Modified siRNA exhibit robust, safe and long-term gene silencing in the retina

Abstract

Purpose : Small interfering RNA (siRNA) is an emerging class of drug that allows to modulate gene expression in different tissues. The siRNA is a large and negatively charged molecule that requires modification for both, efficient gene silencing and effective delivery into the targeted cells. Eyes are an ideal organ for siRNA therapeutics. They are immune privileged, rather small, requiring smaller amounts of siRNA to achieve therapeutic effects, and their transparent nature allows for easier follow-up analyses of therapeutic outcomes. Furthermore, there are more than 200 genes related to retinal diseases that are in unmet need to find treatments. To identify effective siRNAs that target retinal cells, we examined the distribution of 12 modified siRNA in the mouse retina. Disease-related mutations in photoreceptors (PRs) often result in rapid visual impairment. We thus further examined the stability and safety in mouse and pig retinas of one of the modified siRNA that enriches in PRs.

Methods : 12 siRNAs that are modified with either different valency or lipid conjugates, all targeting the ubiquitously expressed Hungtintin (HTT) gene, were intravitreally injected into mouse eyes to examine their retinal distribution by both histological and molecular analyses. A PR-enriched siRNA was injected into both mouse and pig eyes, to establish a dose response curve, and determine long-term efficiency and safety. A scrambled sequence was used as nontargeting controls.

Results : We found that most siRNAs travel efficiently from vitreous to the back of the eye to distribute across the retina. We identified one siRNA that enriches efficiently in PRs. Western blotting analyses showed a dose dependent knockdown of HTT protein levels in mouse and pig. Additionally, knockdown was stable for at least 6 months in the mouse retina and 4 months (longest time point tested) in the pig retina with the PR-enriched siRNA. Finally, neither microglia (IBa1) nor astrocytes (GFAP) activation was detected in long-term treated retinas by histology.

Conclusions : The data suggests that siRNA can be used to target PRs efficiently and safely for the treatment of the many dominant mutations that cause blindness through PR degeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.