Adeno-associated virus capsid as a modular molecular scaffold for the presentation of protein domains

Adeno-associated virus (AAV) capsid engineering has been usually limited to the insertion, substitution, or modification of only a few amino acids within exposed surface loops, restricting the ability to introduce larger functional elements while maintaining capsid assembly and stability. To overcome these limitations, we developed a novel AAV5 capsid scaffold in which the wildtype icosahedral 3-fold protrusions were removed (AAV5 3f DEL), creating an icosahedral platform capable of accommodating larger modular protein assemblies. We validated this approach by integrating twenty trimeric gamma-carbonic anhydrases into the AAV5 3f DEL capsid surface, demonstrating that intact protein domains can be displayed while preserving capsid assembly (AAV5(γ-CA)). The AAV capsid scaffold (AAV5 3f DEL) and the modified capsid (AAV5(γ-CA)) structures were determined using cryo-electron microscopy to 3.2 and 3.8 Å resolution respectively. Furthermore, the AAV5(γ-CA) capsid was demonstrated to be thermostable, with a melting temperature of approximately 65 °C. This approach can be used to expand the AAV engineering platform repertoire and may provide new opportunities for functionalizing AAV based nanoparticles with receptor-binding proteins, catalytic enzymes, and antigenic domains. The ability to decorate AAV capsids with diverse trimeric proteins establishes a versatile framework for potentially developing next-generation gene delivery vectors, targeted therapeutic platforms, and vaccine technologies.