Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery Vectors.

Area of Science:

• Molecular Biology
• Virology
• Gene Therapy

Background:

• Adeno-associated virus (AAV)-based vectors show promise for rare disease gene therapy.
• Neutralizing antibodies against AAV capsids pose a significant barrier to widespread therapeutic use.
• Understanding and evading immune responses is crucial for advancing AAV gene therapy.

Purpose of the Study:

• To map the neutralizing epitope of a novel monoclonal antibody against AAV9.
• To engineer AAV9 capsids to evade antibody neutralization and immune responses.
• To investigate the role of identified capsid residues in AAV9 tropism and immune evasion.

Main Methods:

• Isolation and characterization of a high-titer neutralizing monoclonal antibody (PAV9.1) against AAV9.
• Cryo-electron microscopy to determine the structure of the antibody-AAV9 complex and map the epitope.
• Site-directed mutagenesis of AAV9 capsid residues within the mapped epitope.
• Assessment of antibody binding, neutralization, and vector tropism in vitro and in vivo.
• Evaluation of mutant vector evasion of polyclonal neutralizing antibodies from various species.

Main Results:

• A neutralizing monoclonal antibody (PAV9.1) against AAV9 was generated and characterized.
• The epitope was mapped to specific residues (496-NNN-498 and 588-QAQAQT-592) on the AAV9 capsid.
• Mutations within the epitope significantly reduced PAV9.1 binding and neutralization.
• Epitope mutations conferred a liver-detargeting phenotype in vivo.
• Mutant vectors showed minimal evasion of polyclonal neutralizing sera from mice, macaques, and humans.

Conclusions:

• Targeted mutations at a single epitope can reduce neutralization by specific antibodies.
• Residues involved in antibody binding also influence AAV9 liver tropism.
• Evading the broad repertoire of neutralizing antibodies requires targeting multiple epitopes.
• Designing next-generation AAV vectors necessitates a comprehensive approach to immune evasion.