Abstract
Purpose :
The ocular system is an attractive candidate to test the expanding gene editing toolbox, with numerous diseases and mutations amenable to CRISPR-based techniques. Cas protein expression has been well documented in rod photoreceptors across multiple species, providing valuable pre-clinical models. Limited data suggest expression of Cas in human cone cells, however, there has been no investigation in a relevant mouse model. Successful Cas expression in mouse cone cells would allow further evaluation of therapeutic potential of the CRISPR-Cas system in diseases affecting cone photoreceptors.
Methods :
We were interested in targeting mouse cone cells via the photoreceptor specific G-coupled rhodopsin kinase (GRK1) promoter, which was packaged into adeno-associated virus serotype 5 (AAV5). To isolate transduction of cone photoreceptors, a mouse model which expresses EGFP in cone cells was used. AAV5-GRK1-DsRed and AAV5-GRK1-SaCas9 with a EGFP targeting sgRNA were produced in HEK293T cells. 6-week-old mice received subretinal injections at varying doses between 5E+11 and 1+E13 genome copies/mL. The contralateral eye was injected with PBS as a control. 5-9 weeks post-injection SLO/OCT imaging was performed and repeated at different timepoints to assess GFP expression patterns and health of the photoreceptor layer, respectively. Immunohistochemistry (IHC) analysis was performed to further investigate the efficacy and efficiency of viral vectors within the retina.
Results :
Dose escalation of AAV5-GRK1-SaCas9 and AAV5-GRK1-DsRed showed increasing photoreceptor transduction with IHC. Significant thinning of the photoreceptor layer was evident on IHC and OCT images, mainly associated with higher doses. Although robust transduction of rod photoreceptors was achieved in all doses, no clear colocalization between cone markers and DsRed or anti-HA tag (representing Cas9 binding) was seen. In all doses, IHC showed cone cell survival and cone EGFP expression was present on SLO imaging.
Conclusions :
The combination of AAV5 packaged delivery and the GRK1 promoter is a highly effective method to transduce mouse rod photoreceptors. Neither AAV5-GRK1-DsRed or AAV5-GRK1-SaCas9 showed effective cone transduction in the mouse retina. This might be explained by a higher AAV dose requirement for murine cones compared with rods, or by intrinsic cone mechanisms that suppress specifically SaCas9 and DsRed expression.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.