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Related Experiment Video

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Characterizing Adeno-Associated Virus Capsids with Both Denaturing and Intact Analysis Methods.

Jack P Ryan1, Marius M Kostelic2, Chih-Chieh Hsieh2

  • 1University of North Carolina, Department of Chemistry, Chapel Hill, North Carolina 27599, United States.

Journal of the American Society for Mass Spectrometry
|November 27, 2023
PubMed
Summary

New mass spectrometry methods accurately characterize adeno-associated virus (AAV) capsids and viral proteins (VPs). This improves quality control for gene therapy applications by distinguishing empty and filled capsids and analyzing VP sequences.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Gene Therapy

Background:

  • Adeno-associated viruses (AAVs) are crucial gene delivery vectors for treating human diseases.
  • Diverse AAV serotypes target specific cells and tissues, necessitating precise composition analysis.
  • Current analytical methods struggle with AAV subunit selectivity, intact capsid assessment, and empty/filled capsid differentiation.

Purpose of the Study:

  • To develop and apply advanced analytical workflows for comprehensive AAV capsid characterization.
  • To improve quality control for gene therapy by accurately assessing capsid composition and viral protein (VP) subunits.
  • To differentiate between empty and filled AAV capsids and analyze VP sequences and modifications.

Main Methods:

  • Utilized selective temporal overview of resonant ion (STORI)-based charge detection-mass spectrometry (CD-MS) for intact capsid analysis.
  • Employed liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) for denatured capsid subunit analysis.
  • Applied this combined approach to AAV serotypes 2, 6, and 8.

Main Results:

  • Successfully characterized intact empty and filled capsid ratios for AAV2, AAV6, and AAV8.
  • Analyzed distinct viral protein (VP) sequences and post-translational modifications within the AAV capsids.
  • Demonstrated the capability of the combined analytical workflows to overcome limitations of existing methods.

Conclusions:

  • The integrated STORI-CD-MS and LC-IMS-MS approach provides enhanced selectivity and accuracy for AAV characterization.
  • This methodology is vital for ensuring the quality and efficacy of AAV-based gene therapies.
  • Accurate analysis of AAV capsid composition is critical for precise gene delivery and therapeutic outcomes.