Proteasome inhibitors decrease AAV2 capsid derived peptide epitope presentation on MHC class I following transduction

Abstract

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.

Figure 1

Proteasome inhibitor mediated enhancement of gene expression following AAV2 transduction in vitro. (a) HHL5 cells were transduced with AAV2-CMV-LacZ (MOI 10,000) with the proteasome inhibitors MG-132, lactacystin, or bortezomib at the concentrations indicated. LacZ expression was measured 24 hours after transduction. (b) 293 or (c) Hepa 1-6 cells were transduced with AAV2-CMV-LacZ (MOI 10,000) in the presence of increasing amounts of bortezomib. LacZ expression was measured 24 hours after transduction. All experiments are performed in triplicate and SEM is shown. AAV, Adeno-associated virus; CMV, cytomegalovirus; MOI, multiplicity of infection.

Figure 2

Bortezomib treatment decreases peptide:MHCI complexes on the cell surface and decreases CTL killing. (a) HHL5-B7 cells were incubated with 75 nmol/l bortezomib for 0, 6, or 24 hours. During the last 2 hours the cells were loaded with the capsid-derived VPQYGYLTL peptide epitope (10 µg/ml). After loading, cells were washed and stained with a PE-labeled sTCR specific for the VPQYGYLTL peptide and analyzed by flow cytometry. (b) HHL5-B7 cells were transduced with AAV2 (3 × 10⁵ MOI) for 24 hours in the presence of a range of doses of bortezomib. After 24 hours, the cells were washed and HLA-matched human PBMCs expanded against AAV2 capsid were added at various effector:target ratios (E:T). The percentage of specific lysis was calculated from the release of intracellular lactate dehydrogenase and all samples were normalized to untransduced cells incubated with the appropriate concentration of bortezomib and E:T ratio. CTL assay was performed in triplicate wells and SEM is shown. CTL, cytotoxic T lymphocyte; MHCI, major histocompatibility complex class I; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; sTCR, soluble T-cell receptor.

Figure 3

Capsid-specific TCR cell line. (a) Jurkat/MA cells were transduced with a lentivirus expressing the α and β chains of the TCR specific for the AAV-capsid peptide VPQYGYLTL. Upon engagement of the TCR with the VPQYGYLTL peptide associated with HLA B*0702, the T cell becomes activated and expresses luciferase under control of the NFAT promoter that can be measured as relative light units on a standard luminometer. HHL5-B7 cells were either (b) loaded with VPQYGYLTL peptide for 4 hours or (c) transduced with AAV2 hAAT-hFIX for 24 hours before being washed and incubated with Jurma/VPQ reporter cells at an E:T ratio of 10:1. Cells were harvested after 24-hours incubation and assayed for luciferase expression. All RLU values are normalized by subtracting background Jurma/VPQ values. Experiments were performed in triplicate and (SD) is shown for each data point. AAV, adeno-associated virus; E:T, effector:target; hAAT, human α1-antitrypsin; hFIX, human Factor IX; NFAT, nuclear factor of activated T cells; RLU, relative light unit; TCR, T-cell receptor.

Figure 4

Proteasome inhibitor blocks antigen presentation following AAV transduction. HHL5-B7 cells were either (a) incubated with increasing concentrations of the proteasome inhibitor bortezomib for 24 hours and peptide loaded with VPQYGYLTL peptide for the final 4 hours or (b) transduced with AAV2-hAAT-hFIX (2.5 × 10⁵ MOI) in the presence of increasing amounts of bortezomib for 24 hours. Jurma/VPQ reporter cells were added at an E:T ratio of 10:1 and incubated for 24 hours before being harvested and assayed for luciferase expression. All RLU values are normalized by subtracting background Jurma/VPQ luciferase expression levels. Experiments were performed in triplicate and (SD) is shown for each data point. AAV, adeno-associated virus; E:T, effector:target; hAAT, human α1-antitrypsin; hFIX, human Factor IX; MOI, multiplicity of infection.

Figure 5

Proteasome inhibitors increase hFIX expression in vivo following transduction by AAV2 but not AAV8. C57Bl/6 mice (n = 4) were administered 25 µg of bortezomib at different time points (d0, d7) and transduced with (a) AAV2 (2.5 ×10¹⁰ vg) or (b) AAV8 (2.5 × 10⁹ vg) expressing the hFIX gene under control of the hAAT promoter (hFIX16) on d0. hFIX levels were assayed by ELISA at time-points indicated. SEM is indicated. AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay; hAAT, human α1-antitrypsin; hFIX, human Factor IX; vg, vector genomes.