Suprachoroidal Gene Transfer with Nonviral Nanoparticles in Large Animal Eyes

Abstract

Purpose : AAV vectors have restricted packaging capacity and there are many unanswered questions regarding late immune responses. Nonviral vectors provide an alternative that can address these issues. Previous studies have shown that suprachoroidal injections of biodegradable poly nanoparticles (NPs) provide widespread transgene expression in photoreceptors and RPE cells of rats. However, it is crucial to understand the extent and level of gene expression resulting from suprachoroidal injection of PBAE NPs in eyes that are more similar to human eyes. In this study, the distribution and level of expression of a reporter gene was measured in minipig eyes which resemble human eyes in size and other structural aspects.

Methods : Experiments were done in Göttingen and Yucatan minipigs using PBAE NPs containing GPF expression plasmids. PBAE polymer was synthesized and it was used to condense expression plasmids into NPs. Two plasmids were tested, pCAG-eGFP-Z1 which is a standard plasmid containing bacterial sequences, and pCAG-GFP-nP, which is a miniplasmid from which bacterial sequences were removed. The effect of several variables on the distribution and level of expression after suprachoroidal injection of PBAE NPs was tested, including dose, time after injection, multiple injections, and the impact of minimizing bacterial sequences in the plasmid.

Results : Two weeks after suprachoroidal injection of pCAG-GFP-Z1 in minipig eyes, immunofluorescence and immunohistochemistry of serial sections showed expression of GFP in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after a single injection of 50, 100, or 200 µl of pCAG-GFP-Z1, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 µl and markedly reduced after 3 suprachoroidal injections at different locations around the circumference of the eye. Compared with pCAG-GFP-Z1, injection of the same dose of pCAG-GFP-nP in which bacterial sequences were removed resulted in a trend toward higher expression and also resulted in expression in both the inner and outer retina.

Conclusions : These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-size eye, which is ideal for treatment of inherited retinal degenerations.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.