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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

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.
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fig1: 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.

Mentions: To assess the efficiency of CsCl gradient sedimentation in removing empty virions from fully packaged rAAV particles, we used high-resolution transmission electron microscopy (EM) to examine morphology of negative-stained virions19 in CE (Figure 1a-A) and PE (Figure 1a-B, contaminated with virions containing rAAVhA1AT genomes and Figure 1a-C, contaminated with virions containing rAAVEGFP genomes) AAV8 capsids well as fully packaged rAAV8nLacZ (Figure 1a-D), rAAV8hA1AT (Figure 1a-E), and rAAV8EGFP (Figure 1a-F). It is worth pointing out that the PE AAV8 particles were derived from the empty virion fractions collected from the second CsCl gradient sedimentation in the purification processes for rAAV8hA1AT (Figure 1a-B) and rAAVEGFP (Figure 1a-C) respectively, whereas the CE AAV8 particles (Figure 1a-A) were produced by using AAV8 packaging plasmid and adenoviral helper gene plasmid only for 293 cell transfection. As shown in Figure 1a, while CE and PE particles primarily displayed donut-like shapes of virions without (Figure 1a-A, REV: 100%) or with variable amounts of fully packaged particles (Figure 1a-B, REV: ~60% and Figure 1a-C, REV: >90%), more than 99% of fully packaged virions were observed in all three lots of rAAV8 vectors (Figure 1a-D–E), which was confirmed with a semi-quantitative assessment by counting all empty and full virions in six representative fields at ~92,000× using high-resolution transmission EM (Figure 1b). To test the purity of all the viral preparations, equal amounts of viral particles (~1 × 1010 viral particles) of each sample were analyzed by sliver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which showed that viral protein (VP)1, VP2, and VP3 capsid proteins were the only detectable protein components in all preparations (Figure 1b). Our results documented that the empty virions as well as other impurity proteins of cellular and transgene origins were efficiently removed from rAAV8 vector preparations by the CsCl gradient sedimentation method described by Ayuso et al.18 These highly purified AAV8 vectors and empty virions were next evaluated for transduction efficiency and side effects in mice for liver-directed gene transfer.


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)

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

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

fig1: 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.
Mentions: To assess the efficiency of CsCl gradient sedimentation in removing empty virions from fully packaged rAAV particles, we used high-resolution transmission electron microscopy (EM) to examine morphology of negative-stained virions19 in CE (Figure 1a-A) and PE (Figure 1a-B, contaminated with virions containing rAAVhA1AT genomes and Figure 1a-C, contaminated with virions containing rAAVEGFP genomes) AAV8 capsids well as fully packaged rAAV8nLacZ (Figure 1a-D), rAAV8hA1AT (Figure 1a-E), and rAAV8EGFP (Figure 1a-F). It is worth pointing out that the PE AAV8 particles were derived from the empty virion fractions collected from the second CsCl gradient sedimentation in the purification processes for rAAV8hA1AT (Figure 1a-B) and rAAVEGFP (Figure 1a-C) respectively, whereas the CE AAV8 particles (Figure 1a-A) were produced by using AAV8 packaging plasmid and adenoviral helper gene plasmid only for 293 cell transfection. As shown in Figure 1a, while CE and PE particles primarily displayed donut-like shapes of virions without (Figure 1a-A, REV: 100%) or with variable amounts of fully packaged particles (Figure 1a-B, REV: ~60% and Figure 1a-C, REV: >90%), more than 99% of fully packaged virions were observed in all three lots of rAAV8 vectors (Figure 1a-D–E), which was confirmed with a semi-quantitative assessment by counting all empty and full virions in six representative fields at ~92,000× using high-resolution transmission EM (Figure 1b). To test the purity of all the viral preparations, equal amounts of viral particles (~1 × 1010 viral particles) of each sample were analyzed by sliver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which showed that viral protein (VP)1, VP2, and VP3 capsid proteins were the only detectable protein components in all preparations (Figure 1b). Our results documented that the empty virions as well as other impurity proteins of cellular and transgene origins were efficiently removed from rAAV8 vector preparations by the CsCl gradient sedimentation method described by Ayuso et al.18 These highly purified AAV8 vectors and empty virions were next evaluated for transduction efficiency and side effects in mice for liver-directed gene transfer.

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