An Efficient TetR/TetO-Integrated Packaging System for Fowl Adenovirus 4 Vector Carrying Toxic Transgene.

Area of Science:

• Virology
• Gene Therapy
• Molecular Biology

Background:

• Adenoviral vectors are crucial for gene therapy and vaccines, but pre-existing immunity to human adenovirus type 5 necessitates alternatives.
• Fowl adenovirus 4 (FAdV-4) based vectors, modified for enhanced transduction efficiency in human cells, show promise.
• Producing FAdV-4 vectors with transgenes toxic to viral replication requires specialized packaging systems.

Purpose of the Study:

• To engineer a modified packaging system for efficient production of Fowl adenovirus 4 (FAdV-4) vectors.
• To enable the propagation of FAdV-4 vectors carrying transgenes that are toxic to viral replication.
• To enhance the yield of recombinant FAdV-4 vectors in packaging cells.

Main Methods:

• Developed a colony-competent chicken cell line (LMH-C3532) by adapting cells with conditioned medium.
• Established a tetracycline repressor (tetR)-integrated cell line (LMH-tetR24) using lentiviral transduction.
• Constructed adenoviral plasmids with a tetracycline operator (tetO)-controlled promoter for transgene expression (GFP and HA).

Main Results:

• Successfully rescued, amplified, and purified recombinant FAdV4-CF1K vectors encoding GFP (FAdV4-CF1K-CtG) and HA (FAdV4-CF1K-CtHA).
• The tetR-integrated LMH-tetR24 cells showed significantly higher progeny virus yields (4-10 times for GFP, 3-4 orders of magnitude for HA) compared to control cells.
• Overexpression of the HA transgene in packaging cells negatively impacted FAdV4-CF1K-CtHA replication.

Conclusions:

• A novel FAdV-4 packaging system was established by integrating tetR into packaging cells and TetO into the adenoviral plasmid.
• This system facilitates routine packaging of FAdV-4 vectors.
• The developed system is particularly effective for propagating FAdV-4 vectors carrying toxic transgenes.