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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, non-invasive 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 peptide to selectively target tumors after intravenous administration. Furthermore, the lack of native phage tropism to mammalian cells warrants safe and selective systemic delivery to the tumor microenvironment.In vitroRGD4C.TPA.TNFαtreatment of human medulloblastoma cells generated efficient and selectiveTNFαexpression, subsequently triggering cell death. Combination with the chemotherapeutic drug cisplatin, used clinically against medulloblastoma, resulted in augmented effect through the enhancement ofTNFαgene expression. Systemic administration of RGD4C.TPA.TNFαto 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.TNFαparticle provides selective and efficient systemic delivery ofTNFαto medulloblastoma, yielding a potential TNFα anti-medulloblastoma therapy while sparing healthy tissues from the systemic toxicity of this cytokine. more...

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. more...

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. more...

Background: Reprogrammed cellular metabolism is a cancer hallmark. In addition to increased glycolysis, the oxidation of acetate in the citric acid cycle is another common metabolic phenotype. We have recently developed a novel fluorine-18-labelled trimethylacetate-based radiotracer, [18F]fluoro-pivalic acid ([18F]FPIA), for imaging the transcellular flux of short-chain fatty acids, and investigated whether this radiotracer can be used for the detection of glioma growth. Methods: We evaluated the potential of [18F]FPIA PET to monitor tumor growth in orthotopic patient-derived (HSJD-GBM-001) and cell line-derived (U87, LN229) glioma xenografts, and also included [18F]FDG PET for comparison. We assessed proliferation (Ki-67) and the expression of lipid metabolism and transport proteins (CPT1, SLC22A2, SLC22A5, SLC25A20) by immunohistochemistry, along with etomoxir treatment to provide insights into [18F]FPIA uptake. Results: Longitudinal PET imaging showed gradual increase in [18F]FPIA uptake in orthotopic glioma models with disease progression (p &lt, 0.0001), and high tumor-to-brain contrast compared to [18F]FDG (p &lt, 0.0001). [18F]FPIA uptake correlated positively with Ki-67 (p &lt, 0.01), SLC22A5 (p &lt, 0.001) and SLC25A20 (p = 0.001), and negatively with CPT1 (p &lt, 0.01) and SLC22A2 (p &lt, 0.01). Etomoxir reduced [18F]FPIA uptake, which correlated with decreased Ki-67 (p &lt, 0.05). Conclusions: Our findings support the use of [18F]FPIA PET for the detection and longitudinal monitoring of glioma, showing a positive correlation with tumor proliferation, and suggest transcellular flux-mediated radiotracer uptake. more...

Chondrosarcoma is a cartilage-forming bone tumor, well known for intrinsic resistance to chemotherapy and radiotherapy. We have designed a targeted chondrosarcoma gene therapy using a bacteriophage (phage) particle to deliver therapeutic genes. Phage has no tropism for mammalian cells, allowing engineered phage to be targeted to specific cell surface receptors in cancer. We modified the phage capsid to display the RGD4C ligand on the pIII minor coat proteins to specifically bind to αvβ3 or αvβ5 integrin receptors. The endosomal escape peptide, H5WYG, was also displayed on recombinant pVIII major coat proteins to enhance gene delivery. Finally, a human tumor necrosis factor alpha (TNFα) therapeutic transgene expression cassette was incorporated into the phage genome. First, we found that human chondrosarcoma cells (SW1353) have high expression of αvβ3, αvβ5 integrin receptors, and both TNFα receptors. Targeted particle encoding a luciferase reporter gene efficiently and selectively mediated gene delivery to these cells. When SW1353 cells were treated with the targeted particle encoding a TNFα transgene, significant cell killing was evident and was associated with high expression of TNFα and apoptosis-related genes. In vivo, mice with established human chondrosarcoma showed suppression of tumors upon repetitive intravenous administrations of the targeted phage. These data show that our phage-based particle is a promising, selective, and efficient tool for targeted chondrosarcoma therapy. more...

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 (&alpha, v&beta, 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. more...

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. more...

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive paediatric brainstem tumours, with a dismal prognosis and a 90% mortality rate within 2 years. Over the last 50 years, DIPG patients have seen no increase in survival, owing to progress being hindered by a lack of tissue available for in-vitro study, the critical anatomical location of the pons and the impermeability of the blood-brain barrier (BBB). In this in-vitro study, we describe the use of cancer gene therapy using the targeted-bacteriophage vector to deliver the therapeutic TNFα transgene to DIPG cells, in combination with the novel therapeutic, curcumin, which constitutes the spice turmeric (Curcuma longa). MATERIALS AND METHODS: We evaluated the in-vitro cytotoxicity of curcumin in 2D tissue cultures of primary DIPG cells, with cell-viability measured after treatment with varying concentrations of curcumin for 72 hours. Next, we assessed the gene transduction and cell-killing efficacy of the targeted-hybrid bacteriophage, using the Luciferase and TNFα transgenes, at day 3 post-transduction. Lastly, we assessed the effect of combination therapy by adding concentrations of curcumin to DIPG cells at day 3 post-transduction by the bacteriophage, once again evaluating Luciferase expression and cell-killing. DISCUSSION: Here, we report for the first time, the in-vitro cytotoxicity of curcumin in this DIPG cell line, with significant cell death seen at as low as 37.5μM. Secondly, we show that primary DIPG cells can be successfully transduced with the targeted-modified bacteriophage vector, containing the TNFα gene, leading to TNFα-mediated cell death. Lastly, we show that the combination of curcumin with the targeted-bacteriophage vector led to enhanced DIPG cell death, due to increased bacteriophage-mediated transgene expression. CONCLUSION: We have shown that combination chemovirotherapy of curcumin and the targeted bacteriophage is efficacious in-vitro, warranting further in-vivo work to assess the safety and efficacy after systemic delivery. more...

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive paediatric brainstem tumours, with a dismal prognosis and a 90% mortality rate within 2 years. Over the last 50 years, DIPG patients have seen no increase in survival, owing to progress being hindered by a lack of tissue available for in-vitro study, the critical anatomical location of the pons and the impermeability of the blood-brain barrier (BBB). In this in-vitro study, we describe the use of cancer gene therapy using the targeted-bacteriophage vector to deliver the therapeutic TNFα transgene to DIPG cells, in combination with the novel therapeutic, curcumin, which constitutes the spice turmeric (Curcuma longa). MATERIALS AND METHODS: We evaluated the in-vitro cytotoxicity of curcumin in 2D tissue cultures of primary DIPG cells, with cell-viability measured after treatment with varying concentrations of curcumin for 72 hours. Next, we assessed the gene transduction and cell-killing efficacy of the targeted-hybrid bacteriophage, using the Luciferase and TNFα transgenes, at day 3 post-transduction. Lastly, we assessed the effect of combination therapy by adding concentrations of curcumin to DIPG cells at day 3 post-transduction by the bacteriophage, once again evaluating Luciferase expression and cell-killing. DISCUSSION: Here, we report for the first time, the in-vitro cytotoxicity of curcumin in this DIPG cell line, with significant cell death seen at as low as 37.5μM. Secondly, we show that primary DIPG cells can be successfully transduced with the targeted-modified bacteriophage vector, containing the TNFα gene, leading to TNFα-mediated cell death. Lastly, we show that the combination of curcumin with the targeted-bacteriophage vector led to enhanced DIPG cell death, due to increased bacteriophage-mediated transgene expression. CONCLUSION: We have shown that combination chemovirotherapy of curcumin and the targeted bacteriophage is efficacious in-vitro, warranting further in-vivo work to assess the safety and efficacy after systemic delivery. more...

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix which interacts with hyaluronan at the N-terminal G1 domain, composed of A, B, and B' subdomains. Recently, we generated knock-in mice Cspg2(Delta3/Delta3), whose versican, without the A subdomain, has decreased hyaluronan (HA) binding affinity, thereby exhibiting reduced deposition of versican in the extracellular matrix. Here, we show that the Cspg2(Delta3/Delta3) fibroblasts within 20 passages proliferate more slowly and acquire senescence. Whereas the extracellular matrix of the wild type fibroblasts exhibited a network structure of hyaluronan and versican, that of the Cspg2(Delta3/Delta3) fibroblasts exhibited approximately 35 and approximately 85% deposition of versican and HA, without such a structure. The Cspg2(Delta3/Delta3) fibroblasts showed a substantial increase of ERK1/2 phosphorylation and expression of senescence markers p53, p21, and p16. Treatment of wild type fibroblasts with hyaluronidase and exogenous hyaluronan enhanced ERK1/2 phosphorylation, and treatment with an anti-CD44 antibody that blocks HA-CD44 interaction inhibited the phosphorylation. These results demonstrate that versican is essential for matrix assembly involving hyaluronan and that diminished versican deposition increases free hyaluronan fragments that interact with CD44 and increase phosphorylation of ERK1/2, leading to cellular senescence. more...

Versican/proteoglycan-mesenchymal (PG-M) is a large chondroitin sulfate (CS) proteoglycan of the extracellular matrix (ECM) that is constitutively expressed in adult tissues such as dermis and blood vessels. It serves as a structural macromolecule of the ECM, while in embryonic tissue it is transiently expressed at high levels and regulates cell adhesion, migration, proliferation, and differentiation. Knock-in mouse embryonic (Cspg2(Delta3/Delta3)) fibroblasts whose versican lack the A subdomain of the G1 domain exhibit low proliferation rates and acquire senescence. It was suspected that chondroitin sulfate on versican core protein would be altered when the A subdomain was disrupted, so fibroblasts were made from homozygous Cspg2(Delta3/Delta3) mouse embryos to investigate the hypothesis. Analysis of the resulting versican deposition demonstrated that the total versican deposited in the Cspg2(Delta3/Delta3) fibroblasts culture was approximately 50% of that of the wild type (WT), while the versican deposited in the ECM of Cspg2(Delta3/Delta3) fibroblasts culture was 35% of that of the WT, demonstrating the lower capacity of mutant (Cspg2(Delta3/Delta3)) versican deposited in the ECM. The analysis of CS expression in the Cspg2(Delta3/Delta3) fibroblasts culture compared with wild-type fibroblasts showed that the composition of the non-sulfate chondroitin sulfate isomer on the versican core protein increased in the cell layer but decreased in the culture medium. Interestingly, chondroitin sulfate E isomer was found in the culture medium. The amount of CS in the Cspg2(Delta3/Delta3) cell layer of fibroblasts with mutant versican was dramatically decreased, contrasted to the amount in the culture medium, which increased. It was concluded that the disruption of the A subdomain of the versican molecule leads to lowering of the amount of versican deposited in the ECM and the alteration of the composition and content of CS on the versican molecule. more...

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only, furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer, (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. more...

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface. more...

Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant rAAV2 virus genome carrying the transgene to deliver. In comparison with mammalian viral vectors, the production of AAV/Phage is quicker, simpler and more economical. In addition, the vector is stable at 4oC for many years. The AAV/Phage vector efficiently targeted, delivered, and expressed transgene in cancer cells in vitro. We also proved that the vector selectively targeted gene delivery to tumors after intravenous injection in animal models of cancer. Moreover, in brain tumor models, the vector can penetrate through blood brain barrier and selectively delivers transgene expression to brain tumors. We used our vector as combination therapies with some well-known cancer drugs, such as doxorubicin, and temozolomide. Combination treatment of AAV/Phage vector carrying the Herpes Simplex virus thymidine kinase gene (AAV/Phage-HSVtk.) with doxorubicin increased the targeted cancer cell killing in 2D tissue cultures and 3D tumor spheroids of rat gliosarcoma (9L) and human melanoma (M21) cells. We found that this increase in tumor cell killing was associated with a synergistic effect of doxorubicin on enhancing gene expression by AAV/Phage. We then combined AAV/Phage carrying short hairpin RNA to suppress mTOR gene expression (AAV/Phage-shmTOR) with temozolomide to treat medulloblastoma cells (DAOY). The results exhibited that treatment of medulloblastma with the vector alone efficiently suppresses the expression of mTOR gene, but has no effect on cell killing. Treatment of temozolomide at low dose (500 uM) did not have effect on cell killing, but combination therapies of temozolomide with AAV/Phage-shmTOR significantly increased cell death. Altogether, our results demonstrate that combination of AAV/Phage carrying therapeutic genes with cancer chemotherapeutic drugs is an effective strategy for cancer treatment. In future work, we plan to investigate the efficacy of AAV/Phage and cancer drug combination treatment in pre-clinical models of cancer. more...

Background Gene therapy has been an attractive paradigm for cancer treatment. However, cancer gene therapy has been challenged by the inherent limitation of vectors that are able to deliver therapeutic genes to tumors specifically and efficiently following systemic administration. Bacteriophage (phage) are viruses that have shown promise for targeted systemic gene delivery. Yet, they are considered poor vectors for gene transfer. Recently, we generated a tumor-targeted phage named adeno-associated virus/phage (AAVP), which is a filamentous phage particle whose genome contains the adeno-associated virus genome. Its effectiveness in delivering therapeutic genes to tumors specifically both in vitro and in vivo has been shown in numerous studies. Despite being a clinically useful vector, a multitude of barriers impede gene transduction to tumor cells. We hypothesized that one such factor is the tumor extracellular matrix (ECM). Methods We used a number of tumor cell lines from different species and histological types in 2D monolayers or 3D multicellular tumor spheroid (MCTS) models. To assess whether the ECM is a barrier to tumor cell targeting by AAVP, we depleted the ECM using collagenase, hyaluronidase, or combination of both. We employed multiple techniques to investigate and quantify the effect of ECM depletion on ECM composition (including collagen type I, hyaluronic acid, fibronectin and laminin), and how AAVP adsorption, internalisation, gene expression and therapeutic efficacy are subsequently affected. Data were analyzed using a student’s t test when comparing two groups or one-way ANOVA and post hoc Tukey tests when using more than two groups. Results We demonstrate that collagenase and hyaluronidase-mediated degradation of tumor ECM affects the composition of collagen, hyaluronic acid and fibronectin. Consequently, AAVP diffusion, internalisation, gene expression and tumor cell killing were enhanced after enzymatic treatment. Our data suggest that enhancement of gene transfer by the AAVP is solely attributed to ECM depletion. We provide substantial evidence that ECM modulation is relevant in clinically applicable settings by using 3D MCTS, which simulates in vivo environments more accurately. Conclusion Our findings suggest that ECM depletion is an effective strategy to enhance the efficiency of viral vector-guided gene therapy. more...