Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

Area of Science:

• Biotechnology
• Gene Therapy
• Virology

Background:

• Adenovirus vectors (AdVs) are promising for gene therapy but safety concerns exist with replication-competent AdVs (RC-AdVs).
• Single-cycle AdVs (SC-AdVs) with protease gene deletions (PS) offer enhanced safety by preventing viral particle assembly and shedding.
• Existing complementary cell lines for SC-AdV production are suboptimal and require fetal bovine serum (FBS).

Purpose of the Study:

• To develop and characterize stable cell lines for improved production of protease-deleted SC-AdVs.
• To optimize SC-AdV production in both adherent and suspension cultures, eliminating the need for FBS.
• To assess the scalability of SC-AdV production using the developed cell lines.

Main Methods:

• Generation of stable cell pools and subclones expressing the protease deletion (PS) gene in adherent and suspension cells.
• Optimization of culture conditions including harvesting time, media, and inducer concentration for suspension cell cultures.
• Production and titration of SC-AdV in optimized cell lines at various scales.

Main Results:

• The best adherent cell pool achieved over 3400 infectious viral particles per cell for SC-AdV generation.
• The best suspension subclone, cultured without FBS, yielded over 4000 infectious viral particles per cell.
• Elimination of FBS requirement and successful scale-up production of eight lots of SC-AdV from 2 to 8.2 L.

Conclusions:

• Developed stable adherent and suspension cell lines significantly improve SC-AdV titers.
• Elimination of FBS simplifies scale-up and reduces production costs for SC-AdV therapies.
• Virus backbone and transgene influence viral and infectious particle titers in SC-AdV production.