Preclinical Testing of HUB-101, a Potential Gene Therapy for LCA16

Abstract

Purpose : Lebers Congenital Amaurosis (LCA16) is a monogenic pediatric blindness caused by point mutations in the KCNJ13 gene. This gene encodes an inwardly rectifying potassium channel (Kir7.1) required for retinal pigment epithelial (RPE) function. Gene augmentation, through lentiviral delivery of healthy KCNJ13 ORF, has been shown to rescue Kir7.1 channel function in patient iPSC-RPE cells and a conditional knockout (cKO) mouse. The rescue of RPE cell function in the cKO mouse also resulted in ERG c-wave recovery and preserved retinal integrity. The present study tested in vitrotransgene expression from HUB-101, a gene therapy product in development for Kir7.1 replacement in LCA16.

Methods : Eyes from 4-6-week old wildtype C57BL6 mice were used to culture mature primary RPE cells. After culturing cells in 96-well plates for 0, 7, 14, and 28 days, RNA was isolated and converted to cDNA to assess maturation markers. Maturity of cells was confirmed using qPCR to measure Gapdh, Rpe65, Best1, and Rbp1 gene expressions. HUB-101 is an adeno-associated virus (AAV5) containing KCNJ13 under the control of the VMD2 promotor. Mature RPE cells were transduced with 1x10^8, 1X10^9, or 1X10^10 particles of HUB-101 per well. HUB-101 vehicle served as a negative control. Seven days after transduction of HUB-101, RNA was isolated and converted to cDNA for qPCR assay. Specific molecular expression of HUB-101 in mouse RPE was assessed by measuring hGAPDH, hKCNJ13, and AAV ITR transcripts, along with mGapdh, mKcnj13 serving as experimental controls.

Results : Expression profiles of Rpe65, Best1, and Rbp1 indicated that RPE cells matured as early as 14 days in culture, with characteristic pigmented appearance. The matured primary RPE cells in culture, when transduced with HUB-101, showed dose-dependent expression of both hKCNJ13 and AAV ITR transcripts compared to vehicle-treated controls. In comparison, no changes in expression of mGapdh or mKcnj13 transcripts were detected, as expected.

Conclusions : We established a species-specific detection of candidate gene therapy product as a preclinical validation. Our results further confirm that HUB-101 can transduce mature RPE cells in vitro, with the KCNJ13 gene's dose-dependent expression. These data support the further evaluation of HUB-101 in nonclinical safety and toxicity studies, with the ultimate aims of clinical testing and restoration of sight in patients with LCA16.

This is a 2021 ARVO Annual Meeting abstract.