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

Gao K, Li M, Zhong L, Su Q, Li J, Li S, He R, Zhang Y, Hendricks G, Wang J, Gao G - Mol Ther Methods Clin Dev (2014)

Bottom Line: 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).Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605 ; National Institute for Food & Drug Control, Beijing, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig2: 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.

Mentions: To simulate different clinical scenarios, we first mixed a fixed vector genome copy (GC) dose (3 × 1011 GCs) of highly purified rAAV8nLacZ vector consisting of >99% of fully packaged viral particles with none, (REV: 0%; Figure 2a-B), 1 × 1011 (REV: 25%; Figure 2a-C), 3 × 1011 (REV: 50%; Figure 2a-D), 9 × 1011 (REV: 75%; Figure 2a-E), and 2.7 × 1012 (REV: 90%; Figure 2a-F) viral particles of CE AAV8 virions as dosing vector formulations which were intravenously administrated to adult male C57BL/6 mice. The images of X-gal histochemically stained liver sections from the study animals harvested 5 weeks later revealed that the higher the REV, the stronger repression of nLacZ transduction (Figure 2a). Quantification of nLacZ transduction for each liver section confirmed the REV-dependent inhibition of rAAV8 transduction by CE particles; the inhibition became detectable at a REV of 50% and statistically significant at REVs of 75 and 90%. Such inhibitions led to 29% (REV: 75%; P < 0.05) and 44% (REV: 90%; P < 0.01) reductions in nLacZ transduction as compared with fully packaged rAAV8-nLacZ alone (Figure 2b).


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

Gao K, Li M, Zhong L, Su Q, Li J, Li S, He R, Zhang Y, Hendricks G, Wang J, Gao G - Mol Ther Methods Clin Dev (2014)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: To simulate different clinical scenarios, we first mixed a fixed vector genome copy (GC) dose (3 × 1011 GCs) of highly purified rAAV8nLacZ vector consisting of >99% of fully packaged viral particles with none, (REV: 0%; Figure 2a-B), 1 × 1011 (REV: 25%; Figure 2a-C), 3 × 1011 (REV: 50%; Figure 2a-D), 9 × 1011 (REV: 75%; Figure 2a-E), and 2.7 × 1012 (REV: 90%; Figure 2a-F) viral particles of CE AAV8 virions as dosing vector formulations which were intravenously administrated to adult male C57BL/6 mice. The images of X-gal histochemically stained liver sections from the study animals harvested 5 weeks later revealed that the higher the REV, the stronger repression of nLacZ transduction (Figure 2a). Quantification of nLacZ transduction for each liver section confirmed the REV-dependent inhibition of rAAV8 transduction by CE particles; the inhibition became detectable at a REV of 50% and statistically significant at REVs of 75 and 90%. Such inhibitions led to 29% (REV: 75%; P < 0.05) and 44% (REV: 90%; P < 0.01) reductions in nLacZ transduction as compared with fully packaged rAAV8-nLacZ alone (Figure 2b).

Bottom Line: 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).Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605 ; National Institute for Food & Drug Control, Beijing, China.

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.

No MeSH data available.


Related in: MedlinePlus