Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects

Abstract

Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

Keywords: empty virions; liver gene transfer; rAAV; side-effect.

Figure 1

Efficient removal of empty virions from rAAV8 preparations by CsCl gradient centrifugation. (a) Shown are transmission electron microscopy images of rAAV8nLacZ (D) rAAV8hA1AT (E), and rAAVEGFP (F) vectors as well as completely empty (CE) virions (A) and partially empty (PE) virions from rAAV8hA1AT (B) or rAAV8EGFP (C) production/purification process. The ratio of empty virions was semiquantitatively determined by counting six representative fields using high-resolution electron microscopy. The viral particles with the dimpled (dark) center are the empty virions. Original magnification: ×92,000. (b) Equal amounts of all rAAV8 vector or empty virion samples (1 × 10¹⁰ viral particles) were analyzed by sliver-stained SDS-PAGE. EGFP, enhanced green fluorescent protein; nLacZ, nuclear-targeted LacZ; rAAV, recombinant adeno-associated virus; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 2

Repression of nLacZ liver transduction by increased ratios of empty adeno-associated virus (AAV) particles in C57BL/6 mice. (a) Mice were intravenously injected with rAAV8nLacZ vectors (3 × 10¹¹ GCs/mouse) alone or mixed with AAV8 completely empty (CE) at variable ratios of empty virions (REVs: 25–90%) via tail vein. The liver sections were stained with X-Gal histochemically. Transgene expression was detected by light microscopy at 35-day postinjection. Original magnification: ×100. (b) Quantitative analyses of rAAV8 transduction efficiency. Images from six visual fields were analyzed quantitatively using ImageJ analysis software. Transgene expression was assessed as total area of blue staining (pixel)² per visual field (mean ± SEM). Student’s t-test was used to compare test results with the group received rAAV8 alone, and the differences were determined to be statistically significant. *P < 0.05, **P < 0.01. GC, genome copy; nLacZ, nuclear-targeted LacZ; rAAV, recombinant AAV.

Figure 3

Human α-antitrypsin reporter gene expression from variable doses of rAAV8hA1AT with a fixed ratios of empty virion (REV) of AAV8 completely empty (CE) at different time points in C57BL/6 mice. (a) Mouse sera were collected at different time points after intravenous injection of variable doses (1 × 10⁹ to 1 × 10¹¹ GCs/mouse) of rAAV8hA1AT vectors with or without spiked-in CE AAV8 particles at a fixed REV of 75% (of total particles). The serum hA1AT levels were detected by enzyme-linked immunosorbent assay. (b) Relative serum hA1AT expression of animal groups received different rAAV8 dosing formulations as compared with the groups treated with various dose of fully packaged rAAV8 alone at 2-week postinjection. Student’s t-test was used for comparing the experimental results with those from the groups with various dose of fully packaged rAAV8 alone, and the differences were determined to be statistically significant *P < 0.05. GC, genome copy; rAAV, recombinant adeno-associated virus.

Figure 4

Repression of rAAV8EGFP liver transduction by completely empty (CE) and partially empty (PE) AAV8 particles in BALB/c mice. (a) Adult male BALB/c mice were intravenously injected with rAAV8EGFP vectors (3 × 10¹¹ GCs/mouse) alone or mixed with empty AAV8 particles (9 × 10¹¹ GCs/mouse) from three different sources at a fixed ratio of empty virion (REV: 75%) via tail vein. The liver sections were fixed, and transgene expression was detected by fluorescence microscopy at 4-week postinjection. Original magnification: ×100. (b) Quantitative analyses of rAAV8EGFP transduction efficiency. Images from six visual fields were analyzed quantitatively using ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel)² per visual field (mean ± SEM). Analysis of variance was used to compare test results with those from the group with rAAV8EGFP alone, and the differences were determined to be statistically significant. *P < 0.05, **P < 0.01. GC, genome copy; rAAV, recombinant adeno-associated virus.

Figure 5

Exacerbate liver transaminitis in BALB/c mice by increased ratios of empty adeno-associated virus (AAV) particles in rAAV8EGFP dosing formulations. Mouse sera were collected at different time points after vector perfusion. The serum alanine aminotransferase (ALT) levels were detected by ALT detection kits. The time courses of (a) the ALT levels and (b) comparison of ALT levels of different study groups are presented. Analysis of variance was used for comparing the experimental results with those from the groups with phosphate-buffered saline (PBS) or rAAV8 or AAV8 empty particles alone and was determined to be statistically significant. *P < 0.05, **P < 0.01 versus PBS; ^#P < 0.05, ^##P < 0.01 versus rAAVEGFP alone. Finally, the groups of rAAV8EGFP vector mixed with different AAV8 empty particles were compared with the corresponding AAV8 empty particle alone, respectively, and the P value for each paired comparison is presented. rAAV, recombinant AAV.