GFP Kir7.1 and AAV-CAG-GFP-Kir7.1) efficiently transduced the iPSC-RPE cells. GFP expression was prominent on the apical aspects of cultured iPSC-RPE. Recordings from GFP positive cells measured -999 ± 245 pA of inward current, 10-fold increment, compared to LCA16 iPSC-RPE. Rb⁺ as a charge carrier caused a 6-fold increase in current to -5733 ± 1029 pA, in trasduced LCA16 iPSC-RPE cells only. The recorded V_m of -57 ± 5 mV (n=8) caused a 30 mV hyperpolarization of transduced cells. Both lentiviral and AAV constructs successfully transduced RPE cells in mouse as demonstrated by GFP expression.

Conclusions : We have achieved a functional recovery of Kir7.1 in patient-derived iPSC-RPE cells through gene augmentation. At a cellular level, both current amplitude and membrane potential were normalized upon gene expression. Gene expression in vivo also confirmed the usability of our approach for therapeutic application in cases of LCA16.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.