Modifiers of Adeno-Associated Virus-Mediated Gene Expression in Implication for Serotype-Universal Neutralizing Antibody Assay

Abstract

Adeno-associated virus (AAV)-based gene therapy is undergoing major expansion into clinical practice, with two treatments currently being granted Food and Drug Administration (FDA) approval. However, the presence of pre-existing neutralizing antibodies (NAB) is one of the significant hurdles for the clinical application of AAV vectors that significantly limits the patient population, which benefits from the treatment. A reliable diagnostic to evaluate the patient's seropositivity is required to ensure the effectiveness of the AAV-mediated therapeutic. Here, we describe a simple method for the determination of AAV NAB activity based on our finding that Compound C makes HEK293 cell highly permissive for infection by 10 commonly used AAV serotypes.

Keywords: adeno-associated virus; assay development; neutralizing antibodies; selective inhibitor of AMPK.

Figure 1.

Pretreatment of HEK293 cells with CC significantly increases the infectivity of AAV8-Luc. (A) HEK293 cells were treated with different concentrations of CC and 1 h later infected with AAV8-Luc at MOI = 2,000 vg/cell. *p < 0.05 and **p < 0.01 compared with luciferase activity in untreated cells. (B) 10 μM CC was added to HEK293 at different time points during infection with AAV8-Luc. **p < 0.01 compared with luciferase activity in untreated cells. (C) HEK293 cells were infected with different MOI (100–5,000 vg/cell) of AAV8-Luc in the presence of 10 μM CC or 10 μM CC +20 ng/mL IL-6 + 20 ng/mL TNF-α. Luciferase activity was measured 24 h later. **p < 0.01 for all MOI in the presence of CC compared with infections without pretreatment of HEK293 cells, ***p < 0.01 for MOI in the range 500–5,000 vg/cell in the presence of CC+IL-6+TNF-α compared with the infections in the presence of CC. For MOI = 250 vg/cell, *p < 0.05. (D) HEK293 cells were incubated with different concentrations of CC or with 20 ng/mL IL-6 + 20 ng/mL TNF-α for 48 h. At the end of incubation, cell counting reagent CCK-8 (APExBio, Boston, MA) was added to wells for an additional 1 h. The absorbance was measured at 450 nm. The number of cells in treated wells was compared with the number of cells in non-treated control wells, which was considered as 100% viability. *p < 0.05 and **p < 0.01 compared with non-treated controls. AAV, adeno-associated virus; CC, compound C; CCK-8, cell counting kit-8; IL-6, interleukin-6; MOI, multiplicity of infections; TNF-α, tumor necrosis factor alpha.

Figure 2.

CC enhanced the infectivity of all tested AAV serotypes. (A) HEK293 cells were infected with different AAV-Luc serotypes at MOI 2,000 vg/cell in the presence of 10 μM CC, or CC+IL-6+TNF-α. Luciferase activity was measured 48 h later. For all serotypes, luciferase activity was higher in the presence of CC compared with non-treated cells (p < 0.01). *p < 0.05 and **p < 0.01 for cells infected in the presence of CC+IL6+TNF-α compared with CC only. (B) Comparison of luciferase activity at 24 and 48 h after infection. Cells were infected in the presence of CC as described in (A). *p < 0.05 and **p < 0.01 for 48 h compared with 24 h.

Figure 3.

The performance of NAB assay in the presence of CC does not change NAB titer. The mouse plasma was collected 1 month after injection of AAV2 and analyzed in the presence of NAB by utilizing HEK293 cells either pretreated or non-treated with CC. Although CC significantly increased the values for luciferase activity, it did not affect NAB titer (as demonstrated overlaid vertical dotted lines corresponded to NAB titers for AAV2 measured in the presence and absence of CC). The observed difference is not statistically significant. NAB, neutralizing antibodies.

Figure 4.

Examples of NAB assay for different serotypes performed with HEK293 cell pretreated with CC. For each AAV, serotype mice were injected with 10¹⁰ vg/animal and serum was collected 3 weeks later for NAB assay. (A) Assay for AAV8. (B) Assay for AAV6. (C) Assay for AAV3.