Quantitative and Digital Droplet-Based AAV Genome Titration.

Area of Science:

• Molecular Biology
• Gene Therapy
• Biotechnology

Background:

• Adeno-associated viral vectors (AAV) are key for gene transfer in research and clinical applications.
• Accurate quantification of AAV vector concentration is essential for reproducible experimental results and therapeutic efficacy.
• Existing quantification methods may have limitations in precision and comparability.

Purpose of the Study:

• To provide detailed protocols for quantifying DNase-I protected AAV vector genomes.
• To compare the performance of traditional quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for AAV quantification.
• To highlight the role of surfactants in improving assay accuracy, especially at low vector concentrations.

Main Methods:

• Development and comparison of protocols for AAV genome quantification using TaqMan™ real-time quantitative PCR (qPCR).
• Implementation and validation of droplet digital PCR (ddPCR) for AAV genome quantification.
• Assessment of surfactant (Pluronic® F-68) impact on DNA and AAV adherence during titration.

Main Results:

• Side-by-side comparative data generated using both qPCR and ddPCR methods.
• Demonstration of reduced variability and increased comparability between AAV studies using the presented protocols.
• Evidence supporting the importance of surfactant use for accurate titration, particularly at low AAV concentrations.

Conclusions:

• The developed protocols enable accurate and reproducible quantification of AAV vector genomes.
• Both qPCR and ddPCR are viable methods for AAV quantification, with ddPCR offering potential advantages in precision.
• Optimized titration protocols, including surfactant use, enhance the reliability of AAV vector concentration measurements for gene therapy applications.