Virion Shell Impacts Efficacy of Intraocular Transfer of Viral Interleukin–10 by Adeno–Associated Virus

Abstract

Abstract: : Purpose: Prior work from our group demonstrated that intravitreal injection of a recombinant adeno–associated virus type 2 (AAV2) vector, with transgene transcription controlled by the tetracycline–inducible TetON system, effectively transferred viral interleukin 10 (vIL–10) into intraocular tissues, leading to inhibition of experimental autoimmune uveoretinitis (EAU) in the rat. In this study we compared the effect of packaging the AAV2–TetON–vIL–vector cassette into AAV2 or AAV5 shells. Methods: Lewis rats were injected intravitreally (10 µl) with AAV2/2–GFP or AAV2/5–GFP and killed 2 and 5 weeks (n=2 rats/group) later. Eyes were fixed in 4% paraformaldehyde, cryostat–cut at 10 micron thickness and screened for GFP expression by epifluorescence microscopy. Additional Lewis rats were injected intravitreally with either AAV2/2–TetON–vIL–10 or AAV2/5–TetON–vIL–10 (n=10 rats/group). Five weeks subsequent to injection, transgene expression was induced in 5 rats/group by adding doxycycline (200 mg/kg/day) to the diet. Rats were immunized systemically with S–Ag (30 µg) one week later. Ocular histopathology and S–Ag–specific systemic immune responses were studied 3 weeks post–immunization. Intraocular vIL–10 expression was determined by RT–PCR on whole eyes and ELISA on aqueous/vitreous from a third group of rats treated similarly (n=4 rats/group). Results: Intravitreal injection of AAV2/2–GFP led to GFP expression in both anterior uvea and retina, whereas GFP was detected only in the posterior segment in AAV2/5–GFP–injected rats. There was significant photoreceptor rescue in rats injected with AAV2/2–vIL–10 and treated with doxycycline in comparison to untreated animals (p=0.036). In contrast, AAV2/5–TetON–vIL–10–injected rats experienced EAU of similar severity, irrespective of whether or not they were dosed with doxycycline (p=0.353). Intravitreal AAV2/2–TetON–vIL–10 and AAV2/5–TetON–vIL–10 injections had no effect on the systemic S–Ag lymphocyte proliferation responses of rats. RT–PCR detected vIL–10 in eyes from rats injected with both vectors, but ELISA showed vIL–10 expression in AAV2/2–TetON–vIL–10–injected rats alone (100 pg/mL). Conclusions: Differences in intraocular expression of vIL–10 and effects on EAU following intravitreal injection of AAV2–TetON–vIL–10 in the rat may be achieved by variation in the AAV virion shell.

Keywords: uveitis–clinical/animal model • inflammation • cytokines/chemokines