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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 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.
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fig4: 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.

Mentions: We then repeated our experiment with a fixed GC dose of 3 × 1011 of rAAV8 and a fixed REV of 75% in BALB/c mice which are less tolerogenic to rAAV gene transfer to the liver. In this case, we used EGFP as a cellular reporter gene which is known to be immunotoxic in the BALB/c mouse liver.21 In addition, considering the fact that the only source of empty capsid contamination in clinical vector lots is from the vector production/purification process, we added two more groups of BALB/c mice to receive fully packaged rAAV8EGFP with spiked-in PE AAV8 capsids prepared as described earlier. As shown in Figure 4a, compared with rAAV8EGFP vectors alone (Figure 4a-A), EGFP expression in the livers at 4-week postinjection was clearly repressed by addition of the empty virions from three different sources (i.e., PE from rAAV8EGFP production/purification process, Figure 4a-E; PE from rAAV8hA1AT production/purification process, Figure 4a-F; and CE of AAV8, Figure 4a-G). As expected, while scattered EGFP-positive hepatocytes were visible in the livers of the animals received PE particles from rAAV8EGFP production/purification process (Figure 4a-B), no EGFP signal was detected in the liver sections from the groups received PE particles from rAAV8hA1AT production/purification process (Figure 4a-C) and CE AAV8 particles (Figure 4a-D). Quantification of EGFP expression on liver sections of the study animals at 4-week time point further documented the significant repression of rAAV8EGFP transduction in mouse livers by empty viral particles regardless of the source of the empty particles; addition of 9 × 1011 particles each of PE virions derived from rAAVEGFP and rAAVhA1AT production/purification processes as well as CE AAV8 virions to 3 × 1011 GCs of fully packaged rAAV8EGFP all resulted in a statistically significant 35, 69, and 76% of reduction of EGFP expression, respectively (Figure 4b).


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4255953&req=5

fig4: 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.
Mentions: We then repeated our experiment with a fixed GC dose of 3 × 1011 of rAAV8 and a fixed REV of 75% in BALB/c mice which are less tolerogenic to rAAV gene transfer to the liver. In this case, we used EGFP as a cellular reporter gene which is known to be immunotoxic in the BALB/c mouse liver.21 In addition, considering the fact that the only source of empty capsid contamination in clinical vector lots is from the vector production/purification process, we added two more groups of BALB/c mice to receive fully packaged rAAV8EGFP with spiked-in PE AAV8 capsids prepared as described earlier. As shown in Figure 4a, compared with rAAV8EGFP vectors alone (Figure 4a-A), EGFP expression in the livers at 4-week postinjection was clearly repressed by addition of the empty virions from three different sources (i.e., PE from rAAV8EGFP production/purification process, Figure 4a-E; PE from rAAV8hA1AT production/purification process, Figure 4a-F; and CE of AAV8, Figure 4a-G). As expected, while scattered EGFP-positive hepatocytes were visible in the livers of the animals received PE particles from rAAV8EGFP production/purification process (Figure 4a-B), no EGFP signal was detected in the liver sections from the groups received PE particles from rAAV8hA1AT production/purification process (Figure 4a-C) and CE AAV8 particles (Figure 4a-D). Quantification of EGFP expression on liver sections of the study animals at 4-week time point further documented the significant repression of rAAV8EGFP transduction in mouse livers by empty viral particles regardless of the source of the empty particles; addition of 9 × 1011 particles each of PE virions derived from rAAVEGFP and rAAVhA1AT production/purification processes as well as CE AAV8 virions to 3 × 1011 GCs of fully packaged rAAV8EGFP all resulted in a statistically significant 35, 69, and 76% of reduction of EGFP expression, respectively (Figure 4b).

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