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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

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.
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fig3: 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.

Mentions: In an attempt to further characterize repression of rAAV8 liver transduction by AAV8 CE particles, we performed another experiment in C57BL/6 mice with a fully packaged rAAV vector expressing a quantitative secreted reporter gene hA1AT at variable GC doses (i.e., 3 × 109, 1 × 1010, 3 × 1010, and 1 × 1011 GCs) and a fixed REV at 75%. This experiment generated the following findings. First, at all GC doses tested, only the lowest dose (3 × 109) seemed to show significant repression of hA1AT transduction by AAV8 CE particles at all six time points tested (Figure 3a). Second, we compared hA1AT expressions between the groups of mice received fully packaged rAAV8 only and fully packaged rAAV8 mixed with AAV8 CE particles at a REV of 75% for all four different vector GC doses at the 2-week time point. Although only at the GC dose of 3 × 109, a 64% (P < 0.05) inhibition of hA1AT expression by AAV8 CE particles became statically significant, 33–34% reductions of hA1AT expression were also noticeable in the 1 × 1010 and 3 × 1010 GC dose groups (Figure 3b).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4255953&req=5

fig3: 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.
Mentions: In an attempt to further characterize repression of rAAV8 liver transduction by AAV8 CE particles, we performed another experiment in C57BL/6 mice with a fully packaged rAAV vector expressing a quantitative secreted reporter gene hA1AT at variable GC doses (i.e., 3 × 109, 1 × 1010, 3 × 1010, and 1 × 1011 GCs) and a fixed REV at 75%. This experiment generated the following findings. First, at all GC doses tested, only the lowest dose (3 × 109) seemed to show significant repression of hA1AT transduction by AAV8 CE particles at all six time points tested (Figure 3a). Second, we compared hA1AT expressions between the groups of mice received fully packaged rAAV8 only and fully packaged rAAV8 mixed with AAV8 CE particles at a REV of 75% for all four different vector GC doses at the 2-week time point. Although only at the GC dose of 3 × 109, a 64% (P < 0.05) inhibition of hA1AT expression by AAV8 CE particles became statically significant, 33–34% reductions of hA1AT expression were also noticeable in the 1 × 1010 and 3 × 1010 GC dose groups (Figure 3b).

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