Tolerability and tropism of recombinant adeno-associated virus vectors in the African green monkey (Chlorocebus sabaeus) anterior chamber

Abstract

While many studies have investigated the use of recombinant adeno-associated vectors (rAAV) in the posterior chamber for treatment of inherited retinal diseases, fewer studies have looked at rAAV's ability to transduce cells within the anterior chamber. This study focuses on evaluating the tropism and tolerability of three rAAV serotypes-rAAV2/6, rAAV2/9, and rAAV2/2[MAX]-expressing a green fluorescent protein (GFP) reporter following intracameral injection in the non-human primate (NHP) African green monkey (Chlorocebus sabaeus) model. Injection of high dose (1 × 10¹² vg/eye) rAAV vector resulted in transient inflammation characterized by aqueous flare and cellular infiltrate that resolved without intervention in all serotypes. Post-mortem histology revealed widespread expression of GFP in cells of the trabecular meshwork and iris in high dose rAAV2/6, rAAV2/9, and particularly rAAV2/2[MAX] eyes, indicating that rAAV vectors of these serotypes have broad tropism for cells of the anterior chamber and may facilitate the treatment of blinding disorders, such as glaucoma.

Figure 1.. Slit-lamp biomicroscopy of the anterior chamber for indications of inflammation.

All eyes except for the uninjected control (A) exhibited a transient phase of inflammation immediately after vector administration (B-D). In most animals this phase resolved by day 7. One high dose treated eye in each treated group (B342, B354, and B361) exhibited a second phase of inflammation that occurred at days 14 and 28 possibly coinciding with gene expression. In MAX treated eyes, both high and low dose treated eyes in subject B361 showed a second phase of inflammation (D). Total clinical scores compiled from multiple parameters showed increase incidence of clinical findings high dose treated eyes (F-H). Most instances resolved by day 42 without intervention.

Figure 2.. Intraocular pressures as measured by rebound tonometry.

A transient increase in IOP was seen immediately following vector injection at day 0 (B-D). One MAX treated animal (B361) exhibited a decrease in IOP at day 28 that resolved by day 35 (D).

Figure 3.

Average percent change in retinal thickness from baseline to day 42 for each treatment group. A representative cSLO and OCT scan indicates the examined areas (A). Retinal thicknesses stayed largely consistent throughout the study with most deviations staying within 9% of baseline measurements. Exceptions to this trend are seen in the control eye (B), rAAV2/6 low treated group (D), and both rAAV2/2[MAX] treated groups (G, H).

Figure 4.. Representative fundus photography at baseline and 42 days.

No changes were found between any eyes at baseline vs. 42 days in either the control (A,B), Low dose treated (C, E, G, I, K, M) or high dose treated eyes (D, F, H, J, L, N).

Figure 5.. Average corneal thicknesses as determined by pachymetry.

Corneal thickness remained fairly constant with minor fluctuations throughout the study. One low dose treated rAAV2/6 eye (B347 OS) (B) showed the largest decrease in thickness at day 42. An increase in thickness was noted at day 7 in both control and rAAV2/9 treated eyes in subject B353 (A, C). All other measurements remained similar to baseline (A-D).

Figure 6.. Representative brightfield and confocal microscopy of the control and high dose treated eyes.

Transduction of the iris, and iridocorneal angle was found in rAAV2/6 (B,F) and rAAV2/9 (C,G) treated eyes, while the most transduction was found in rAAV2/2[MAX] treated eyes (D,H). A small amount of non-specific staining is seen in the control eye (A). Scale bar = 100μm