89. Expanding the Toolkit of Protease-Activatable Viruses to Optimize Their Versatility and Efficiency

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viral vectors. However, its wide adoption as a safe and effective gene therapy treatment for disease may rely on targeting the vector to deliver transgenes to desired cell populations. To this end, our group has developed a protease-activatable virus (PAV) based on AAV that responds to elevated protease activity commonly found in many diseased tissue microenvironments. Small peptide “locks” were inserted into the viral capsid near its primary receptor binding domain, interfering with the virus-cell receptor interaction and preventing binding and transduction. The peptide locks are flanked by two proteolytically cleavable peptides, which are cleaved off in the presence of matrix metalloproteinases (MMPs), restoring virus-receptor interaction and allowing transduction to resume. Cleavage sequences were chosen from literature based on well-characterized linear peptide substrates. Initial testing demonstrated the desired MMP-activated cellular binding and transduction behavior, but it was discovered that cleavage efficiency and transduction activity of the PAV variants were uncorrelated with the reported specificity constants of the cleavage sequences. We therefore devised several ways to tune the sensitivity and specificity of our PAV. For example, by controlling the ratio of wild type to MMP-activatable protein subunits in the self-assembling 60-mer capsid, we can modulate the level of transduction achieved by the PAVs to attain targeted gene delivery to a range of human pathologies. A new lock format was also designed to allow for more predictable kinetics. This new “linearizable” lock format is modified by replacing one of the two flanking MMP cleavage motifs with the enterokinase cleavage motif, DDDDK, on the C-terminal side of the lock. When the enterokinase-linearized PAVs (ePAVs) are pretreated with enterokinase, the peptide locks are cleaved on one side, allowing the peptides to assume a more flexible conformation protruding from the capsid. The newly linearized peptide locks are cleaved with greater efficiency and kinetics that more closely correlate with expected activity. The PAV platform was also expanded into several AAV serotypes to allow for more diverse clinical applications. Our PAVs have demonstrated targeted delivery to several cancer models, including ovarian and pancreatic cancers, as well as ischemic tissue resulting from myocardial infarction.