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

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

Validation of the Thai Osteoporosis Foundation and Royal College of Orthopaedic Surgeons of Thailand Clinical Practice Guideline for bone mineral density measurement in postmenopausal women

AbstractObjectiveThe primary objective of this study was to determine the sensitivity, specificity, and predictive values of the Thai Osteoporosis Foundation (TOPF) and Royal College of Orthopaedic Surgeons of Thailand (RCOST) Clinical Practice Guideline for bone mineral density (BMD) measurement for the detection of postmenopausal osteoporosis. Its secondary objective was to find better indicators to detect postmenopausal osteoporosis.MethodsPostmenopausal women were enrolled in this study between June and December 2014. The clinical risk factors following TOPF and RCOST Clinical Practice Guideline for BMD measurement were collected. Bone mineral density was measured using dual energy X-ray absorptiometry.ResultsFour hundred postmenopausal women were enrolled in the study. The mean age of the studied population was 66.16 ± 6.04 years. Twenty-seven percent of the participants had either osteoporosis of the lumbar spine, femoral neck, or total hip, of which 13.3% had osteoporosis at the lumbar spine, 21.3% had osteoporosis at the femoral neck, and 2.5% had osteoporosis of the total hip. The sensitivity and specificity for detecting osteoporosis of the whole TOPF and RCOST guideline were 96.2% and 16.7%, 98.8% and 18.7%, 90.0% and 15.1%, and 97.2% and 19.5% at the lumbar spine, femoral neck, total hip, and any sites, respectively. Multiple logistic regression analysis revealed that only OSTA ≤−1, osteopenia on X-ray and low trauma fracture after age of 40 years were significant clinical risk factors in the detection of postmenopausal osteoporosis. The Receiver Operating Characteristics (ROC) curve was used to obtain the optimum probability value of osteoporosis at any sites which revealed that the probability value of 0.2222236 would have a sensitivity of 67% and specificity of 62% as the optimal cut point to detect osteoporosis. A simple flow diagram of “OSTA ≤−1”, “Osteopenia on X-ray” and “A history of low trauma fracture after age of 40 years” was developed as a better trade-off guideline for BMD measurement.ConclusionsThis study revealed that the TOPF and RCOST guideline for BMD measurement provided a high true positive rate of disease detection but with an expense of high false positive rate. The simple flow diagram was proposed as a more appropriate guideline for BMD measurement in postmenopausal women

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

Bio-electrospraying and aerodynamically assisted bio-jetting the model eukaryotic Dictyostelium discoideum: assessing stress and developmental competency post treatment

Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ) have recently been established as important novel biospray technologies for directly manipulating living cells. To elucidate their potential in medical and clinical sciences, these bio-aerosol techniques have been subjected to increasingly rigorous investigations. In parallel to these studies, we wish to introduce these unique biotechnologies for use in the basic biological sciences, for handling a wide range of cell types and systems, thus increasing the range and the scope of these techniques for modern research. Here, the authors present the analysis of the new use of these biospray techniques for the direct handling of the simple eukaryotic biomedical model organism Dictyostelium discoideum. These cells are widely used as a model for immune cell chemotaxis and as a simple model for development. We demonstrate that AABJ of these cells did not cause cell stress, as defined by the stress-gene induction, nor affect cell development. Furthermore, although BES induced the increased expression of one stress-related gene (gapA), this was not a generalized stress response nor did it affect cell development. These data suggest that these biospray techniques can be used to directly manipulate single cells of this biomedical model without inducing a generalized stress response or perturbing later development

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

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-alpha

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-a is responsible for accelerated Mtb growth, and TNF-a neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-a immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-a concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-a secretion.</p

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)