701. Successful In Vivo Re-Administration of AAV with the Use of Two-Step AAV Injection

Abstract

Introduction: The major challenge for a successful re-administration of AAV vectors is the presence of neutralizing antibodies (NABs) directed against AAV developed after the first administration of AAV vectors. Those serotype-specific neutralizing antibodies directed towards the viral capsid proteins prevent efficient gene transfer with AAV of the same serotype. The generation of a humoral immune response does not permit the use of “vector of choice” more than once which is a concern for life-long disorders for which re-administration has to be considered. Currently different methods are being explored in order to circumvent the presence of anti-AAV NABs at re-administration. One of those methods is based on the principle of antibody adsorption in AAV-based gene delivery. It was first observed in mice by Scallan et al. who reported that the administration of AAV vectors in the presence of empty AAV capsids significantly reduced the neutralization of AAV vectors by anti-AAV circulating NAB (Scallan et al. 2006). Additionally, Mingozzi et al. have also shown in mice and non-human primates that injection of therapeutic AAV vector together with empty AAV capsids allows liver transduction in the presence of high titers of anti-AAV NABs (Mingozzi et al. 2013). Goal: The goal of our study was to decrease the anti-AAV NABs to a level that would allow successful repeated gene delivery with the same AAV serotype. Study design and results: We have designed an approach to decrease the levels of circulating NABs in vivo by mean of anti-AAV NABs adsorption. In our study, for the re-administration of AAV in the presence of anti-AAV NABs, a two-step AAV vector injection was used. The first dose of AAV vector was used as a “decoy” AAV that captured circulating anti-AAV NABs. To allow a proper binding of circulating anti-AAV NABs by “decoy” AAVs, we delayed the second AAV injection. In our proof of concept experiments, mice were initially immunized with AAV5-GFP. Two weeks later the re-administration of AAV5 was performed. The re-administration procedure consisted of a first “decoy” AAV5-GFP injection followed by an injection with AAV5-hSEAP. In a first experiment, mice were injected with a “decoy” AAV5-GFP followed by AAV5-hSEAP at 30, 60 or 120 minutes after the first injection. A significant decrease of total anti-AAV5 antibody levels after injection of the “decoy” AAV5-GFP vector was observed. In the second study, we used a similar procedure with a 30 min time frame between ”decoy” AAV5-GFP and AAV5-hSEAP injection, using three different doses of the “decoy” AAV5-GFP. The decrease of total anti-AAV5 antibodies 30 min after “decoy” AAV5-GFP injection was inversely proportional to the dose of the “decoy” AAV5-GFP used and to the hSEAP activity levels that were achieved after AAV5-hSEAP injection. Conclusions: Based on hSEAP transgene activity levels, we were able to determine a cut-off level of anti-AAV5 antibodies that allow a successful re-administration of the AAV5. Overall, the two-step AAV injection approach as such is promising for AAV re-administration.