Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Adeno-associated viruses (AAVs) are crucial gene therapy vectors.
• Understanding AAV charge heterogeneity is vital for vector quality control and efficacy.
• Existing methods often lack the resolution to dissect multiple contributing factors simultaneously.

Purpose of the Study:

• To develop and validate a comprehensive workflow for analyzing AAV charge heterogeneity.
• To identify and quantify the contributions of capsid protein composition, post-translational modifications, and packaged DNA to AAV charge variants.
• To provide a deeper understanding of the factors driving AAV capsid heterogeneity.

Main Methods:

• Imaged capillary isoelectric focusing (icIEF)-based fractionation of intact AAV9 capsids.
• Analytical icIEF and charge detection mass spectrometry (CD-MS) for capsid integrity assessment.
• Capillary electrophoresis (CE) immunoassays for capsid protein ratio and deamidation analysis.
• CE-immunoassay and high-resolution CD-MS for ssDNA content analysis.

Main Results:

• Successfully fractionated AAV9 capsids based on isoelectric points (pIs).
• Quantified capsid protein ratios and deamidation levels across different fractions.
• Characterized the ssDNA content within each capsid fraction.
• Demonstrated the distinct contributions of these factors to overall AAV charge heterogeneity.

Conclusions:

• The described workflow effectively dissects multiple contributors to AAV charge heterogeneity from a single sample.
• This methodology enhances the characterization of AAV vector quality.
• Provides critical insights for optimizing AAV production and therapeutic applications.