Abstract
Purpose :
The therapeutic potential of the CRISPR/Cas9 mediated gene editing in ophthalmic diseases is uncertain due to the lack of a comprehensive understanding of the toxicity and immune responses associated with the exogenous expression of Cas9. The purpose of this study is to evaluate for signatures of off-target immune responses and cytotoxicity associated with long-term Cas9 expression in the retina and retinal pigment epithelium (RPE).
Methods :
Rosa26-Cas9 knock-in mice (RD8 negative), that constitutively express Cas9, were evaluated using color fundoscopy, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography (ERG) across different age groups and compared to age-matched wild-type (WT) controls. RPE integrity was examined using ZO-1 immunofluorescence of RPE/choroid flat-mounts. In vitro evaluation of Cas9 toxicity in RPE cells was performed using MTT and annexin/PI assays. A panel of 84 genes associated with mouse innate and adaptive immunity was screened in neural retina and RPE/choroid of Cas9 and WT mice using Taqman PCR arrays. Statistical tests used included Mann-Whitney U, ANOVA, and Fisher's exact.
Results :
Cas9 was expressed abundantly in the retina of Rosa26-Cas9 mice compared to WT mice, and fundus imaging revealed a normal-appearing posterior segment in young and aged (up to 12 months) mice. Full-field ERG responses were largely normal, but significant reduction was observed at the highest flash intensity in both a-wave and b-wave amplitudes of Cas9 mice compared to that of controls (p-value = 0.008). ZO-1 staining reveals intact RPE with regular hexagonal tiling even in the aged mice. Plasmid enforced expression of Cas9 in ARPE-19 cells demonstrated no loss of cell viability. Of the 84 genes tested in RPE/choroid of Cas9 and WT mice, IL6 was significantly upregulated (17.20 fold, p=0.0012) in Cas9 mice.
Conclusions :
The Rosa-26 Cas9 knock-in mice appear phenotypically normal even up to 1 year. Mildly reduced full-field bright-flash ERG recordings were observed. Overall, long-term Cas9 expression does not appear to significantly reduce retinal cell viability. The altered expression of IL6 observed in the RPE/choroid of Cas9 mice implies the unexpected consequences and off-targeting events that CRISPR/Cas9 might cause. Further investigations of the mechanism by which IL6 is upregulated are underway and should be fully considered before employing this technology.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.