Post-Packaging pH-Responsive Peptide Conjugation Enhances Productive Transduction by Antibody-Guided Adeno-Associated Viral Vectors

Adeno-associated virus vectors are widely used for gene delivery, yet their functional efficiency is limited by multiple intracellular bottlenecks. Here, we develop a modular vector engineering strategy that combines antibody-guided cellular entry with site-specific capsid modification to enhance post-entry processes. By systematically introducing surface-exposed cysteine residues into a heparan sulfate-binding-deficient capsid, we identify a permissive position that enables efficient chemical conjugation without compromising capsid assembly while revealing strong serotype-dependent constraints on capsid engineering. Using this optimized backbone, we implement antibody-guided delivery and conjugate a membrane-inserting peptide through polyethylene glycol linkers to enhance the post-entry efficiency. Peptide conjugation significantly increases transgene expression across multiple cell types without increasing viral uptake, indicating improved intracellular processing. Dose−response analyses further reveal a threshold-dependent behavior, in which functional enhancement becomes evident only above a critical vector input, a phenomenon consistently observed across distinct antigen−antibody pairs and extended to therapeutic transgenes. Together, these findings define a flexible and generalizable capsid modification strategy that improves productive transduction independently of intrinsic capsid tropism and provides a practical framework for next-generation gene delivery vectors.