Abstract
Purpose :
Cone dystrophy with supernormal rod response (CDSRR) is a rare autosomal hereditary retinopathy that affects 1 out of every 100,000 people due to mutations in the KCNV2 gene. This gene encodes for the voltage-gated potassium channel Kv8.2, which is a regulatory component of the delayed rectifier potassium channel in the photoreceptors. Several complete gene knock out mouse models reveal molecular mechanism of the disease. We sought to develop a nonsense mutation, Glu-156-to-Ter (E156X), knock in mouse for disease modeling and therapeutic development.
Methods :
A targeted c.466 G>T, p.E156X Kcnv2 mutation was achieved through delivery of CRISPR-Cas9 RNP to the mouse zygote. To confirm the single nucleotide substitution, Sanger sequencing and Restriction Fragment Length Polymorphism (RFLP) were performed on genomic DNA isolated from newly generated mice. We used Espion-E3 system at 6 weeks to record full field electroretinography (ERG) to characterize visual outcome. Standard scotopic and photopic (ISCEV) stimulus was used to record responses to compare a-, and b-wave amplitudes and implicit times. For measurement of c-wave we used a 25 cd.s/m2 flash stimulus. Optical Coherence Tomography (OCT) was used to examine retinal structure, and OptoMotor Response (OMR) to measure visual acuity (VA).
Results :
Genotyping validated the expected base change in the target codon, GAG to TAG, resulting in the nonsense mutation in the Kcnv2 gene without any off-target alterations. When compared to wild-type (WT) and het (E156X-/+) mice, OCT images revealed the absence of EZ layer in the homozygous (E156X+/+) mutant retina. At 6 weeks of age, OMR demonstrated no statistical difference in VA between WT, E156X-/+, and E156X+/+ mice. In comparison to WT mice, full-field ERG in the E156X+/+ mice exhibited a reduced and delayed a-wave amplitude with a flattened peak. The E156X+/+ mice had a higher b-wave amplitude at lower flash intensities, but their overall response was delayed at all flash intensities. The c-wave amplitude was noticeably reduced.
Conclusions :
The supernormal rod response in the E156X+/+ mice revealed by increased amplitude b-wave is consistent with Cone dystrophy caused by clinically relevant KCNV2 nonsense mutation. We report a novel CDSRR mouse model for understanding retinal dystrophy progression and possible therapeutic interventions for clinical translation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.