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Cited by 155Virus-based gene therapy researchRNA Interference and Gene DeliveryViral Infectious Diseases and Gene Expression in Insects

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Production of adeno-associated virus vectors for in vitro and in vivo applications

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Kimura, Toyokazu, Ferran, Beatriz, Tsukahara, Yuko, Shang, Qifan, Desai, Suveer, Fedoce, Alessandra, Pimentel, David Richard, Luptak, Ivan, Adachi, Takeshi, Ido, Yasuo, Matsui, Reiko, Bachschmid, Markus Michael·2019·Scientific Reports
DOI 10.1038/s41598-019-49624-wPMC PMC6753157
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Abstract

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 10 10 –10 11 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

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Figure 1 Comparison of AAV production using different media formulations and time points. The graph represents (A) pH, (B) glucose concentration, and (C) viral titers of AAV2-DJ-shGlrx-mVenus in the cell culture medium of HEK293T cells at days 3 and 5 post-transfection with viral plasmids. HEK293T cells were cultured for AAV production in supplemented DMEM as described in the table below. Statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical differences (n = 8; ns = not significant; **P < 0.01; ***P < 0.001; SEM) are compared with medium containing 2% FBS as a control. (D) The graph shows titers of the released AAV2-DJ-shGlrx-mVenus and AAV2-DJ/8-shGlrx-mVenus into the cell culture medium at different days post-transfection. The medium was composed of DMEM, 1% FBS, 1x Glutamax, 10 mM HEPES and addition of 0.075% sodium bicarbonate. On day 3, the medium was changed (n = 8; SEM).
Figure 1 Comparison of AAV production using different media formulations and time points. The graph represents (A) pH, (B) glucose concentration, and (C) viral titers of AAV2-DJ-shGlrx-mVenus in the cell culture medium of HEK293T cells at days 3 and 5 post-transfection with viral plasmids. HEK293T cells were cultured for AAV production in supplemented DMEM as described in the table below. Statistical analysis was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical differences (n = 8; ns = not significant; **P < 0.01; ***P < 0.001; SEM) are compared with medium containing 2% FBS as a control. (D) The graph shows titers of the released AAV2-DJ-shGlrx-mVenus and AAV2-DJ/8-shGlrx-mVenus into the cell culture medium at different days post-transfection. The medium was composed of DMEM, 1% FBS, 1x Glutamax, 10 mM HEPES and addition of 0.075% sodium bicarbonate. On day 3, the medium was changed (n = 8; SEM).CC BY · PubMed Central
Figure 2 Flowchart of AAV purification. The helper-free AAV system comprises three types of plasmids (ITR-containing plasmid, AAV Rep-Cap plasmid, pHelper). Triple plasmid co-transfection with PEI into HEK293T cells was performed in five T150 flasks (day 0). On day 3 and day 5, 150 ml of medium were collected and subjected to AAV purification. On day 5, cells were detached with 0.5 M EDTA (pH 8.0), collected, and subjected to lysis in citrate buffer for further purification. AAVs were isolated from both cell lysate and medium, and the purification took 2 days, from day 5 to day 6. Cell lysate and medium were purified in the same manner after the PEG/NaCl precipitation. After aqueous two-phase partitioning, an iodixanol discontinuous gradient purification step was performed.
Figure 2 Flowchart of AAV purification. The helper-free AAV system comprises three types of plasmids (ITR-containing plasmid, AAV Rep-Cap plasmid, pHelper). Triple plasmid co-transfection with PEI into HEK293T cells was performed in five T150 flasks (day 0). On day 3 and day 5, 150 ml of medium were collected and subjected to AAV purification. On day 5, cells were detached with 0.5 M EDTA (pH 8.0), collected, and subjected to lysis in citrate buffer for further purification. AAVs were isolated from both cell lysate and medium, and the purification took 2 days, from day 5 to day 6. Cell lysate and medium were purified in the same manner after the PEG/NaCl precipitation. After aqueous two-phase partitioning, an iodixanol discontinuous gradient purification step was performed.CC BY · PubMed Central
Figure 3 AAV iodixanol discontinuous gradient purification and purity assessment. (A) Serotype-independent purification of AAVs using a discontinuous iodixanol gradient and ultracentrifugation. The 15% iodixanol layer was underlayered with denser iodixanol solutions to create the step gradient. The 25% and 54% iodixanol layers included phenol red for better visualization. Complete AAVs concentrated in the 40% layer. For elution of the iodixanol gradient, a small hole was drilled into the bottom of the centrifuge tube using a 25 G needle and another 25 G needle was inserted at the top. Fractions of 1 ml for the 54% layer, three of 400 μl for the 40% layer, and 1 ml for the 25% interface were collected. (B) Actual iodixanol step gradient before and after centrifugation. Visible interphase (arrow) between the 40% and 25% layers consists of empty and incomplete AAV2-DJ/8 particles, which may contain fragments of viral ssDNA. (C) Silver-stained protein gel of AAV2-DJ/8 samples at different stages of purification. Box denotes the area of the viral capsid proteins VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa). The purification procedure, using PEG precipitation, chloroform extraction, and aqueous two-phase extraction gradually separated contaminating proteins. The final discontinuous iodixanol gradient ultracentrifugation yielded highly purified AAV particles in fractions 2–4. The viral genome copy number measured with qPCR was high in fractions 2–4 while fraction 5 contained ~10 times lower copy numbers, likely due to viral particles containing ssDNA fragments carried over from the 40% layer.
Figure 3 AAV iodixanol discontinuous gradient purification and purity assessment. (A) Serotype-independent purification of AAVs using a discontinuous iodixanol gradient and ultracentrifugation. The 15% iodixanol layer was underlayered with denser iodixanol solutions to create the step gradient. The 25% and 54% iodixanol layers included phenol red for better visualization. Complete AAVs concentrated in the 40% layer. For elution of the iodixanol gradient, a small hole was drilled into the bottom of the centrifuge tube using a 25 G needle and another 25 G needle was inserted at the top. Fractions of 1 ml for the 54% layer, three of 400 μl for the 40% layer, and 1 ml for the 25% interface were collected. (B) Actual iodixanol step gradient before and after centrifugation. Visible interphase (arrow) between the 40% and 25% layers consists of empty and incomplete AAV2-DJ/8 particles, which may contain fragments of viral ssDNA. (C) Silver-stained protein gel of AAV2-DJ/8 samples at different stages of purification. Box denotes the area of the viral capsid proteins VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa). The purification procedure, using PEG precipitation, chloroform extraction, and aqueous two-phase extraction gradually separated contaminating proteins. The final discontinuous iodixanol gradient ultracentrifugation yielded highly purified AAV particles in fractions 2–4. The viral genome copy number measured with qPCR was high in fractions 2–4 while fraction 5 contained ~10 times lower copy numbers, likely due to viral particles containing ssDNA fragments carried over from the 40% layer.CC BY · PubMed Central
Figure 4 mRNA and protein levels in primary hepatocytes and C2C12 cells infected with AAV2-DJ-shGlrx-Venus. Glrx mRNA levels in primary hepatocytes (A), and C2C12 cells (B) after 6 and 4 days post infection, respectively, with either AAV2-DJ-shControl-mVenus (shControl) or AAV2-DJ-shGlrx-mVenus (shGlrx). Glrx mRNA expression was normalized to Actb mRNA, and the fold-change was compared to shControl (n = 6–7; SD). (C) Glrx and Venus protein levels in C2C12 cells from (B). β-tubulin served as a loading control. shCT: C2C12 cells infected with AAV2-DJ-shControl-mVenus.
Figure 4 mRNA and protein levels in primary hepatocytes and C2C12 cells infected with AAV2-DJ-shGlrx-Venus. Glrx mRNA levels in primary hepatocytes (A), and C2C12 cells (B) after 6 and 4 days post infection, respectively, with either AAV2-DJ-shControl-mVenus (shControl) or AAV2-DJ-shGlrx-mVenus (shGlrx). Glrx mRNA expression was normalized to Actb mRNA, and the fold-change was compared to shControl (n = 6–7; SD). (C) Glrx and Venus protein levels in C2C12 cells from (B). β-tubulin served as a loading control. shCT: C2C12 cells infected with AAV2-DJ-shControl-mVenus.CC BY · PubMed Central
Figure 5 Relative plasma luciferase activity. Relative plasma luciferase activity of AAV2-DJ/8-GLuc (1.5 × 1011 vg/µl) tail vein injected male mice compared to PBS injected controls. Blood was collected from the tail vein at 1, 2, and 3 weeks after injection, and luciferase activity was measured. n = 4 per condition, SEM.
Figure 5 Relative plasma luciferase activity. Relative plasma luciferase activity of AAV2-DJ/8-GLuc (1.5 × 1011 vg/µl) tail vein injected male mice compared to PBS injected controls. Blood was collected from the tail vein at 1, 2, and 3 weeks after injection, and luciferase activity was measured. n = 4 per condition, SEM.CC BY · PubMed Central
Figure 6 mRNA and protein expression of Glrx in mouse liver. (A) Glrx mRNA expression levels in livers of C57BL/6J mice two weeks after administration of AAV2-DJ/8-shControl-mVenus (shControl) or AAV2-DJ/8-shGlrx-mVenus (shGlrx). Gene expression was measured by RT-qPCR and the statistical analysis was performed using the two-tailed Mann-Whitney test. Statistical differences (n = 6–7; P < 0.05; SD) were compared to shControl. (B) Western blot analysis of the liver samples described in (A). The membrane (dotted line indicates the cut) was probed for mVenus and Glrx expression 2 weeks post AAV injection. (C) Liver sections from an AAV2-DJ/8-shGlrx-mVenus injected mouse showed a strong fluorescence signal of mVenus (top row, red color) in cytoplasm and nuclei. Nuclei were counterstained with Hoechst 33342. The mVenus signal was most intense around the triad (T) and faded towards (arrow) the central vein (C), showing zoning across the liver acinus. Neither liver sections of AAV2-DJ/8-shGlrx-mVenus injected mice, incubated with secondary antibody alone (middle row), nor saline-injected mice exhibited a significant mVenus signal at identical exposure time settings. Scale bar denotes 100 µm.
Figure 6 mRNA and protein expression of Glrx in mouse liver. (A) Glrx mRNA expression levels in livers of C57BL/6J mice two weeks after administration of AAV2-DJ/8-shControl-mVenus (shControl) or AAV2-DJ/8-shGlrx-mVenus (shGlrx). Gene expression was measured by RT-qPCR and the statistical analysis was performed using the two-tailed Mann-Whitney test. Statistical differences (n = 6–7; P < 0.05; SD) were compared to shControl. (B) Western blot analysis of the liver samples described in (A). The membrane (dotted line indicates the cut) was probed for mVenus and Glrx expression 2 weeks post AAV injection. (C) Liver sections from an AAV2-DJ/8-shGlrx-mVenus injected mouse showed a strong fluorescence signal of mVenus (top row, red color) in cytoplasm and nuclei. Nuclei were counterstained with Hoechst 33342. The mVenus signal was most intense around the triad (T) and faded towards (arrow) the central vein (C), showing zoning across the liver acinus. Neither liver sections of AAV2-DJ/8-shGlrx-mVenus injected mice, incubated with secondary antibody alone (middle row), nor saline-injected mice exhibited a significant mVenus signal at identical exposure time settings. Scale bar denotes 100 µm.CC BY · PubMed Central
Figure 7 AAV2-6 mVenus transduced skeletal muscle. AAV2-6-shGlrx-mVenus IM injection induced in mVenus protein expression after 2 weeks but inhibition of Glrx expression occurred only after 6 weeks. (A) mVenus and Glrx expression in AAV-injected muscles. Mice were euthanized at 3 and 6 weeks after IM injection. shCT: AAV expressing short hairpin control. shGlrx: AAV expressing short hairpin RNA to Glrx. At 6 weeks, shGlrx inhibited Glrx expression to half of the control. Ponceau staining is shown as protein loading control. (B) Upper panel: The AAV-injected muscle turned yellow by mVenus expression and was swollen (arrow) compared to the non-injected muscle at 3 weeks. Lower panel: After 6 weeks, the muscle size appeared similar to the non-injected muscle. (C) mVenus and Glrx expression in other organs at 4 weeks after AAV IM injection. mVenus only expressed in the injected gastrocnemius muscle (I), but the AVV did not leak into the non-injected muscle (N), heart (H), or liver (L). AAV-shGlrx-injected muscles did not decrease Glrx protein expression at 4 weeks post-injection.
Figure 7 AAV2-6 mVenus transduced skeletal muscle. AAV2-6-shGlrx-mVenus IM injection induced in mVenus protein expression after 2 weeks but inhibition of Glrx expression occurred only after 6 weeks. (A) mVenus and Glrx expression in AAV-injected muscles. Mice were euthanized at 3 and 6 weeks after IM injection. shCT: AAV expressing short hairpin control. shGlrx: AAV expressing short hairpin RNA to Glrx. At 6 weeks, shGlrx inhibited Glrx expression to half of the control. Ponceau staining is shown as protein loading control. (B) Upper panel: The AAV-injected muscle turned yellow by mVenus expression and was swollen (arrow) compared to the non-injected muscle at 3 weeks. Lower panel: After 6 weeks, the muscle size appeared similar to the non-injected muscle. (C) mVenus and Glrx expression in other organs at 4 weeks after AAV IM injection. mVenus only expressed in the injected gastrocnemius muscle (I), but the AVV did not leak into the non-injected muscle (N), heart (H), or liver (L). AAV-shGlrx-injected muscles did not decrease Glrx protein expression at 4 weeks post-injection.CC BY · PubMed Central

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Authors
Kimura, T., Ferran, B., Tsukahara, Y., Shang, Q., Desai, S., Fedoce, A., Pimentel, D. R., Luptak, I., Adachi, T., Ido, Y., Matsui, R., & Bachschmid, M. M.
Affiliations
  • Boston University (US)
  • National Defense Medical College Hospital (JP)
Corresponding author
  • Matsui, Reiko
  • Bachschmid, Markus Michael
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2019-09-19
Volume
9
Issue
1
Pages
13601-13601
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-019-49624-w
PMC
PMC6753157
OpenAlex
W2973390883
Cited by
155
References
70
Citations by year
2026: 18 · 2025: 30 · 2024: 27 · 2023: 27 · 2022: 23 · 2021: 20 · 2020: 10
FWCI
7.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • American Heart Association (15FTF25890062, 16GRNT27660006)
  • School of Medicine, Boston University (1UL1TR001430)
  • National Defense Medical College
  • Ministry of Education, Culture, Sports, Science and Technology
  • Ministry of Defense
  • National Institutes of Health (NIH T32, 15FTF25890062, UL1TR001430)
  • Japan Society for the Promotion of Science (18H02815)
  • National Institute on Aging (R03AG051857)
  • National Heart, Lung, and Blood Institute (T32HL007224, R01HL133013)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK103750)
  • National Center for Advancing Translational Sciences
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Keywords
adeno-associated virus vectors, AAV production, HEK293T cells, polyethylene glycol precipitation, aqueous two-phase extraction, viral titer, in vivo transduction, glutaredoxin 1, shRNA, liver, skeletal muscle, gene therapy
MeSH terms
Proof of Concept Study, Dependovirus, Animals, Cell Line, Genetic Vectors, Humans, Liver, Male, Polyethylenes, Chemical Precipitation, Transduction, Genetic, Down-Regulation, Muscle, Skeletal, Viral Load, RNA, Small Interfering, Glutaredoxins, HEK293 Cells

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