Doxorubicin Improves Cancer Cell Targeting by Filamentous Phage Gene Delivery Vectors

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.

Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles

“…GFP lacking the RGD4C mutation, as control for targeting, that we produced with a non‐targeted (NT) M13KO7 helper phage. We subsequently subjected the cell‐lysate to qPCR with primers specific to the AAV‐2 ITRs, 2‐h post‐transduction, as previously reported (Aurnhammer et al , 2012 ; Tsafa et al , 2016 , 2020 ). RGD4C.TPA.…”

“…Quantification of the ITR cis elements in the cytoplasm and nucleus of cells treated with TPA or AAVP particles was carried out using a modified protocol from previous reports (Aurnhammer et al , 2012 ; Tsafa et al , 2016 , 2020 ). The nuclear and cytosolic fractions isolated from TPA or AAVP‐transduced cells were diluted (1 in 20) in TE buffer, then used as templates for qPCR analysis by using primers specifics for the ITR elements (Table EV2 ).…”

“…These factors contribute significantly to poor uptake and induction of gene expression observed in bacteriophage vectors. Previous studies on the AAVP have explored a number of strategies to enhance the relatively low transduction efficiency when compared to conventional mammalian viruses (Kia et al , 2013 ; Przystal et al , 2013 ; Yata et al , 2014 , 2015 ; Donnelly et al , 2015 ; Tsafa et al , 2016 , 2020 ; Campbell et al , 2018 ). Furthermore, alternative studies have explored removing parts of the phage genome, albeit at the cost of packaging efficiency (Chasteen et al , 2006 ).…”

The Increase in the Drug Resistance of Acute Myeloid Leukemia THP-1 Cells in High-Density Cell Culture Is Associated with Inflammatory-like Activation and Anti-Apoptotic Bcl-2 Proteins

“…The increase in drug resistance in leukemic cells in high-density cultures was marked earlier [ 50 , 51 ]; however, the mechanism of this phenomena remains unclear. The results of our work show the great increase in the resistance of different AML cells (lines HL-60, THP-1, MV411, U937) in high-density cultures to inhibitors of topoisomerases, etoposide and topotecan [ 52 , 53 ]; an antimetabolite, cytarabine [ 54 ]; DNA targeting antitumor drugs doxorubicin and cisplatin [ 55 , 56 , 57 ]; as well as to antitumor recombinant protein izTRAIL [ 58 ]. It was important that the increase in the drug resistance of AML cells in high-density cell culture was reversible and that the transfer of the AML cells from high-density to low-density cultures decreased the resistance to an initial state.…”

Bacteriophages as Solid Tumor Theragnostic Agents

“…The suicidal gene cassette codes for an HSVtk enzyme that is known to inhibit cell doubling and subsequently induce target cell death. The authors established an efficient synergistic cancer cell killing by the combined effect of Dox and the suicidal gene by utilizing the phage vector [ 107 ]. In yet another study by the same group, the investigators customized the phage capsid to display the RGD4C ligand on the pIII minor coat proteins for targeting purposes and a human tumor necrosis factor alpha (TNFα) therapeutic transgene was fused into the phage genome.…”