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

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Therapy

Background:

• Current Good Manufacturing Practice (GMP)-grade adeno-associated virus (AAV) vector production relies on a triple-plasmid system requiring transient transfection of mammalian cells.
• Optimizing the ratios of these three plasmids is crucial but complex, impacting production efficiency and purity.

Purpose of the Study:

• To develop a simplified, single-plasmid system for efficient and consistent GMP-grade AAV vector manufacturing.
• To improve AAV production yields, reduce impurities, and enhance the overall cost-effectiveness of the process.

Main Methods:

• Engineered a single plasmid, AAVone, integrating adenovirus helper genes (E2A, E4orf6, VA RNA), AAV packaging genes (rep, cap), and the vector transgene cassette.
• Compared the AAVone system with the traditional triple-plasmid system for AAV production in terms of yield, purity, and efficiency.

Main Results:

• The AAVone system demonstrated a 2- to 4-fold increase in AAV yields with reduced batch-to-batch variation.
• AAVs produced using AAVone showed similar in vitro and in vivo transduction efficiency but with significantly lower DNA impurities and non-functional genomes.
• The system requires less DNA input, maintains favorable full:empty particle ratios, and eliminates the risk of replication-competent AAV contaminants.

Conclusions:

• The AAVone single-plasmid system offers a highly efficient, straightforward, cost-effective, and consistent platform for GMP-grade AAV vector manufacturing.
• This platform simplifies the production process, reduces impurities, and is well-suited for large-scale gene therapy vector production.