Date of Award

2022

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Chemical Engineering (PhD)

Administrative Home Department

Department of Chemical Engineering

Advisor 1

Caryn L. Heldt

Committee Member 1

Tony N. Rogers

Committee Member 2

Lei Pan

Committee Member 3

Jeremy Goldman

Abstract

Gene therapy is a therapeutic intervention designed to correct single gene disorders. AAV has been identified as a suitable vector for delivering therapeutic genes. However, the use of AAV has been hampered by manufacturing challenges inclusive of low virus recovery, and the presence of AAV without the gene of interest (empty capsids). To solve these problems, we characterized the charge and hydrophobicity of AAV, and surrogate viruses using chemical force microscopy (CFM). CFM uses a modified atomic force microscope (AFM) probe to measure the adhesion force between a virus particle and a functional chemistry.The virus particles to be measured are covalently bound on a gold coated glass slide.

CFM revealed the hydrophobic interaction of was used to characterize the hydrophobicity of non-enveloped porcine parvovirus (PPV) enveloped bovine viral diarrhea virus (BVDV) increased with rising sodium chloride concentration but not non-enveloped porcine parvovirus (PPV) while the inclusion of polyethylene glycol (PEG) improved the hydrophobic interaction of PPV and BVDV. Ethanol enhanced PPV hydrophobic interaction but not for BVDV. Hydrophobic dye absorption to PPV and BVDV correlated to the CFM results when ethanol was added. This is the first evaluation of virus hydrophobicity using CFM.

The charge and hydrophobicity of AAV empty and full capsids assessed by the CFM has been utilized to interpret previously unknown interactions of the anion exchange (AEX) chromatogram. Although, AEX is designed to be solely dependent on electrostatic, hydrophobic interactions seemed to prevail for AAV at lower conductivity levels. CFM may be used in the future to optimize buffers, develop and choose AEX ligands.

The isoelectric point (IEP) of SARS-CoV-2 was first experimentally established using CFM. Understanding viral transmission and adherence requires deciphering the structural, surface, and functional features of each viral protein. Viral RNA sequence modeling and protein crystals has been insufficient in determining the IEP. Thus, we experimentally measured the IEP of SARS-CoV-2 and compared it to variations of interest (VOIs).

With the novel CFM approach presented in this study, viral surfaces can be appropriately characterized, and a predictive model can be designed for selecting the solution conditions for virus purification.

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