AAV Production Using Baculovirus Expression Vector System

  • Quentin Sandro
  • Karima Relizani
  • Rachid BenchaouirEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1937)


Gene transfer and gene therapy are powerful approaches for many biological research applications and promising avenues for the treatment of many genetic or cancer diseases. The most efficient gene transfer tools are currently derived from viruses. Among them, the recombinant adeno-associated viruses (AAVs) are vectors of choice for many fundamental and therapeutic applications. The increasing number of clinical trials involving AAVs demonstrates the need to implement production and purification processes to meet the quantitative and qualitative demands of regulatory agencies for the use of these vectors in clinical trials. In this context, the rise of production levels on an industrial scale appeared essential. The introduction, in 2002, of an AAV process using a baculovirus expression vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in expanding the AAV production in insect cells has been to switch the process to bioreactor systems, which are the ideal equipment for scaling up. We describe here a method for producing AAV vectors using the BEVS which can be easily used by research laboratories wishing to overcome the difficulties associated with the scaling up of production levels. The method provides sufficient quantities of AAV vectors to initiate preclinical projects in large animal models or for research projects where a single batch of vectors will consolidate the repeatability and reproducibility of in vitro and especially in vivo experimental approaches.

Key words

AAV vectors Baculovirus Production Purification Upstream process Downstream process 


  1. 1.
    Gaudet D, Methot J, Dery S et al (2013) Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial. Gene Ther 20:361–369CrossRefGoogle Scholar
  2. 2.
    Grimm D, Kern A, Rittner K et al (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 9:2745–2760CrossRefGoogle Scholar
  3. 3.
    Salvetti A, Oreve S, Chadeuf G et al (1998) Factors influencing recombinant adeno-associated virus production. Hum Gene Ther 9:695–706CrossRefGoogle Scholar
  4. 4.
    Xiao X, Li J, Samulski RJ (1998) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72:2224–2232PubMedPubMedCentralGoogle Scholar
  5. 5.
    Dias Florencio G, Precigout G, Beley C et al (2015) Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors. Mol Ther Methods Clin Dev 2:15024CrossRefGoogle Scholar
  6. 6.
    Chahal PS, Schulze E, Tran R et al (2014) Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery. J Virol Methods 196:163–173CrossRefGoogle Scholar
  7. 7.
    Martin J, Frederick A, Luo Y et al (2013) Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production. Hum Gene Ther Methods 24:253–269CrossRefGoogle Scholar
  8. 8.
    Ye GJ, Scotti MM, Thomas DL et al (2014) Herpes simplex virus clearance during purification of a recombinant adeno-associated virus serotype 1 vector. Hum Gene Ther Clin Dev 25:212–217CrossRefGoogle Scholar
  9. 9.
    Urabe M, Ding C, Kotin RM (2002) Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 13:1935–1943CrossRefGoogle Scholar
  10. 10.
    Wasilko DJ, Lee SE, Stutzman-Engwall KJ et al (2009) The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. Protein Expr Purif 65:122–132CrossRefGoogle Scholar
  11. 11.
    Cecchini S, Virag T, Kotin RM (2011) Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures. Hum Gene Ther 22:1021–1030CrossRefGoogle Scholar
  12. 12.
    Buclez PO, Dias Florencio G, Relizani K et al (2016) Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system. Mol Ther Methods Clin Dev 3:16035CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Quentin Sandro
    • 1
  • Karima Relizani
    • 1
    • 3
  • Rachid Benchaouir
    • 1
    • 2
    • 3
    Email author
  1. 1.University of Versailles Saint-Quentin en YvelinesMontigny-le-BretonneuxFrance
  2. 2.Centre Scientifique de MonacoMonacoMonaco
  3. 3.SQY Therapeutics SARLNoisy-le-RoiFrance

Personalised recommendations