Development of cell-based assay for detecting replication-competent adeno-associated virus by qPCR

Area of Science:

• Gene therapy vector manufacturing
• Molecular biology
• Virology

Background:

• Recombinant adeno-associated virus (AAV) is a key vector for gene therapy.
• AAV manufacturing via triple-transfection can potentially generate replication-competent AAV (rcAAV) contaminants.
• Detection of rcAAV is critical for product safety and regulatory compliance due to potential adverse effects.

Purpose of the Study:

• To develop a sensitive, cell-based assay for detecting rcAAV in AAV preparations.
• To establish a reliable method for quality control in gene therapy vector manufacturing.
• To provide a means for improving process development and ensuring product safety.

Main Methods:

• Development of a cell-based assay using HEK293 cells.
• Transduction of cells with AAV8 in the presence of helper adenovirus 5 over three rounds.
• Quantitative PCR (qPCR) using the rep2 gene as a marker for rcAAV detection.

Main Results:

• An optimized 2 mL scale assay was established.
• The assay minimizes false-positive results.
• A reportable result sensitivity of 1 rcAAV per 10^6 vector genomes was achieved.

Conclusions:

• The developed assay is sensitive and specific for detecting rcAAV contaminants.
• This method enhances the safety and efficacy of gene therapy products.
• The assay approach is adaptable to other AAV serotypes, supporting broader application in process development.