Membrane-bound MMP-14 protease-activatable adeno-associated viral vectors for gene delivery to pancreatic tumors.

Adeno-associated virus' (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This "provector" was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.

Constructing and evaluating caspase-activatable adeno-associated virus vector for gene delivery to the injured heart.

Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or "switching ON" the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.