(W1130-05-26) Empty Capsids can Enhance the Transduction Efficiency of AAVs by Inhibiting the Effect of NAbs

Purpose: The purpose of this study was to investigate, in an in vitro cell-based assay, the potential utility of empty capsids of AAV2 in overcoming the negative effect of neutralizing antibody (NAb) on AAV2-mediated transduction.

Methods: Transduction efficiency was measured using HEK293 cells transduced with AAV2-luciferase (AAV2-CMV-Luc) viruses. The feasibility of using empty capsids to overcome the negative impact of NAbs was assessed by exposing NAbs to empty AAV2 capsids prior to viral transduction. The assay procedure is briefly described as follows: HEK293 cells were plated into 96-well plates at a density of 3,000 cells per well and incubated at 37°C for 24 hr, after which cells were transduced with AAV2 at a 1×10⁴ MOI. The neutralizing effects of NAbs on AAV transduction was examined using a monoclonal antibody against AAV2. Prior to transduction, AAV2 virus and corresponding empty capsids (AAV: empty capsid = 1:10, 1:30, and 1:100) were mixed with the antibody, in a serial diluted manner, and pre-incubated for 1 hour before adding the mixture to the cells. Twenty-four hours after transduction, a luciferase substrate was added into each well and the luminescence of the reaction product was recorded on a BMG CLARIOstar multi-mode plate reader.

Results: NAb at 1:100 and 1:500 dilutions completely or nearly completely inhibited AAV2 transduction on HEK293 cells (100% and 98.6%, respectively); this inhibitory effect decreased as NAb was further diluted and was 26.9% at 1:4000 dilution. The inhibition percentage was appreciable decreased when NAb was treated with empty capsids of AAV2 (virus: empty capsid = 1:100) at all NAb dilutions, e.g., from 100% to 89.6% at 1:100; 98.6% to 27.3% at 1:500; and 75.6% to 2.38% at 1:1,000, suggesting that capsids adsorbed or bound the antibodies against AAV2, which led to enhanced transduction efficiency of HEK293 cells. In addition, the transduction enhancement caused by empty capsids decreased with decreasing dose of added capsids.

Conclusion: This study demonstrated a profound inhibitory effect of NAb on the AAV2-mediated transduction of HEK293 cells. It appears that AAV2 empty capsids were able to interact with NAbs and significantly restore the transduction efficiency of AAV2-CMV-Luc in HEK293 cells in a dose-dependent manner, and the magnitude of transduction enhancement depended on antibody concentration. Our results indicate that the empty capsids of AAV can be a potential bio-product to enhance the efficacy of AAV-mediated gene therapy in patients previously infected with AAVs.