Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity

Abstract

Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

Keywords: PEGylation; adeno-associated virus 2; genetic code expansion; selective conjugation.

Figure 1

Diagram of precision PEGylation of adeno-associated virus 2 (AAV-2) to protect against recognition and degradation by neutralizing antibodies and proteases. The AAV-2 vector was site-specifically labeled with azide-bearing amino acids and then modified with polyethylene glycols (PEGs) via a bioorthogonal reaction to achieve preferable serum stability.

Figure 2

Feasibility of site-specific PEGylation of AAV capsid protein VP1 and AAV vectors by incorporation of NAEK (Nε-2-azideoethyloxycarbonyl-L-lysine, an azide moiety). (A) Analysis of NAEK-dependent expression of VP1 proteins by Western blotting. GAPDH was used as an internal control; (B) Western blotting analysis of site-specific PEGylation of VP1 proteins with the S452+1 mutant chosen as a representative; (C) Transmission electron microscope (TEM) images of naked or PEGylated rAAV2 vectors (a, b: unmodified particles; c: particle modified with 20-kD PEG).

Figure 3

Comparing the effect of NAEK incorporation at different sites on the production and infectivity of rAAV2 vectors. (A) Viral packaging efficiency of site-specific NAEK rAAV2 mutants was measured by qPCR as the number of packaged genome copies/mL and normalized to that of the wild type; (B) The viral infectivity of site-specific NAEK mutants of rAAV2 vectors, which was calculated as the ratios of functional titers (transgene expression detected by luciferase assay on HT-1080 cells) to genomic titers and finally normalized to that of the wild-type group. Data represent the average values and standard deviations from triplicate experiments.

Figure 4

The effect of PEGylation on rAAV2 infectivity and stability in human serum. (A) Effect of PEGylation on in vitro transduction efficiency of rAAV2; (B) Effect of site-specific PEGylation on rAAV2 stability in human serum in vitro. PEGylated/naked rAAV2 vectors were incubated in pooled human serum/PBS and then used to infect HT1080 cells, as measured by an increase in fluorescence. The retained activity was calculated by an equation described in Section 4.8 and normalized to that of the wild-type control. All quantitative data shown are average values with standard deviations from triplicate experiments. A two-sample t-test was used to determine the statistical differences among the groups at the same time point. *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 5

In vitro evaluation for PEGylated rAAV2 at sites Q325+1, S452+1, and R585+1 using 5-kD, 10-kD, 20-kD, and 40-kD PEG. (A) Transduction efficiency of rAAV2 modified with PEG of different molecular weights; (B) Effect of PEGylation size on the in vitro stability of rAAV2 in pooled human serum; (C) Site-specific PEGylation at Q325+1, S452+1, and R585+1 protects rAAV2 from recognition by antibodies in vitro. The affinity between anti-AAV polyclonal antibody and rAAV2 vectors modified/not modified with 20-kD PEG was determined by the enzyme-linked immunosorbent assay. Optical densities at 450 nm are shown. All quantitative data shown are average values with standard deviations from triplicate experiments. A two-sample t-test was used to determine statistical differences. *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 6

In vivo evaluation of PEGylated rAAV2 elimination in blood and antibody inducement using a Sprague Dawley rat model. (A) Blood clearance curve of PEGylated rAAV2. The genomic titer of retained rAAV2 in the blood at each time point was measured by quantitative PCR after a single dose injection of rAAV2, then normalized to that of wild type (WT) at 1 min; (B) Antibody induction experiment of PEGylated rAAV2 for a one-month period involving repeated stimulation. The level of VP1-specific antibodies in the blood at each time point was determined by an ELISA assay and presented in the form of normalized optical densities at 450 nm. All quantitative data shown are the average values with standard deviations from triplicate experiments. A two-sample t-test was used to determine the statistical difference among the groups at the same time point. *** p < 0.001, ** p < 0.01, * p < 0.05.