pH-responsive virus-drug conjugates for targeted PD-L1 silencing and enhanced cancer immunotherapy.

Clinical efficacy of virotherapy is often limited by poor transduction and off-target effects. To overcome these hurdles, we engineered a multi-stage, pH-responsive virus-drug conjugate (VDC) for targeted, cancer immunotherapy. Our VDC comprises a recombinant adeno-associated virus serotype 2 (AAV2) encoding a programmed death-ligand 1 (PD-L1) short hairpin RNA (shRNA), which is chemically conjugated to the transduction-enhancing doxorubicin (Dox) with minimally cytotoxic dosage. This AAV2-Dox is shielded by a polyethylene glycol (PEG) layer attached via a pH-sensitive benzoic-imine linker, designed to be stable at physiological pH but cleavable within the acidic tumor microenvironment. AAV2-Dox produced an equivalent enhancement of transgene expression at a much lower concentration (0.005 µg mL) than Dox alone (0.02 µg mL). The pH-sensitive PEG shield successfully renders the VDC inert at pH 7.4 while restoring full transduction and therapeutic efficacy upon acid-triggered unshielding. Mice treatment with the PEGylated AAV2-Dox leads to efficient PD-L1 knockdown, which enhanced T-cell infiltration and polarization toward M1 macrophages. This study establishes a smart VDC platform that integrates a transduction enhancer with a gene therapy payload under the control of an acidic tumor microenvironment-activated stealth shield. This strategy provides a new paradigm for developing safer and more potent nanomedicines for combinatorial cancer immunotherapy.