Abstract
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 (αvβ3) 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.
Highlights
Gene therapy has been attempted against cancer for the past 28 years, with more than 67% of clinical trials of gene therapy designed to treat cancer patients [1]
We have investigated various steps of gene delivery upon RGD4C/AAVP vector treatment of cells and found that the main extracellular limiting steps are vector diffusion through the extracellular matrix (ECM) and binding to the cell surface receptors caused by repulsion between the negatively charged phage capsid and mammalian cell surface membranes [21,22]
At day four post-vector transduction, we observed more green fluorescent protein (GFP) expression in 9L and M21 tumor cells treated with a combination of RGD4C/AAVP-GFP and doxorubicin than in cells transduced with the RGD4C/AAVP-GFP vector alone (Figure 1B)
Summary
Gene therapy has been attempted against cancer for the past 28 years, with more than 67% of clinical trials of gene therapy designed to treat cancer patients [1]. We have further refined the RGD4C/AAVP delivery technology and combined the vector with drug treatment, such as collagenase and hyaluronidase enzymes for increased diffusion through the ECM, cationic polymers to alter the phage electrostatic charge, chloroquine for endosomal escape, proteasome blocking agents and histone deacetylase inhibitors [21,22,23,24,25]. All these studies showed dramatic enhancement of gene transfers. We investigated the effects on vector cellular trafficking, since doxorubicin can alter the intracellular trafficking of eukaryotic viral vectors [30], and uncovered that destabilization of the nuclear envelope is one mechanism by which doxorubicin might enhance gene delivery
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