Graham, N.; Kumar, S.; Rainaldi, J.; Yang, S.; Portell, A.; Santoso, B.; Mali, P.

Precise control over viral tropism remains a major challenge in the development of gene delivery technologies. We present MATCH (Modulation of AAV Tropism through Conjugation to Homing proteins), a modular biochemical method that enables programmable, receptor-guided retargeting of adeno-associated viruses (AAVs) through site-specific covalent protein conjugation. By incorporating a SpyTag peptide motif into selected AAV capsid loops, MATCH allows one-step, stoichiometrically defined attachment of recombinant SpyCatcher-linked targeting proteins to the viral surface. Using mosaic AAV-DJ and AAV9 capsids with controlled SpyTag incorporation, we achieve efficient assembly and tunable ligand display. MATCH-AAVs conjugated to an anti-CD3 single-chain antibody efficiently activate and transduce resting human T cells within mixed PBMC populations in vitro, achieving transduction levels of up to ~58% of total PBMCs. Conjugation to transferrin receptor (TfR1)-binding proteins yielded enhanced brain transduction in vivo, with murine TfR1-targeted MATCH-AAV9 exhibiting up to an 84-fold increase in brain expression relative to wild-type AAV9. Human TfR1-targeted vectors similarly enabled robust, receptor-dependent transduction both in vitro and in humanized mouse models. Both TfR-targeted vectors enabled widespread transduction of the parenchyma, consistent with TfR1-mediated crossing of the blood-brain barrier. Finally, we establish a streamlined one-pot Mix-and-MATCH production strategy in which capsid and targeting ligands are co-expressed during vector generation, yielding functional, targeted AAVs at titers comparable to conventional production. This simple and generalizable synthetic-biology approach provides a versatile toolkit for rational AAV tropism engineering, offering a scalable route to custom vector design for research and therapeutic applications.