# Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines

## TL;DR

This study evaluates the effectiveness of delivering CRISPR/Cas9 ribonucleoproteins to the eye in mice and pigs, showing significant differences in genome editing efficiency between species.

## Contribution

The study highlights the importance of species-specific considerations in preclinical testing of genetic therapies for the eye.

## Key findings

- Subretinal delivery of Cas9 eRNPs achieved up to 12% base editing in mice but only 1.5% in minipigs.
- Editing primarily occurred near the injection site in both species.
- The results emphasize the lack of translation between small and large animal models for genetic therapies.

## Abstract

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of BE-eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in preclinical studies for the development of genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.