Adeno-associated virus capsid antigen presentation is dependent on endosomal escape
Chengwen Li, Yi He, Sarah Nicolson, Matt Hirsch, Marc S. Weinberg, Ping Zhang, Tal Kafri, R. Jude Samulski
Chengwen Li, Yi He, Sarah Nicolson, Matt Hirsch, Marc S. Weinberg, Ping Zhang, Tal Kafri, R. Jude Samulski
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Research Article Genetics

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

Abstract

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials.

Authors

Chengwen Li, Yi He, Sarah Nicolson, Matt Hirsch, Marc S. Weinberg, Ping Zhang, Tal Kafri, R. Jude Samulski

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Figure 7

Inhibition of capsid antigen cross-presentation with proteasome inhibitor in 293 and Cos7 cells.

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Inhibition of capsid antigen cross-presentation with proteasome inhibito...
(A) Toxicity of bortezomib on 293/H-2Kb and Cos-7/H-2Kb cells. 293/H-2Kb cells (2 × 105 cells) or Cos-7/H-2Kb cells (2 × 105 cells) were seeded in 24-well plates and incubated with bortezomib. Cell viability was measured 24 hours later. (B) Effect of bortezomib on AAV2 transduction in 293/H-2Kb cells and Cos-7/H-2Kb cells. 293 cells or Cos-7 cells were incubated with inhibitors for 1 hour, and then 1 × 104 particles per cell of AAV2/luc vector were added to the culture and cells were incubated for 24 hours, then harvested for luciferase analysis. *P < 0.05 when compared with AAV2 without bortezomib treatment. (C) Effect of bortezomib on antigen presentation. After 293/H-2Kb cells or Cos-7/H-2Kb cells were incubated with bortezomib for 1 hour, they were treated with 2 × 1010 AAV2-OVA/AAT vector for 24 hours, then fixed with 1% paraformaldehyde and washed. Next, OT-1 spleen cells were added to HepG2/H-2Kb cell culture medium. Activation of OT-1 spleen cells was determined by flow cytometry for detection of CD8 and CD69 expression (C). **P < 0.01 when compared with AAV2-OVA without bortezomib treatment. Data represent the average of 3 individual experiments and SD.