Targeted treatment of chondrosarcoma with a bacteriophage-based particle delivering a secreted tumor necrosis factor-related apoptosis-inducing ligand.

Chondrosarcoma (CS) is a malignant cartilage-forming bone tumor that is inherently resistant to chemotherapy and radiotherapy, leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvß3 and αvß5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human CS cells (SW1353), but not in primary normal chondrocytes. The RGD4C.PDP-Luc particle carrying a luciferase reporter gene, Luc, effectively and selectively mediated gene delivery to SW1353 cells, but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP-sTRAIL encoding a human sTRAIL, resulted in the expression of TRAIL and subsequent cell death without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP-sTRAIL to mice with established human CS resulted in a decrease in tumor size and tumor viability. Altogether, RGD4C.PDP-sTRAIL can be used to target systemic treatment of CS with the sTRAIL.

Doxorubicin Improves Cancer Cell Targeting by Filamentous Phage Gene Delivery Vectors.

Merging targeted systemic gene delivery and systemic chemotherapy against cancer, chemovirotherapy, has the potential to improve chemotherapy and gene therapy treatments and overcome cancer resistance. We introduced a bacteriophage (phage) vector, named human adeno-associated virus (AAV)/phage or AAVP, for the systemic targeting of therapeutic genes to cancer. The vector was designed as a hybrid between a recombinant adeno-associated virus genome (rAAV) and a filamentous phage capsid. To achieve tumor targeting, we displayed on the phage capsid the double-cyclic CDCRGDCFC (RGD4C) ligand that binds the alpha-V/beta-3 (αvß3) integrin receptor. Here, we investigated a combination of doxorubicin chemotherapeutic drug and targeted gene delivery by the RGD4C/AAVP vector. Firstly, we showed that doxorubicin boosts transgene expression from the RGD4C/AAVP in two-dimensional (2D) cell cultures and three-dimensional (3D) tumor spheres established from human and murine cancer cells, while preserving selective gene delivery by RGD4C/AAVP. Next, we confirmed that doxorubicin does not increase vector attachment to cancer cells nor vector cell entry. In contrast, doxorubicin may alter the intracellular trafficking of the vector by facilitating nuclear accumulation of the RGD4C/AAVP genome through destabilization of the nuclear membrane. Finally, a combination of doxorubicin and RGD4C/AAVP-targeted suicide gene therapy exerts a synergistic effect to destroy human and murine tumor cells in 2D and 3D tumor sphere settings.

Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer.

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction.

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP.