Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus.

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Recombinant adeno-associated virus (rAAV) vectors are crucial for gene therapy.
• Current Good Manufacturing Practices (GMP) production often uses a three-plasmid transfection system.
• A two-plasmid system offers practical advantages but is less frequently employed for GMP production.

Purpose of the Study:

• To evaluate the utility of a two-plasmid cotransfection system for GMP production of rAAV vectors.
• To compare the performance of the two-plasmid system against the traditional three-plasmid system.
• To assess the impact of the packaging system on vector yield, purity, and in vivo bioactivity.

Main Methods:

• Generated GMP-compatible two-plasmid and three-plasmid transfection systems.
• Packaged reporter genes and various therapeutic cargos using multiple AAV capsid variants.
• Performed side-by-side in vivo comparisons in mice via intravenous and intramuscular injections.
• Evaluated vector expression, transduction efficiency in muscle and liver, and cargo bioactivity.

Main Results:

• Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods across all tested capsid variants.
• In vivo studies demonstrated equivalent expression and transduction in muscle and liver tissues.
• Packaged cargos using the two-plasmid system exhibited bioactivity equivalent to those from the triple transfection system.

Conclusions:

• The two-plasmid cotransfection system is a viable and effective alternative for GMP production of AAV vectors.
• This system demonstrates utility for midrange (1E12-1E16) GMP-compatible packaging across several AAV capsids.
• The findings support the broader adoption of the two-plasmid system in biopharmaceutical manufacturing.