Structural characterization and epitope mapping of the AAVX affinity purification ligand

Area of Science:

• Biochemistry
• Structural Biology
• Gene Therapy

Background:

• Adeno-associated virus (AAV) vectors are crucial for human gene therapies, necessitating high-purity preparations.
• Scalable affinity chromatography, using resins like POROS CaptureSelect AAVX, is vital for downstream processing of AAV vectors.
• The AAVX ligand, a camelid single-domain antibody, captures diverse recombinant AAV capsids.

Purpose of the Study:

• To determine the structural basis of AAVX ligand binding to the AAV8 capsid.
• To elucidate the epitope recognized by the AAVX ligand at high resolution.
• To guide future AAV capsid engineering for improved purification.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to resolve the AAV8 capsid-AAVX ligand complex.
• High-resolution structural analysis was performed at 2.3 Ångström resolution.
• Comparative analysis with previously characterized ligands (e.g., AVB) was conducted.

Main Results:

• The AAVX ligand binds to the AAV8 capsid near the 5-fold axis.
• The binding interaction does not conform to the capsid's icosahedral symmetry.
• Cross-reactivity across different AAV capsids is mediated by interactions with conserved DE and HI loop peptide backbones.

Conclusions:

• Understanding the AAVX epitope provides critical structural information for AAV vector purification.
• The findings facilitate the rational design of novel AAV capsids with enhanced affinity for antibody-based purification.
• This research supports the development of more efficient and reliable gene therapy manufacturing processes.