Structural and chemical changes between empty and full AAV capsids - Project Summary
AAV is a popular gene therapy vector because it has large tissue tropism and low toxicity. However, AAV is
inefficient at packaging its genome, and this leaves many empty or partially full capsids that need to be
removed in manufacturing. Empty capsids can increase the immunogenicity and toxicity of the AAV, especially
when high doses are required. This project will take a structural biology approach to understand how the
presence of the genome in AAV affects the capsid quaternary structure and the resulting effects on capsid
surface chemistry and structural integrity. This will provide better methods to remove empty capsids or reduce
the production of empty capsids, thus providing a safer AAV product for patients. Molecular changes induced
by AAV genome packaging will be determined and integrated with changes in viral conformational dynamics
and physicochemical properties. This novel integration of single particle force measurements using an atomic
force microscopy (AFM) with surface residue charge distributions from amide hydrogen-deuterium exchange
(HDXMS) and native mass spectrometry (MS) will lead to insight into genome interaction with AAV capsids and
how the presence of the genome changes the capsid structure, chemistry, and integrity. An unprecedented
view into how the structure of a viral capsid reacts to different manufacturing conditions and cellular trafficking
conditions that occur during AAV production will be developed by completing the following aims:
Aim 1: Ascertain the difference in the charge and hydrophobicity of AAV capsids. AAV2 and AAV8 will be used
as model systems with three different genome sizes. Chemical force microscopy (CFM) a specialized AFM
technique, will measure the changes in charge and hydrophobicity of AAV capsids under relevant
manufacturing and cellular trafficking conditions.
Aim 2: Identify the contributions of capsid protein-DNA and protein-protein interactions on AAV viral particle
dynamics. Comparison of empty, partially full, and full AAV will reveal contributions of DNA on intrinsic
dynamics using HDXMS and native MS. Further, these measurements will also map interaction interfaces of
AAV capsid with encapsulated DNA in full and partially full AAV.
Aim 3: Determine the physical rigidity and brittleness difference between AAV capsids. Nanoindentation, an
AFM technique, will be used to determine the effects of DNA on AAV capsid strength.
Upon completion of this work, a data driven hypothesis on how AAV interacts with its genome and how the
capsid structure changes with different genome sizes will be developed. The effect of solution conditions,
which vary greatly during the virus life cycle and manufacturing cycle, will be elucidated. Descriptions of DNA
packaging in AAV and the structural changes that occur due to DNA packaging will be completed. This
information will improve production, quality control, and safety of AAV and bring more lifesaving AAV therapies
to market.
