Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP.

Slides:



Advertisements
Similar presentations
Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector  Wei Huang, Travis McMurphy,
Advertisements

Molecular Therapy - Methods & Clinical Development
Volume 3, Issue 6, Pages (June 2001)
Volume 7, Issue 1, Pages (January 2003)
Volume 20, Issue 2, Pages (February 2012)
Human Gene Therapy: Adeno associated virus (aav)
Volume 13, Issue 4, Pages (April 2006)
Molecular Therapy - Methods & Clinical Development
PHAPI, CAS, and Hsp70 Promote Apoptosome Formation by Preventing Apaf-1 Aggregation and Enhancing Nucleotide Exchange on Apaf-1  Hyun-Eui Kim, Xuejun.
Molecular Therapy - Methods & Clinical Development
Volume 21, Issue 12, Pages (December 2013)
Volume 3, Issue 5, Pages (May 2008)
Molecular Therapy - Methods & Clinical Development
Improved system for helper-dependent adenoviral vector production
Aquaporin 3 Colocates with Phospholipase D2 in Caveolin-Rich Membrane Microdomains and Is Downregulated Upon Keratinocyte Differentiation  Xiangjian Zheng,
Volume 61, Issue 1, Pages S9-S15 (January 2002)
ATP-Dependent Positive Supercoiling of DNA by 13S Condensin: A Biochemical Implication for Chromosome Condensation  Keiji Kimura, Tatsuya Hirano  Cell 
Multiple PKCδ Tyrosine Residues Are Required for PKCδ-Dependent Activation of Involucrin Expression—a Key Role of PKCδ-Y311  Ling Zhu, Chaya Brodie, Sivaprakasam.
Molecular Therapy - Methods & Clinical Development
Volume 3, Issue 5, Pages (May 2008)
Vaccinia virus as a subhelper for AAV replication and packaging
Molecular Therapy - Methods & Clinical Development
Volume 14, Issue 1, Pages (July 2006)
M.Brandon Parrott, Michael A. Barry  Molecular Therapy 
Proteolytic Mapping of the Adeno-associated Virus Capsid
Volume 20, Issue 2, Pages (February 2012)
Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells
A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae  Yang Wu, Liangyu Jiang,
Volume 12, Issue 6, Pages (December 2005)
Volume 2, Issue 3, Pages (September 2000)
Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector  Donna J Palmer, Nathan.
A Robust System for Production of Superabundant VP1 Recombinant AAV Vectors  Qizhao Wang, Zhongren Wu, Junping Zhang, Jenni Firrman, Hongying Wei, Zhengjing.
Molecular Therapy - Methods & Clinical Development
Xiuwu Zhang, Chuan-Yuan Li  Molecular Therapy 
Incorporation of the B18R Gene of Vaccinia Virus Into an Oncolytic Herpes Simplex Virus Improves Antitumor Activity  Xinping Fu, Armando Rivera, Lihua.
Molecular Therapy  Volume 20, (May 2012) DOI: /S (16)
Volume 23, Issue 10, Pages (October 2015)
Volume 26, Issue 4, Pages (April 2018)
Molecular Therapy - Methods & Clinical Development
Thermal Stability as a Determinant of AAV Serotype Identity
Volume 16, Issue 5, Pages (May 2008)
Molecular Therapy - Methods & Clinical Development
Volume 16, Issue 6, Pages (June 2008)
Volume 15, Issue 2, Pages (February 2007)
Md Nasimuzzaman, Danielle Lynn, Johannes CM van der Loo, Punam Malik 
Mathieu Nonnenmacher, Harm van Bakel, Roger J Hajjar, Thomas Weber 
Volume 6, Issue 5, Pages (November 2002)
PiggyBac Transposon-mediated Gene Transfer in Human Cells
Molecular Therapy - Methods & Clinical Development
Volume 18, Issue 3, Pages (March 2010)
Volume 26, Issue 2, Pages (February 2018)
372. Targeted Mutagenesis of Ubiquitin-Binding Lysine Residues on the Adeno- Associated Virus (AAV)2 Capsid Improves Its Transduction Efficiency    Molecular.
Effect of Genome Size on AAV Vector Packaging
Volume 4, Issue 6, Pages (December 2001)
Volume 17, Issue 1, Pages (January 2009)
Volume 20, Issue 3, Pages (March 2012)
Molecular Therapy - Methods & Clinical Development
Volume 24, Issue 4, Pages (April 2016)
Volume 15, Issue 7, Pages (July 2007)
Volume 22, Issue 2, Pages (February 2014)
Wei Ding, Liang N. Zhang, Charles Yeaman, John F. Engelhardt 
Jiamiao Lu, Feijie Zhang, Mark A Kay  Molecular Therapy 
A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect Cells  Richard.
Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes  Zekun Wang, Fang Cheng, John F. Engelhardt,
Proteolytic Mapping of the Adeno-associated Virus Capsid
Zhijian Wu, Aravind Asokan, R. Jude Samulski  Molecular Therapy 
High-level production of replication-defective human immunodeficiency type 1 virus vector particles using helper-dependent adenovirus vectors  Yani Hu,
Biao Dong, Hiroyuki Nakai, Weidong Xiao  Molecular Therapy 
Volume 9, Issue 4, Pages (April 2004)
Molecular Therapy - Methods & Clinical Development
Presentation transcript:

Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector  Joshua C Grieger, Stephen M Soltys, Richard Jude Samulski  Molecular Therapy  Volume 24, Issue 2, Pages 287-297 (February 2016) DOI: 10.1038/mt.2015.187 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Optimization of triple transfection plasmid ratios and transfection cocktail volumes. (a) Varying plasmid ratios of XX680, pXR2 helper, and TReGFP plasmid were explored to determine optimal plasmid ratio for rAAV vector production. (b) Varying transfection cocktail volumes were tested to determine optimal transfection conditions with PEI Max to DNA ratio of 2:1 and an incubation time of 10–14 minutes. Percent volume of transfection cocktail is in reference to percent of final cell culture volume. These parameters were optimized using the SFM4Transfx-293 media (HyClone). Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Cell density transfection optimization experiments and impact of cell culture age on vector production. (a) 1 × 106 and 2 × 106 viable cells/ml were transfected with 1, 1.5, and 2 μg of plasmid DNA (2:1.5:1 XX680:pXR:TR plasmid). (b) Low, medium, and high passage cells were transfected using optimized parameters to determine if cell culture age impacted transfection efficiency and rAAV production. Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Characterization of elution peak fractions of adeno-associated virus (AAV) after ion exchange chromatography. (a) Silver stain and negative stain transmission electron microscopy (TEM) images of elution peak fractions of AAV using nonoptimized elution conditions for rAAV. Arrows represent the major contaminant (Ferritin) on the silver stain gel and the TEM image. (b) Silver stain and negative stain TEM images of elution peak fractions of AAV using optimized elution conditions for rAAV. Silver stain and TEM images illustrate physical separation of the virions and the ferritin contaminants. Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Production and purification of single-stranded and self-complementary recombinant adeno-associated virus (rAAV) serotypes 1–6, 8, and 9 using optimized production and purification conditions. (a) Silver stain image of single-stranded serotypes 1–6, 8, and 9 postpurification. (b) Southern blot of alkaline agarose gel of self-complementary rAAV1–6, 8, and 9. (c) Negative stain transmission electron microscopy (TEM) images of rAAV serotype 1 (left image), serotype 2 (middle image), and empty rAAV2 capsids (right image). TEM images were also taken of serotypes 3–6, 8, and 9 (images not shown). Empty particles (indicated by arrows) can be distinguished based on the electron dense center within the particle. Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Continuous production and harvesting of recombinant adeno-associated virus (rAAV) 8 and rAAV9 from the suspension HEK293 culture medium. Total yield represents all media time-points and 120-hour cell pellet rAAV yields combined. Forty-eight hour cell pellet control represents the yield from a standard harvest at 48 hours post-transfection as described in this report. Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Depiction of recombinant adeno-associated virus (rAAV) manufacturing technology. (1) Suspension HEK293 cells in the WAVE bioreactor 20/50EHT are transfected. (2) Approximately 48 hours post-transfection, cells are harvested into centrifuge bottles and pelleted at low-speed centrifugation. (3) Cell pellets are resuspended and then lysed using a sonicator. (4) Lysate is incubated with Benzonase for 45 minutes at 37 °C. (5) Lysate is clarified using low-speed centrifugation. (6) Clarified lysate is loaded onto iodixanol gradient and ultracentrifuged. (7) rAAV collected from the 40%/60% iodixanol interface are loaded onto AKTA FPLC and purified using anion exchange chromatography. (8) rAAV chromatography peak fractions are dialyzed into final formulation buffer. (9) Sterile filtration through 0.2 μm filter. Molecular Therapy 2016 24, 287-297DOI: (10.1038/mt.2015.187) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions