A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

Area of Science:

• Molecular Biology
• Gene Therapy Vector Characterization

Background:

• Recombinant adeno-associated virus (rAAV) is a key vector in gene therapy, requiring precise genome titer quantification for clinical use and product analysis.
• Traditional quantitative PCR (qPCR) methods for AAV genome titer determination lack optimal precision.
• Droplet digital PCR (ddPCR) offers high accuracy but faces limitations in availability, throughput, and cost.

Purpose of the Study:

• To develop and validate an improved qPCR method for accurate and precise AAV genome titer determination.
• To address the limitations of traditional qPCR and the accessibility issues of ddPCR in AAV characterization.

Main Methods:

• Optimization of qPCR by utilizing AAV reference material instead of plasmid DNA as the standard.
• Implementation of a "digestion-free" protocol by incorporating 5% Tween 20 in sample and standard preparations.
• Validation of the improved method across various AAV serotypes, purification states, and transgene payloads.

Main Results:

• The optimized qPCR method demonstrated high accuracy, precision, and robustness.
• The new assay simplifies AAV genome titer determination compared to traditional methods.
• The method is suitable for various AAV vectors and can be adapted for high-throughput automation.

Conclusions:

• The developed "digestion-free" qPCR method with AAV reference material offers a significant improvement for accurate AAV genome titer quantification.
• This enhanced assay is readily adoptable by gene therapy researchers and facilitates reliable AAV product characterization.
• The method provides a more accessible and efficient alternative for precise AAV vector analysis.