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ORIGINAL RESEARCH article

Front. Bioeng. Biotechnol.

Sec. Bioprocess Engineering

Impact of multiplicity of infection and baculovirus co-infection ratio on recombinant adeno-associated virus vector production in insect cells

Provisionally accepted
Mels SchramaMels Schrama1*Linda A. De JongLinda A. De Jong2Stefan KintStefan Kint1Pranav PuriPranav Puri3Femke HoeksemaFemke Hoeksema3Gorben P. PijlmanGorben P. Pijlman2Monique M. van OersMonique M. van Oers2Rene H. WijffelsRene H. Wijffels1Dirk E. MartensDirk E. Martens1
  • 1Bioprocess Engineering, Wageningen University and Research, Wageningen, Netherlands
  • 2Laboratory of Virology, Wageningen University and Research, Wageningen, Netherlands
  • 3VectorY Therapeutics, Amsterdam, Netherlands

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Recombinant adeno-associated virus vectors (rAAVs) play an important role in gene therapy for the effective delivery of therapeutic genes into target cells. The Baculovirus Expression Vector System (BEVS) has gained significant attention for its versatility and scalability as an rAAV production platform. The existing Dual-Bac system uses two separate baculovirus constructs (Bac-GOI-ITR and Bac-Rep-Cap), each carrying essential genetic elements for rAAV production in insect cells. This study investigated how two infection parameters of the Dual-Bac system, the total Multiplicity of Infection (MOI) and the baculovirus co-infection ratio, influence rAAV production efficiency. Different budded baculovirus (BV) concentrations were used to explore the effects on assembled rAAV capsid and encapsidated transgenic genome yields. An excess of Bac-Rep-Cap in synchronous co-infection produced high-quality rAAVs, whereas increasing the ratio of Bac-GOI-ITR to Bac-Rep-Cap resulted in more empty rAAV capsids. The optimal BV ratio varied with the total MOI used for co-infection, and when applying a BV ratio of one, MOI variations had a minimal impact on rAAV quality. These findings highlight the importance of optimizing the MOI and BV ratio to enhance rAAV yield and quality, contributing to more robust gene therapy production.

Keywords: recombinant AAV, Baculovirus expression vector system, Co-infection, Gene Therapy, Multiplicity of Infection

Received: 04 Aug 2025; Accepted: 03 Nov 2025.

Copyright: © 2025 Schrama, De Jong, Kint, Puri, Hoeksema, Pijlman, van Oers, Wijffels and Martens. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Mels Schrama, mels.schrama@wur.nl

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Supplementary Material