Abstract
Purpose :
Based on recent developments in genome editing, it is now feasible to edit a mutation in retinal cells with the CRISPR platform. A limitation to bringing this to therapeutic use is its potential for off target effects due to continuous expression of Cas9 protein. There is currently an unmet need for an effective protein delivery system for retinal cells in vivo. We hypothesize that a cell penetrating peptide (PODb) may be utilized as a delivery vehicle for Cas9 protein in vivo.
Methods :
To localize the delivery of Cas9 protein in the murine retina, Cas9-GFP fusion protein was co-injected with PODb via intravitreal injection in C57BL/6J mice. To test delivery of functional Cas9 protein, two gRNAs were designed to target the tdTomato stop cassette in Ai9 reporter mice. First, Cas9 protein was complexed with gRNAs and tested in fibroblasts derived from Ai9 mice in vitro. Subsequently, two distinct Cas9 ribonucleoprotein (RNP) complexes were delivered to the Ai9 murine retina after co-injection with PODb peptide. C57BL/6J mice were sacrificed 4 hours after injection while Ai9 mice were sacrificed 10 days after injections. Frozen sections of eyes were examined by fluorescence microscopy for Cas9-GFP uptake and tdTomato expression.
Results :
After intravitreal injection of Cas9-GFP fusion protein and PODb peptide, we observed localization of GFP in the ONL, INL, and GCL of the retina in C57BL/6J mice. This indicates that a 160kD protein can be delivered to the retinal layers when injected with PODb peptide. Intravitreal injection of PODb and Cas9 RNPs showed reduced functional uptake in the retina compared to Cas9-GFP uptake as the Tdtomato expression was observed only in the INL of the retina. For more efficient uptake of Cas9 RNP subretinal injections were performed and tdTomato expression was observed in the RPE layer and ONL layer of the Ai9 reporter mouse.
Conclusions :
There is efficient Cas9-GFP uptake in the murine retinal layers after intravitreal co-injection with PODb. Delivery of functional Cas9 RNP was achieved to the INL after intravitreal injection and in the RPE layer and ONL after subretinal injection in the Ai9 reporter mouse. The efficient uptake of Cas9 RNP to the photoreceptors after intravitreal co-injection with PODb needs to be further optimized. We are developing this to be a complete therapeutic platform and apply it to a mouse model of a retinal degenerative disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.