Biodistribution of ARMMs as Non-Viral Vehicles for Therapeutic Payloads by Sub-Retinal Administration in Minipigs and NHP

Abstract

Purpose : Therapeutic delivery of genetic medicines to the back of the eye has been a challenge in effective management of ocular diseases. We developed a novel non-viral platform comprising an engineered class of extracellular vesicles called Arrestin domain containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs) for delivery of therapeutic payloads. Using this platform, we previously demonstrated delivery of fluorescent proteins and nuclear genome modifiers in murine ocular tissue. However, the biodistribution characteristics of ARMMs in higher mammals is not known. Here we present our findings on cellular uptake of ARMMs delivered by subretinal administration in adult Gottingen minipigs and African Green Monkeys.

Methods : Minipigs were subretinally injected with ARMMs loaded with Green Fluorescent Protein (GFP). A time-course analysis was performed by harvesting eyes at 6-, 12-, and 24-hours postdosing (n=4 eyes /group), by immunohistochemistry (IHC). GFP staining was used as proxy for ARMMs uptake. Co-staining of GFP with the following cell type-specific markers was examined: RPE65 for Retinal Pigment Epithelium (RPE), Rhodopsin for Rod and L/M Opsin for Cone photoreceptors. Eyes from uninjected animals were used as negative controls. A similar study was conducted in Green monkeys using ARMMs loaded with mCherry protein. to assess biodistribution in a non-human primate (NHP) species. Eyes (n=4 /group) were harvested at 6- and 24-hours post-administration and tissue sections were assayed by fluorescent IHC to assess the colocalization of mCherry with RPE65, rhodopsin and Arrestin3.

Results : In both minipigs and NHPs, ARMMs rapidly and robustly transfected the RPE as assessed by the presence of a strong payload-specific signal at all timepoints. The neural retina demonstrated a time-dependent movement of the signal from the photoreceptor outer segments towards the inner segments (IS). Both rods and cones were transfected and thesignal primarily localized in theIS.. No payload signal was detected in the inner retinal layers. . In the NHP eye, ~80% of the coneswere transfected.

Conclusions : Our data demonstrate the propensity of ARMMs to transfect the RPE and photoreceptors, and consequently uniquely position them as non-viral delivery vehicles for genome editors to these cell types with the potential to significantly modify disease.

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