An Expanded Toolbox for Versatile Chemical Editing of Adeno-Associated Virus

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy Vector Engineering

Background:

• Adeno-associated virus (AAV) is a leading vector for gene therapy.
• Current methods for modifying AAV capsids with noncanonical amino acids (ncAAs) are limited, primarily using a single azide-containing ncAA.
• Expanding ncAA incorporation broadens the possibilities for AAV engineering.

Purpose of the Study:

• To develop new platforms for incorporating diverse ncAAs into AAV capsids.
• To establish design principles for AAV capsid tolerance to various side chains.
• To utilize expanded ncAA chemistry for precise AAV capsid modification and functionalization.

Main Methods:

• Demonstrated successful incorporation of numerous ncAAs into AAV capsids using four distinct platforms.
• Identified design principles governing capsid tolerance to structurally diverse side chains.
• Employed bioorthogonal conjugation chemistry for site-specific AAV capsid modification.

Main Results:

• Developed an expanded toolbox for ncAA incorporation into AAV capsids.
• Achieved ultrafast conjugation of an anti-HER2 nanobody to AAV capsids via a tetrazine-containing ncAA, enabling selective infection of HER2+ cells.
• Demonstrated optimized capsid PEGylation to reduce immunogenicity without compromising infectivity.
• Successfully incorporated and orthogonally labeled two distinct ncAAs on the AAV capsid for dual functionalization.

Conclusions:

• These advances significantly expand the range of chemistries introducible into AAV capsids.
• The developed platform offers powerful new tools for probing and engineering AAV properties.
• This work paves the way for improved and more versatile AAV-based gene therapy vectors.