The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction.
ABSTRACT: 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.
Project description:Bacteriophage (phage), which are viruses that infect bacteria only, have shown promise as vehicles for targeted cancer gene therapy, albeit with poor efficiency. Recently, we generated an improved version of phage vectors by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) efficiently delivered systemically administered therapeutic genes to various tumor targets by displaying an integrin tumor-targeting ligand on the phage capsid. However, inherent limitations in bacteriophage mean that these AAVP vectors still need to be improved. One of the limitations of AAVP in mammalian cells may be its susceptibility to proteasomal degradation. The proteasome is upregulated in cancer and it is known that it constitutes a barrier to gene delivery by certain eukaryotic viruses. We report here that inhibition of proteasome improved targeted reporter gene delivery by AAVP in cancer cells in vitro and in tumors in vivo after intravenous vector administration to tumor-bearing mice. We also show enhanced targeted tumor cell killing by AAVP upon proteasome inhibition. The AAVP particles persisted significantly in cancer cells in vitro and in tumors in vivo after systemic administration, and accumulated polyubiquitinated coat proteins. Our results suggest that the proteasome is indeed a barrier to tumor targeting by AAVP and indicate that a combination of proteasome-inhibiting drugs and AAVP should be considered for clinical anticancer therapy.
Project description: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 (?v?3) 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.
Project description:Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno-associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the ?v?3 integrin receptor. Second, genes are expressed from a tumor-activated and temozolomide (TMZ)-induced promoter of the glucose-regulated protein, Grp78. Here we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP-Grp78. Firstly, in vitro we showed that TMZ increases endogenous Grp78 gene expression and boosts transgene expression from the RGD4C/AAVP-Grp78 in human GBM cells. Next, RGD4C/AAVP-Grp78 targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP-Grp78 targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.
Project description:Glioblastoma multiforme (GBM) is the most lethal primary intracranial malignant neoplasm in adults and most resistant to treatment. Integration of gene therapy and chemotherapy, chemovirotherapy, has the potential to improve treatment. We have introduced an intravenous bacteriophage (phage) vector for dual targeting of therapeutic genes to glioblastoma. It is a hybrid AAV/phage, AAVP, designed to deliver a recombinant adeno-associated virus genome (rAAV) by the capsid of M13 phage. In this vector, dual tumor targeting is first achieved by phage capsid display of the RGD4C ligand that binds the ?<sub>v</sub>?<sub>3</sub> integrin receptor. Second, genes are expressed from a tumor-activated and temozolomide (TMZ)-induced promoter of the glucose-regulated protein, <i>Grp78</i> Here, we investigated systemic combination therapy using TMZ and targeted suicide gene therapy by the RGD4C/AAVP-<i>Grp78</i> Firstly, <i>in vitro</i> we showed that TMZ increases endogenous <i>Grp78</i> gene expression and boosts transgene expression from the RGD4C/AAVP-<i>Grp78</i> in human GBM cells. Next, RGD4C/AAVP-<i>Grp78</i> targets intracranial tumors in mice following intravenous administration. Finally, combination of TMZ and RGD4C/AAVP-<i>Grp78</i> targeted gene therapy exerts a synergistic effect to suppress growth of orthotopic glioblastoma.
Project description:The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.
Project description:Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.
Project description: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.
Project description:In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-? (AAVP-TNF-?) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-? alone (AAVP-TNF-? plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-? plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-? production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-? and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-? and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.
Project description:Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.
Project description:p53 gene (TP53) replacement therapy has shown promising results in cancer gene therapy. However, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) can knock out the mutated TP53 (mutTP53), but due to its large size, many viral vectors are not suitable or require implemented strategies that lower the therapeutic efficiency. Here, we introduced a bacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the feasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells. First, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette. Next, we investigated any negative impact on vector titers via quantitative polymerase chain reaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and immunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted bacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence without any negative impact on the active or total number of bacteriophage particles. Then, we detected expression of the Cas9 in human lung adenocarcinoma cells in a targeted and efficient manner. Finally, we proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9 phage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage for TP53 replacement therapy in lung cancer.