Bioorthogonal Chemical Engineering of rAAV Capsid: Advancing Gene Therapy Targeting Using Proteins

Area of Science:

• Bioconjugation Chemistry
• Gene Therapy Vector Engineering
• Viral Vector Modification

Background:

• Recombinant Adeno-Associated Virus (rAAV) is a key vector for gene therapy.
• Current rAAV modification methods have limitations in precision and functionality.
• Bioorthogonal chemistry offers a precise strategy for modifying viral vectors.

Purpose of the Study:

• To develop a bioorthogonal chemical conjugation strategy for rAAV capsids.
• To enable the attachment of diverse functionalities, including proteins, to rAAV.
• To enhance tissue-specific targeting for improved gene therapy applications.

Main Methods:

• Chemical modification of lysine or tyrosine residues on rAAV capsids with azide or dibenzylcyclooctyne (DBCO) anchors.
• Utilizing strain-promoted azide-alkyne cycloaddition (SPAAC) for bioorthogonal conjugation.
• Characterization using biochemical assays, mass spectrometry, and in vitro transduction assays.

Main Results:

• Demonstrated anchor-dependent labeling efficiency of rAAV capsids.
• Confirmed efficient SPAAC reactions for appending fluorescein, biotin, and carbohydrates.
• Successfully conjugated rAAV with nanobodies targeting CD62L and CD45, showing promising in vitro targeting capabilities.

Conclusions:

• The developed bioorthogonal strategy enables precise chemical modification of rAAV capsids.
• This approach facilitates the attachment of functional molecules, including targeting nanobodies.
• The findings hold significant potential for advancing rAAV capsid engineering and tissue-specific gene therapy.