An Improved Method for Determining the Infection Titer of Replication-Competent Adeno-Associated Virus

Area of Science:

• Gene Therapy
• Virology
• Molecular Biology

Background:

• Recombinant adeno-associated virus (rAAV) is a key vector in gene therapy.
• Manufacturing can lead to replication-competent AAV (rcAAV) contamination, posing safety risks.
• Accurate quantification of rcAAV is crucial for assay reliability and gene therapy safety.

Purpose of the Study:

• To develop an optimized Tissue Culture Infectious Dose 50 (TCID50) assay for precise quantification of infectious rcAAV particles.
• To improve the reliability and reduce variability in rcAAV titer determination.
• To establish a standardized method for calibrating rcAAV reference materials.

Main Methods:

• Developed a tailored TCID50 assay for rep2-based rcAAV.
• Optimized viral infection conditions, qPCR systems, and standard curve preparation.
• Utilized serial dilutions of rcAAV in cell lysate for standard curve generation, aligning with test sample matrices.

Main Results:

• The optimized standard curve showed excellent linearity (R² > 0.99) and sensitivity (LOQ ≈ 38 copies).
• Achieved high reproducibility, enabling robust endpoint qPCR analysis.
• Significantly improved TCID50 assay precision with an inter-assay coefficient of variation (CV) of 11.4%.

Conclusions:

• The refined TCID50 assay provides a reliable method for quantifying infectious rcAAV titers.
• This assay enables accurate calibration of rcAAV reference standard materials.
• Standardization of rcAAV testing is facilitated, enhancing gene therapy product safety.