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Updated: Jun 4, 2026
Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System
Published on: January 13, 2022
Plug-in hybrid baculovirus expression vector for high-yield recombinant adeno-associated virus gene therapy vector
Linda A de Jong1, Jelmer Willems2, Adriaan H de Wilde2
1Laboratory of Virology, Wageningen University & Research, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands.
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In vivo gene therapy is rapidly advancing to treat patients with a wide range of genetic disorders. Recombinant adeno-associated virus (rAAV) vectors provide long-term gene expression, low immunogenicity, and adaptable tissue tropism. Production of rAAV in insect cells with the Baculovirus Expression Vector System currently depends on infection with multiple baculoviruses encoding AAV genes Rep, Cap, and the therapeutic gene. In this research article, we present simplified rAAV production using a single baculovirus vector with improved genetic stability, called BAC6Rep, with AAV Rep locked into the baculovirus genome. Robust AAV Rep and Cap expression was maintained upon serial passaging, and high rAAV yields (up to 3.5e11 genome copies/ml) were obtained at high and low multiplicities of infection. Next-generation sequencing revealed a significantly improved genetic stability of BAC6Rep, which can now be used as a plug-in hybrid vector for flexible insertion of different AAV Cap and gene of interest tailored to the therapeutic target.
