Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness Pawan K. Shahi, Dalton Hermans, Divya Sinha, Simran.

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Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness Pawan K. Shahi, Dalton Hermans, Divya Sinha, Simran Brar, Hannah Moulton, Sabrina Stulo, Katarzyna D. Borys, Elizabeth Capowski, De-Ann M. Pillers, David M. Gamm, Bikash R. Pattnaik The American Journal of Human Genetics Volume 104, Issue 2, Pages 310-318 (February 2019) DOI: 10.1016/j.ajhg.2018.12.019 Copyright © 2019 American Society of Human Genetics Terms and Conditions

Figure 1 hiPSC-RPE Cells Derived from an Affected Individual Show the LCA16 Phenotype (A and B) Bright-field images of a mature hiPSC-RPE cell monolayer derived from (A) an unaffected family member showing a heterozygous sequence, and (B) an LCA16-affected proband with the TAG sequence. A pedigree chart shows sources of hiPSCs. (C) Normal karyotype in the affected individual’s hiPSC line showing no chromosomal abnormality. (D) Control, LCA16, and wild-type hiPSC-RPE cells and hfRPE cells showing Nhe1 digestion product (lanes marked +Nhe1) and undigested (lanes marked −Nhe1). The full-length Kir7.1 sequence is 1,083 base pairs (bp) in length, and the digested products are 925 and 158 bp in length. Note that the PCR product for both wild-type hiPSC-RPE cells and hfRPE cells (+Nhe1 lanes) runs at a slightly higher molecular weight in the presence of Nhe1. (E) RPE-specific gene expression in hiPSC-RPE cells. The American Journal of Human Genetics 2019 104, 310-318DOI: (10.1016/j.ajhg.2018.12.019) Copyright © 2019 American Society of Human Genetics Terms and Conditions

Figure 2 Impaired Kir7.1 Protein Expression in LCA16 hiPSC-RPE Cells (A) Electron micrograph of a representative LCA16 iPSC-RPE cell. (B) Comparison of average mitochondria (Mit.) count within 10 μm2 of the cell. (C) Evaluation of average length of RPE apical processes (AP). (D) Immunofluorescence localization of Kir7.1 (red), ZO-1 (green), and DAPI (blue) in control hiPSC-RPE cells. Both the lower and side panels reveal a polarized distribution of Kir7.1 in reference to ZO-1 and DAPI (confocal z stack images). (E) Localization of Kir7.1 (red), ZO-1 (green), and DAPI (blue) in LCA16 hiPSC-RPE cells. (F) Immunoblot showing the expression of RPE-cell-specific proteins in both tissue samples. Using a C-terminal-specific antibody against Kir7.1, we detected Kir7.1 protein in whole-cell lysates from the control hiPSC-RPE cells but not in those from the LCA16 hiPSC-RPE cells. The American Journal of Human Genetics 2019 104, 310-318DOI: (10.1016/j.ajhg.2018.12.019) Copyright © 2019 American Society of Human Genetics Terms and Conditions

Figure 3 LCA16 hiPSC-RPE Cells Exhibit Aberrant Physiology (A and B) Phagosome (red) localization within (A) control hiPSC-RPE cell and (B) LCA16 iPSC-RPE cell samples. (C) Plot of the average phagosome count within a fixed 200 μm2 area in the control cells and LCA16 hiPSC-RPE cells after 4 hr of feeding and a subsequent 48 hr digestion period or after 1 day of feeding followed by 6 days of digestion. (D) Plot of the average current-voltage (I/V) curve for Kir7.1 currents in the presence of normal external K+ (black) or high external Rb+ (light blue) in control hiPSC-RPE cells. (E) An average I/V curve in the presence of normal K+ (red) or high Rb+ (light blue) in LCA16 hiPSC-RPE cells. (F) Average plot of an inward current amplitude measured at −150 mV. Color representation as shown in (D) and (E). (G) Comparison of the average membrane potential of the control (black) cells to depolarized LCA16 (red) hiPSC-RPE cells. The American Journal of Human Genetics 2019 104, 310-318DOI: (10.1016/j.ajhg.2018.12.019) Copyright © 2019 American Society of Human Genetics Terms and Conditions

Figure 4 Putative Kir7.1 Loss-of-Function Rescue Through Nonsense Suppression (A–C) (A) Average I/V relationship before (red) and after (dark blue) treatment with NB84. The Rb+ current measured is shown as a light-blue trace. Evaluation of the average inward current measured at −150 mV (B) and of the membrane potential (C) to demonstrate the effect of NB84. (D) Immunoblot showing detection of eGFP fusion proteins, via anti-GFP antibody, in cell lysates from CHO cells transfected with empty vector (GFP), wild-type Kir7.1 coding sequence (WT), or Trp53∗ coding sequence (Mut). A partial restoration of the full-length protein product was observed in the latter after NB84 treatment (Mut + NB84). The American Journal of Human Genetics 2019 104, 310-318DOI: (10.1016/j.ajhg.2018.12.019) Copyright © 2019 American Society of Human Genetics Terms and Conditions

Figure 5 hiPSC-RPE Cells Regain Kir7.1 Function after Gene Augmentation (A–C) Plot of the average I/V curve for Kir7.1 currents measured in LCA16 hiPSC-RPE GFP-positive cells expressing a normal copy of the human Kir7.1 clone (A). Both K+ (green) and Rb+ (light blue) traces are shown. Average plots of the (B) current amplitudes measured at −150 mV and (C) membrane potential show rescue after gene augmentation. (D) Immunoblot analysis of lysate from control hiPSC-RPE cells (Ctrl), LCA16 hiPSC-RPE cells (LCA16), or LCA16 hiPSC-RPE cells infected with lentivirus coding for the eGFP-WT Kir7.1 fusion protein (LV) via anti-GFP antibody. (E) Cultured LCA16 hiPSC-RPE cells showing wild-type Kir7.1 (green), ZO-1 (red), and DAPI (blue) proteins. Z stack planes are shown in the lower panel. The American Journal of Human Genetics 2019 104, 310-318DOI: (10.1016/j.ajhg.2018.12.019) Copyright © 2019 American Society of Human Genetics Terms and Conditions