06/17/2025
By Brandon Hoyle

The Department of Biomedical and Nutritional Sciences in the Zuckerberg College of Health Sciences invites you to attend a doctoral dissertation defense by Brandon Hoyle titled "Analytical Methods to Characterize Adeno-Associated Virus (AAV) Serotypes: Assessing Viral Load, Surface Charge Properties, Post-Translational Modifications, and Their Transduction Efficiency and Impact."

Candidate: Brandon Hoyle
Degree: Doctoral, Pharmaceutical Sciences
Defense Date: Wednesday, June 25, 2025
Time: 10 - 11 a.m.
Location: Dugan Hall, 110B, South Campus
Thesis/Dissertation Title: Analytical Methods to Characterize Adeno-Associated Virus (AAV) Serotypes: Assessing Viral Load, Surface Charge Properties, Post-Translational Modifications, and Their Transduction Efficiency and Impact.

Committee:

  • Dhimiter Bello, Sc.D., M.Sc. (Chair)
  • Jonathan Hill, Ph.D. (industry on site advisor)
  • Soumita Das, Ph.D.
  • Jonghan Kim, Ph.D.

Brief Abstract: 

Adeno-Associated Virus (AAV) vectors are being used in the gene therapy field due to their potential to treat a range of genetic diseases. AAVs are comprised of a capsid protein encapsulating a Deoxyribonucleic Acid (DNA) transgene of interest (GOI). Existing biomanufacturing methods yield AAVs with varying levels of purity, viral load (empties, partially full or full capsids), and surface charge properties. Despite progress, major questions remain regarding the role of empty and partially full capsids, their post-translational modifications, and surface charge, on the AAVs transduction efficiency, and their engagement of the immune system in vivo. In this thesis, a versatile approach was presented for investigating the charge heterogeneity of AAV capsid proteins of a variety of AAV serotypes to enable generation of sufficient depth in AAV characterization to interrogate the interplay of biomanufacturing methods and post-translational modifications on AAV treatment efficacy in vitro. This method employs Imaged Capillary Isoelectric Focusing (icIEF) coupled with native fluorescence imaging detection and has undergone exhaustive validation. The distinctive profiles generated for each AAV serotype serve as valuable indicators for both identity confirmation and stability assessment. It was shown that thermal stress and pH conditions play a role in increasing acidic charged variants over time, affecting the charge heterogeneity of AAVs, which can be serotype specific. These PTMs have biological consequences reflected in the diminished expression of the protein of interest in vitro. The findings of this thesis have relevant implications and translational value and can guide further development of more robust biomanufacturing platforms and AAV characterization techniques for safer and more effective gene therapies.