Protease-activatable viruses (PAV) based on adeno-associated virus have previously been generated for gene delivery to pathological sites characterized by elevated extracellular proteases. "Peptide locks", composed of a tetra-aspartic acid motif flanked by protease cleavage sequences, were inserted into the virus capsid to inhibit virus-host cell receptor binding and transduction. In the presence of proteases, the peptide locks are cleaved off the capsid, restoring the virus' ability to bind cells and deliver cargo. Although promising, questions remained regarding how the peptide locks prevented cell binding. In particular, it was unclear if the tetra-amino acid (4AA) motif blocks receptor binding via electrostatic repulsion or steric obstruction. To explore this question, we generated a panel of PAVs with lock designs incorporating altered 4AA motifs, each wielding various chemical properties (negative, positive, uncharged polar, and hydrophobic) and characterized the resultant PAV candidates. Notably, all mutants display reduced receptor binding and decreased transduction effciency in the absence of proteases, suggesting simple electrostatics between heparin and the D4 motif do not play an exclusive role in obstructing virus-receptor binding. Even small hydrophobic (A4) and uncharged polar (SGGS) motifs confer a reduction in heparin binding compared to the wild type. Furthermore, both uncharged polar N4 and Q4 mutants (comparable in size to the D4 and E4 motifs respectively, but lacking the negative charge) demonstrate partial ablation of heparin binding. Collectively, these results support a possible dual mechanism of PAV lock operation, where steric hindrance and electrostatics make nonredundant contributions to the disruption of virus-receptor interactions. Finally, because of high virus titer production and superior capsid stability, only the negatively charged 4AA motifs remain viable design choices for PAV construction. Future studies probing the structure-function relationship of PAVs will further expand its promise as a gene delivery vector able to target diseased tissues exhibiting elevated extracellular proteases.
Keywords: adeno-associated virus; enzyme-responsive; gene delivery; matrix metalloproteinases; stimulus-responsive; synthetic virology.