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Pawan K Shahi, Sabrina Stulo, Dalton James Hermans, De-Ann M Pillers, Bikash R Pattnaik; KCNJ13 Gene Augmentation Therapy to Treat Blindness due to Kir7.1 Defects. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4091. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the KCNJ13 gene, encoding the inwardly rectifying potassium Kir7.1 ion-channel in the RPE, cause autosomal dominant snowflake vitreoretinal degeneration (SVD) and autosomal recessive Leber’s congenital amaurosis (LCA16). Molecular and biophysical analysis of the mutant protein revealed a non-functional protein product. With a goal of restoring retinal function via gene augmentation therapy, we tested the efficacy of Kir7.1 channel functional rescue using an in vitro model of CHO cells expressing disease – associated mutations.
We cloned human Kir7.1-WT (wild-type) and W53X (mutant) into Flip-In™ expression vector (ThermoFisher Scientific) and transfected them into Chinese Hamster Ovary (CHO) cells to express the protein products. Expression of Kir7.1 was verified through PCR and Restriction fragment length polymorphism (RFLP) analysis. Whole-cell configuration of patch-clamp technology was used to measure Kir7.1 current. Protein expression was confirmed by Western Blotting. N-terminal GFP-fused Kir7.1 wildtype was cloned into an AAV serotype 2 gene expression vector, packaged, transduced to CHO cells expressing the W53X mutant channel with a MOI of 100 for 6 hrs and analyzed within 1-2 weeks.
Sequencing and RFLP analysis confirmed stable integration human Kir7.1-WT and W53X into CHO-K1 lines. We measured 9.6 ± 0.94 fold (n=10) increase in inward current on replacing the extracellular K+ with Rb+ in Kir7.1-WT expressing cells. Higher Rb+ conductivity is a known feature of the Kir7.1 channel. The W53X stable cell line, in contrast, registered only 1.84 ± 0.04 fold increase (n=9, P=3.32E-07) in Rb+ conductance, due to the truncated Kir7.1 protein. When W53X CHO cells were transduced with the AAV carrying the GFP fused functional Kir7.1 gene, a distinct Kir7.1 inward current was observed as the current was amplified 9.64 ± 3.62 fold after Rb+ treatment (n=7, P= 3.37E-08).We observed a complete recovery of membrane potential from -35 mV to -54 mV. We also visualized GFP expression on the cell membrane by confocal microscopy and identified a 70 kDa Kir7.1 protein band through Western Blot analysis.
We showed successful functional restoration of the Kir7.1 channel in an in vitro CHO stable cell model expressing the W53X LCA16 mutant channel. This proof-of-principle approach shows the potential of augmenting a normal gene into cells with diseases caused by KCNJ13 mutations.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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