AAVone: A cost-effective, single-plasmid solution for efficient AAV production with reduced DNA impurities

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy Manufacturing

Background:

• Current Good Manufacturing Practice (GMP)-grade adeno-associated virus (AAV) vector production relies on a multi-plasmid transfection system.
• This traditional triple-plasmid approach necessitates complex optimization of plasmid ratios and can lead to batch variability and impurities.

Purpose of the Study:

• To develop an improved, single-plasmid system for efficient and consistent AAV vector manufacturing.
• To simplify the production process while maintaining or enhancing vector quality and yield.

Main Methods:

• Development of an "all-in-one" single plasmid (AAVone) integrating adenovirus helper genes, AAV packaging genes (rep and cap), and the vector transgene cassette.
• Comparison of the AAVone system with the traditional triple-plasmid system in terms of yield, purity, transduction efficiency, and safety.

Main Results:

• The AAVone system demonstrated a 2- to 4-fold increase in AAV yields compared to the triple-plasmid system.
• AAV vectors produced using AAVone exhibited reduced DNA impurities, fewer non-functional genomes, and no risk of replication-competent AAV generation.
• The system showed low batch-to-batch variation and simplified the overall production workflow.

Conclusions:

• The AAVone single-plasmid system offers a more efficient, cost-effective, and consistent method for producing GMP-grade AAV vectors.
• This platform is highly suitable for large-scale manufacturing, potentially accelerating gene therapy development and delivery.