Stronger together: Analytical techniques for recombinant adeno associated virus

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy

Background:

• Recombinant adeno-associated virus (rAAV) vectors are increasingly used for gene therapy, evidenced by recent FDA approvals.
• High production and therapy costs necessitate improvements in manufacturing and quality control (QC) for rAAVs.
• Accurate analysis of encapsidated DNA is crucial for rAAV quality and efficacy.

Purpose of the Study:

• To evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two capillary electrophoresis (CE) modes for analyzing DNA within rAAV vectors.
• To assess the suitability of these methods for characterizing viral genome content and determining genome titer.
• To establish a comprehensive analytical strategy for robust rAAV quality control.

Main Methods:

• Transmission electron microscopy (TEM) for structural visualization.
• Analytical ultracentrifugation (AUC) for size and homogeneity assessment.
• Capillary gel electrophoresis (CGE) for viral genome sizing.
• Capillary zone electrophoresis (CZE) for simplified genome titer estimation.

Main Results:

• rAAV vectors contain complex DNA mixtures, including fragments smaller and larger than the target transgene.
• CGE offers insights into rAAV filling but is not suitable for absolute titer determination due to lack of established calibration.
• A simplified CZE method with minimal preparation and rapid run times (<5 min) is proposed for genome titer estimation.
• Each analytical technique alone can yield ambiguous results; combined use is essential for a clear view of rAAV interior DNA.

Conclusions:

• A multi-analytical approach combining TEM, AUC, CGE, and CZE is necessary for comprehensive characterization of rAAV encapsidated DNA.
• This integrated strategy enhances the accuracy and reliability of rAAV quality control, addressing current manufacturing challenges.
• Simultaneous application of diverse methods provides a clear view of rAAV interior composition, crucial for therapeutic development and application.