Orthogonal approaches to AAV vector characterization: Validating quantitative TEM for partially filled particles.

Area of Science:

• Gene Therapy
• Virology
• Biotechnology

Background:

• Adeno-associated viruses (AAVs) are key vectors in gene therapy.
• Empty or partially filled capsids in AAV preparations reduce therapeutic efficacy.
• Accurate characterization of AAV vector content is essential for quality control.

Purpose of the Study:

• To present and validate quantitative electron microscopy (QuTEM) for AAV capsid characterization.
• To compare QuTEM with analytical ultracentrifugation (AUC), mass photometry (MP), and SEC-HPLC.
• To assess the effectiveness of these methods in evaluating AAV vector content.

Main Methods:

• Quantitative electron microscopy (QuTEM) to distinguish AAV capsids by internal density.
• Comparison of QuTEM with AUC, MP, and SEC-HPLC.
• Production and evaluation of self-complementary (scAAV) and single-stranded (ssAAV) AAV vectors.

Main Results:

• QuTEM reliably quantified AAV populations with high concordance to MP and AUC data.
• QuTEM provided superior granularity by visualizing native viral capsids.
• The study evaluated encapsidation efficiency for scAAV and ssAAV vectors.

Conclusions:

• QuTEM is a robust and potentially gold-standard method for AAV characterization.
• Integrated analytical approaches are vital for optimizing AAV production and ensuring gene therapy consistency.
• Accurate AAV capsid quantification is critical for therapeutic efficacy.