Distinct infectivity and neutralization antibody responses in the highly homologous AAV Go.1 and AAV5

Abstract

Introduction: Goat-derived adeno-associated virus (AAV) vectors, such as AAV Go.1, represent a novel platform for gene therapy due to their unique origin and potential advantages in transduction efficiency and immune evasion. However, their therapeutic potential and biological properties remain underexplored.

Methods: In this study, we developed a recombinant AAV (rAAV) Go.1 by replacing the goat AAV rep gene with the standard AAV2-rep gene to improve packaging efficiency. We compared the transduction efficiency of rAAV Go.1 with that of AAV5, a closely related serotype with 95% genome similarity, both in vitro and in vivo. Additionally, we assessed immune evasion properties by evaluating resistance to neutralization using sera from rAAV5-immunized mice and human volunteers. To further enhance transduction efficiency, we introduced site-specific mutations in the VP1 unique (VP1u) region and VP1/2 common region.

Results: The rep gene modification led to a significantly higher packaging efficiency for rAAV Go.1 compared to the original goat AAV. rAAV Go.1 exhibited markedly higher transduction efficiency than AAV5 in both in vitro and in vivo models. Furthermore, rAAV Go.1 demonstrated a 4-fold increase in resistance to neutralization by sera from rAAV5-immunized mice. A study involving 20 healthy volunteers revealed that high-titer neutralizing antibodies had a more pronounced inhibitory effect on rAAV5 compared to rAAV Go.1. Mutagenesis studies identified key modifications that enhanced viral properties: K32R, K91R, and K122R mutations in the VP1u region significantly improved viral production, while K137R (VP1u) enhanced transduction efficiency in vitro and in vivo.

Discussion: Our findings highlight the potential of rAAV Go.1 as an improved gene therapy vector with superior transduction efficiency and enhanced immune evasion. The identified VP1 mutations further optimize viral properties, making rAAV Go.1 a promising candidate for future therapeutic applications.

Keywords: cell transfection; gene therapy; neutralizing antibody; packaging efficiency; rAAV Go.1.

Figure 1

Study design and virus production. (A) Overview the utilized the rep/cap helper plasmid, pAAV2/Go1 contain AAV2 P5 promoter (green arrow) and rep element. The pAAV Go/Go1 derived from AAV Go1 P5 promoter (red arrow) and rep gene; (B) Diagram of packing vector containing AAV2-ITRs, the cytomegalovirus (CMV)-driven luciferase and EGFP transgenes; (C) SDS-PAGE gels with silver staining and Western blotting. Anti-AAV VP1/VP2/VP3 (clone B1) antibody was used to visualized different viral proteins (VP) in Western blotting; (D) Comparison of genome titers of two rAAVs which were determined by qPCR. The data were shown using the mean + SEM for three independent experiments.

Figure 2

Comparison of the transduction in cells and animals between rAAV Go.1 and rAAV5. (A,B) HEK293T or HeLa cells were incubated with rAAV5 or rAAV Go.1 at a MOI of 10⁵, respectively. At 48 h post-infection, the green cells with were visualized by an Olympus microscope, and the representative confocal images from three independent experiments are illustrated (Scale bar: 100 μm). (B) The luciferase activities are presented as mean + SD for three independent experiments. (C) In vivo imaging of luciferase activity was performed 3 weeks after administration of rAAV5 and rAAV Go.1. Representative ventral views of the results are shown. (D) Representative image of the explanted kidneys and lungs scanned with IVIS.

Figure 3

Inhibitory effect of anti-AAV5 antibodies on AAV Go.1 Transduction. (A) Serum from the treated mice was collected 4 weeks after an injection of 1 × 10¹¹ vg rAAV5 through lateral tail-vein. The rAAV5/luc vectors in 0.5 μl were incubated with equal volumes of different concentration of mouse serum or PBS. The mixture was then added into HEK293T cells. After 48 h of infection, the green fluorescent cells were visualized by an Olympus microscope (Scale bars: 100 μm). (B) Results of the in vitro NAb assay showed the inhibition of AAV5 and AAV Go.1 transduction in HEK293T cells by mouse serum. The AAV/luc vectors (20 μl) were incubated with equal volumes of different concentration of mouse or PBS. The mixture was then used to infect HEK293T cells in a 96-well plate. Luciferase activity was measured 48 h later. The inhibition was calculated as 100% minus the ratio of luciferase activity from the serum group to that of the PBS group. The dates are shown using mean + SEM for two independent experiments.

Figure 4

In vitro neutralizing antibody assay for AAV Go.1 and AAV5 using human serum (A) Neutralizing antibodies (NAbs) were determined using an in vitro transduction inhibition assay. The stacked histogram shows the distribution of serum samples with varying levels of AAV neutralizing activity. (B) The NAb testing for AAV5 and AAV Go.1 was performed with two human serum samples (No 16 and No 18). The dates were shown using mean + SEM for three independent experiments.

Figure 5

Essential role of lysine residues in the N terminus of VP1 for virus production (A) The structure of the VP1 unique VP1u (red) and VP1/2 common region (green) in model. (B) Sequence of VP with the positions of the lysine-arginine exchanges highlighted. (C) Quantitative PCR results showing the production levels of wild type and different mutant viruses. The dates were shown using mean + SEM for three independent experiments.

Figure 6

In vitro and in vivo transduction efficiency of AAV Go.1 and its mutant vectors. (A) HEK293T cells were infected with 10³ vg/cell of different AAV Go.1 and various mutant vectors. The green cells were visualized by an Olympus microscrope (Scale bars: 100 μm). (B) At 48 h post-infection, cells were lysis and analyzed by luciferase activities. Dates are presented as mean + SEM from three independent experiments. (C) In vivo imaging of luciferase activity was performed 3 weeks after administration of rAAV Go.1 and its mutant K136R. Representative ventral views of the results are shown. (D) Graph showing Average Radiant Efficiency of IVIS imaged mice.