Homologous Recombination Offers Advantages Over Transposition-Based Systems to Generate Recombinant Baculovirus for Adeno-Associated Viral Vector Production. Biotechnol J 2020;:e2000014.Abstract.
Viral vectors have a great potential for gene delivery, but manufacturing at a pharmaceutical scale is a big challenge for the industry. The baculovirus-insect cell system is one of the most scalable platforms to produce clinical-grade recombinant Adeno-Associated Virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and still suffers technical constraints. Moreover, werecently showed that baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raised concerns about safety for clinical applications due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, weinvestigated a faster and safer method based on homologous recombination (HR). First, weconfirmed the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes. Strikingly, wefound that the exogenous cassette showed increased stability over passages when using the HR system. Finally, wetested these materials to produce rAAV vectors. The baculoviruses originated from both systems lead to high rAAV vector genome yields, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for the manufacturing of rAAV vectors. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products. This article is protected by copyright. All rights reserved.