Multiparametric domain insertional profiling of Adeno-Associated Virus VP1

Area of Science:

• Molecular Biology
• Biotechnology
• Virology

Background:

• Adeno-associated virus (AAV) has broad tropism and immunogenicity, posing challenges for gene therapy applications.
• Previous AAV engineering efforts focused on specific capsid regions, limiting comprehensive analysis.

Approach:

• A large-scale domain insertion dataset was generated by inserting structured protein domains into the AAV-DJ VP1 capsid protein.
• Multiple AAV fitness phenotypes were determined for these engineered capsids.
• Positional, domain-type, and fitness phenotype dependencies of insertion permissibility were analyzed.

Key Points:

• AAV capsids demonstrate significant robustness in accommodating large domain insertions.
• Insertion permissibility is strongly dependent on insertion site, domain type, and resulting fitness.
• Identified engineerable hotspots facilitate covalent attachment of binding scaffolds for tropism redirection.

Conclusions:

• This comprehensive survey reveals critical insights into AAV capsid structural biology and engineering.
• New engineerable hotspots offer alternative strategies for redirecting AAV tropism in gene therapy.
• Findings pave the way for improved AAV vector design for enhanced gene delivery.