Minimal Immune Responses Following Subretinal Delivery of AAV2 Demonstrate Safety, Efficacy and Allow Long-Term Transgene Expression in C57Bl/6 and RPE65-/- Mice

Abstract

Purpose:: Adeno-associated virus-2 (AAV2) vectors show considerable promise for gene transfer to the eye. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene.

Methods:: We studied anti-vector immune responses following delivery of GFP or RPE65, mutations in which are involved in early onset retinal degeneration.

Results:: Subretinal administration of AAV2-RPE65 (1x10¹¹ or 5x10¹¹ vg/ml) or AAV2-GFP (1x10¹¹) results in only limited immune responses in a small proportion of C57Bl/6 and Rpe65^-/- mice. Cells from draining lymph nodes showed limited T-cell proliferation following co-culture with AAV2 in vitro, although FACS showed only low levels of activation markers. Analysis showed no upregulation of T_H1 or T_H2 cytokines or T-cell proliferation in response to co-culture of splenocytes from injected or control mice with AAV2. Neutralising antibodies were present only in the serum of a proportion of mice receiving the higher dose of AAV2-RPE65, and no NAb were detected in the ocular fluids of mice receiving either dose of AAV2-RPE65 or the serum of mice receiving the lower dose. Importantly, this response is insufficient to inhibit transgene expression. Furthermore, ELISA showed no difference in IgG, IgA and IgM titre in serum or ocular fluids against the RPE65 transgene product or AAV2 capsid proteins between treated and control mice. Neither fundus nor histological examination showed evidence of retinal toxicity or leukocyte infiltration compared to uninjected control eyes (n=103) and ERG amplitudes remained in the normal range (n=36). Finally, AAV-GFP was administered in the right eye of C57Bl/6 mice, then administered in the left eye 9 months later. After 3 months, GFP expression was detected in both eyes in the complete absence of inflammation, demonstrating that repeat administration of vector and transgene expression is achievable.

Conclusions:: These data suggest that AAV2 is well tolerated in the eye and while a limited immune response may develop, this does not appear to inhibit transgene expression.

Keywords: gene transfer/gene therapy • inflammation • retina