Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery

Area of Science:

• Molecular Biology
• Biotechnology
• Virology

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy, but require optimization for tissue tropism, efficiency, and immune evasion.
• Current methods for AAV capsid evolution are limited in scope and structural guidance.

Purpose of the Study:

• To present a detailed protocol for structure-guided evolution of novel AAV capsids.
• To enhance AAV vector performance for gene transfer, particularly in the central nervous system (CNS).
• To provide a toolkit for developing improved AAV vectors for research and human gene therapies.

Main Methods:

• Development of structure-guided AAV capsid libraries using divergent serotypes.
• Implementation of two distinct library evolution strategies: adenovirus-enabled infectious cycling and noninfectious cross-species cycling.
• In vivo evaluation of lead AAV vector candidates in animal models over approximately 95 days.

Main Results:

• The protocol facilitates the evolution of AAV capsids with altered tissue tropism and enhanced transduction efficiency.
• Demonstrated improvement in CNS gene transfer efficiency using the described evolution strategies.
• The workflow is adaptable for tissue-specific capsid selection and can be integrated with other library strategies.

Conclusions:

• The presented structure-guided protocol offers a robust method for AAV capsid engineering.
• This approach expands the AAV vector toolkit, enabling precise capsid modifications for advanced gene therapies.
• The protocol supports the development of AAV vectors with improved efficacy and safety profiles for diverse therapeutic applications.