# Genetically Encoded SpyTag Enables Modular AAV Retargeting via SpyCatcher-Fused Ligands for Targeted Gene Delivery

## TL;DR

This paper introduces a new method for retargeting AAV gene therapy vectors using a modular SpyTag/SpyCatcher system, enabling precise and flexible delivery to specific cell types.

## Contribution

The first genetically encoded SpyTag system for modular AAV retargeting without altering the vector genome or production process.

## Key findings

- SpyTag insertion into AAV2 capsid enabled post-assembly coupling with SpyCatcher-fused ligands for targeted transduction.
- DARPin-based targeting achieved high specificity for cancer cell lines with minimal off-target effects.
- The platform supports large or complex ligands and enables high-throughput preclinical evaluation.

## Abstract

Recombinant adeno-associated
viral (rAAV) vectors are
a leading
platform for in vivo gene therapy, valued for their
excellent safety, broad serotype diversity, and scalable production.
Targeted delivery through capsid display of ligands holds great promise,
yet current retargeting strategies often rely on extensive capsid
re-engineering and restrict the use of ligands incompatible with intracellular
expression systems. Here, we present a modular AAV retargeting platform
that, for the first time, employs the SpyTag/SpyCatcher system via
genetic integration into the AAV2 capsid. SpyTag is a small peptide
that forms a covalent, irreversible bond with its protein partner,
SpyCatcher, allowing site-specific ligand coupling under physiological
conditions. Inserting SpyTag into surface-exposed capsid sites enabled
postassembly functionalization of AAVs with SpyCatcher-fused targeting
proteins. As proof of concept, we used SpyCatcher fusions with designed
ankyrin repeat proteins (DARPins) specific for EGFR, EpCAM, and HER2.
This conferred highly specific transduction of corresponding cancer
cell lines with minimal off-target activity. Therapeutic potential
was demonstrated by delivering a suicide gene, inducing selective
cancer cell killing upon prodrug administration. This “one-fits-all”
platform allows rapid and flexible retargeting without significantly
altering the underlying vectors genome or production process. It supports
the incorporation of large or complex ligands not amenable to genetic
fusion and facilitates high-throughput preclinical evaluation strategies.
By uniting capsid engineering with modular ligand display, our approach
provides a scalable and versatile framework for precision gene delivery,
broadening the applicability of rAAV in both therapeutic and discovery
settings.