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

Transmorphic phage-guided systemic delivery of TNFα gene for the treatment of human pediatric medulloblastoma

Medulloblastoma is the most common childhood brain tumor with an unfavorable prognosis and limited options of harmful treatments that are associated with devastating long-term side effects. Therefore, the development of safe, noninvasive, and effective therapeutic approaches is required to save the quality of life of young medulloblastoma survivors. We postulated that therapeutic targeting is a solution. Thus, we used a recently designed tumor-targeted bacteriophage (phage)-derived particle, named transmorphic phage/AAV, TPA, to deliver a transgene expressing the tumor necrosis factor-alpha (TNFα) for targeted systemic therapy of medulloblastoma. This vector was engineered to display the double-cyclic RGD4C ligand to selectively target tumors after intravenous administration. Furthermore, the lack of native phage tropism in mammalian cells warrants safe and selective systemic delivery to the tumor microenvironment. In vitro RGD4C.TPA.TNFα treatment of human medulloblastoma cells generated efficient and selective TNFα expression, subsequently triggering cell death. Combination with the chemotherapeutic drug cisplatin used clinically against medulloblastoma resulted in augmented effect through the enhancement of TNFα gene expression. Systemic administration of RGD4C.TPA.TNFα to mice-bearing subcutaneous medulloblastoma xenografts resulted in selective tumor homing of these particles and consequently, targeted tumor expression of TNFα, apoptosis, and destruction of the tumor vasculature. Thus, our RGD4C.TPA.TNFα particle provides selective and efficient systemic delivery of TNFα to medulloblastoma, yielding a potential TNFα anti-medulloblastoma therapy while sparing healthy tissues from the systemic toxicity of this cytokine

Targeting human osteoarthritic chondrocytes with ligand directed bacteriophage-based particles

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs

Launching a Novel Preclinical Infrastructure: Comparative Oncology Trials Consortium Directed Therapeutic Targeting of TNFα to Cancer Vasculature

Background: Under the direction and sponsorship of the National Cancer Institute, we report on the first pre-clinical trial of the Comparative Oncology Trials Consortium (COTC). The COTC is a novel infrastructure to integrate cancers that naturally develop in pet dogs into the development path of new human drugs. Trials are designed to address questions challenging in conventional preclinical models and early phase human trials. Large animal spontaneous cancer models can be a valuable addition to successful studies of cancer biology and novel therapeutic drug, imaging and device development. Methodology/Principal Findings: Through this established infrastructure, the first trial of the COTC (COTC001) evaluated a targeted AAV-phage vector delivering tumor necrosis factor (RGD-A-TNF) to αV integrins on tumor endothelium. Trial progress and data was reviewed contemporaneously using a web-enabled electronic reporting system developed for the consortium. Dose-escalation in cohorts of 3 dogs (n = 24) determined an optimal safe dose (5 x 1012 transducing units intravenous) of RGD-A-TNF. This demonstrated selective targeting of tumor-associated vasculature and sparing of normal tissues assessed via serial biopsy of both tumor and normal tissue. Repetitive dosing in a cohort of 14 dogs, at the defined optimal dose, was well tolerated and led to objective tumor regression in two dogs (14%), stable disease in six (43%), and disease progression in six (43%) via Response Evaluation Criteria in Solid Tumors (RECIST). Conclusions/Significance: The first study of the COTC has demonstrated the utility and efficiency of the established infrastructure to inform the development of new cancer drugs within large animal naturally occurring cancer models. The preclinical evaluation of RGD-A-TNF within this network provided valuable and necessary data to complete the design of first-in-man studies

Thermoresponsive bacteriophage nanocarrier as a gene delivery vector targeted to the gastrointestinal tract.

The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine-glycine-aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that, in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract

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 (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

Progression in MCF-7 Breast Cancer Cell Tumorigenicity: Compared Effect of FGF-3 and FGF-4.

The transforming properties of fibroblast growth factor 3 (FGF-3) were investigated in MCF7 breast cancer cells and compared to those of FGF-4, a known oncogenic product. The short form of fgf-3 and the fgf-4 sequences were each introduced with retroviral vectors and the proteins were only detected in the cytoplasm of the infected cells, as expected. In vitro, cells producing FGF-3 (MCF7.fgf-3) and FGF-4 (MCF7.fgf-4) displayed an amount of estrogen receptors decreased to around 45% of the control value. However, MCF7.fgf-3 cell proliferation remained responsive to estradiol supply. The sensitivity of the MCF7.fgf-4 cells, if existant, was masked by the important mitogenic action exerted by FGF-4. In vivo, the MCF7.fgf-3 and MCF7.fgf-4 cells gave rise to tumors under conditions in which the control cells were not tumorigenic. Supplementing the mice with estrogen had the paradoxical effect of totally suppressing the start of the FGF-3 as well as the FGF-4 tumors. Tumorigenicity in the presence of matrigel was similar for MCF7.fgf-3 and control cells and was increased by estrogen supplementation. Once started, the MCF7.fgf-4 tumors grew with a characteristic high rate. Remarkably, FGF-4 but not FGF-3, stimulated the secretion of vascular endothelial growth factor (VEGF165) without altering the steady-state level of its mRNA, suggesting a possible regulation of VEGF synthesis at the translational level in MCF7 cells. The increased VEGF secretion is probably involved in the more aggressive phenotype of the MCF7.fgf-4 cells while a decreased dependence upon micro-environmental factors might be part of the increased tumorigenic potential of the MCF7.fgf-3 cells.Peer reviewe

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

Bacteriophage (phage) have attractive advantages as delivery sys-tems compared to mammalian viruses, but have been consideredpoor vectors because they lack evolved strategies to confrontand overcome mammalian cell barriers to infective agents. Wereasoned that improved efficacy of delivery might be achievedthrough structural modification of the viral capsid to avoid pre-and post-internalization barriers to mammalian cell transduction.We generated multifunctional hybrid AAV/phage (AAVP) particlesto enable simultaneous display of targeting ligands on the phage’sminor pIII proteins and also degradation-resistance motifs on thevery numerous pVIII coat proteins. This genetic strategy of directedevolution, bestows a next-generation of AAVP particles that fea-ture resistance to fibrinogen adsorption or neutralizing antibodies,and ability to escape endolysosomal degradation. This results insuperior gene transfer efficacyin vitroand also in preclinicalmouse models of rodent and human solid tumors. Thus, the uniquefunctions of our next-generation AAVP particles enable improvedtargeted gene delivery to tumor cells

Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles

Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNFα) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses

Efficacy of systemic temozolomide-activated phage-targeted gene therapy in human glioblastoma.

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 αvβ3 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.. This study was supported by a grant G0701159 of the UK Medical Research Council (MRC) and the Brain Tumour Research Campaign (BTRC). Keittisak Suwan is funded by the Children with Cancer UK (13/147 and 16/230). AMC is funded by ISCIII-FEDER (CP13/00189). JP received a stipend from the BTRC. EOA acknowledges support from Imperial College NIHR Biomedical Research Centre award (WSCC_P62585), Cancer Research UK grant (C2536/A16584), Medical Research Council grant (MC-A652- 5PY80), and Experimental Cancer Medicine Centres grant (C37/A7283)