Targeted treatment of chondrosarcoma with a bacteriophage-based particle delivering a secreted tumor necrosis factor-related apoptosis-inducing ligand.

Chondrosarcoma (CS) is a malignant cartilage-forming bone tumor that is inherently resistant to chemotherapy and radiotherapy, leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvß3 and αvß5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human CS cells (SW1353), but not in primary normal chondrocytes. The RGD4C.PDP-Luc particle carrying a luciferase reporter gene, Luc, effectively and selectively mediated gene delivery to SW1353 cells, but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP-sTRAIL encoding a human sTRAIL, resulted in the expression of TRAIL and subsequent cell death without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP-sTRAIL to mice with established human CS resulted in a decrease in tumor size and tumor viability. Altogether, RGD4C.PDP-sTRAIL can be used to target systemic treatment of CS with the sTRAIL.

Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvß3 and αvß5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvß5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

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.

Masculinization of Red Tilapia (Oreochromis spp.) Using 17α-Methyltestosterone-Loaded Alkyl Polyglucosides Integrated into Nanostructured Lipid Carriers.

The aim of the present study was to optimize a masculinization platform for the production of all-male red tilapia fry by oral administration of 30 and 60 ppm of MT and alkyl polyglucoside nanostructured lipid carriers (APG-NLC) loaded with MT, respectively, for 14 and 21 days. The characterization, encapsulation efficiency and release kinetics of MT in lipid-based nanoparticles were assessed in vitro. The results showed that the MT-loaded nanoparticles were spherical, ranging from 80 to 125 nm in size, and had a negative charge with a narrow particle distribution. The APG-NLC loaded with MT provided higher physical stability and encapsulation efficacy than the NLC. The release rate constants of MT from MT-NLC and MT-APG-NLC were higher than those of free MT, which is insoluble in aqueous media. There was no significant difference in survival between the fish administered MT or the those fed orally with MT-APG-NLC fish. According to the logistic regression analysis, the sex reversal efficacy of MT-APG-NLC (30 ppm) and MT (60 ppm), resulted in significantly higher numbers of males after 21 days of treatment compared with the controls. The production cost of MT-APG-NLC (30 ppm) after 21 days of treatment was reduced by 32.9% compared with the conventional MT treatment group (60 ppm). In all the treatments, the length-weight relationship (LWR) showed negatively allomeric growth behavior (b < 3), with a relative condition factor (Kn) of more than 1. Therefore, MT-APG-NLC (30 ppm) would seem to be a promising, cost-effective way to reduce the dose of MT used for the masculinization of farmed red tilapia.

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 (TNF α ) 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.

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.

Effective Detection and Monitoring of Glioma Using [18F]FPIA PET Imaging.

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 < 0.0001), and high tumor-to-brain contrast compared to [18F]FDG (p < 0.0001). [18F]FPIA uptake correlated positively with Ki-67 (p < 0.01), SLC22A5 (p < 0.001) and SLC25A20 (p = 0.001), and negatively with CPT1 (p < 0.01) and SLC22A2 (p < 0.01). Etomoxir reduced [18F]FPIA uptake, which correlated with decreased Ki-67 (p < 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.

Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.

Bacteriophage-mediated therapy of chondrosarcoma by selective delivery of the tumor necrosis factor alpha (TNFα) gene.

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 αvß3 or αvß5 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 αvß3, αvß5 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.

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 (αvß3) 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.

Initial Steps for the Development of a Phage-Mediated Gene Replacement Therapy Using CRISPR-Cas9 Technology.

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.

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

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.

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 αvß3 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.

Efficacy of arginine depletion by ADI-PEG20 in an intracranial model of GBM.

Glioblastoma multiforme (GBM) remains a cancer with a poor prognosis and few effective therapeutic options. Successful medical management of GBM is limited by the restricted access of drugs to the central nervous system (CNS) caused by the blood brain barrier (BBB). We previously showed that a subset of GBM are arginine auxotrophic because of transcriptional silencing of ASS1 and/or ASL and are sensitive to pegylated arginine deiminase (ADI-PEG20). However, it is unknown whether depletion of arginine in peripheral blood in vivo has therapeutic activity against intracranial disease. In the present work, we describe the efficacy of ADI-PEG20 in an intracranial model of human GBM in which tumour growth and regression are assessed in real time by measurement of luciferase activity. Animals bearing intracranial human GBM tumours of varying ASS status were treated with ADI-PEG20 alone or in combination with temozolomide and monitored for tumour growth and regression. Monotherapy ADI-PEG20 significantly reduces the intracranial growth of ASS1 negative GBM and extends survival of mice carrying ASS1 negative GBM without obvious toxicity. The combination of ADI-PEG20 with temozolomide (TMZ) demonstrates enhanced effects in both ASS1 negative and ASS1 positive backgrounds.Our data provide proof of principle for a therapeutic strategy for GBM using peripheral blood arginine depletion that does not require BBB passage of drug and is well tolerated. The ability of ADI-PEG20 to cytoreduce GBM and enhance the effects of temozolomide argues strongly for its early clinical evaluation in the treatment of GBM.

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.

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector.

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.

Restoring the p53 'Guardian' Phenotype in p53-Deficient Tumor Cells with CRISPR/Cas9.

With an increasing prevalence in the human population, cancer has become one of the most investigated fields of medicine. Among the potential targets for cancer therapy is the tumor suppressor gene TP53, which is found in a mutated state in approximately 50% of human cancers and is often associated with poor prognosis. We propose a novel, highly tumor-specific delivery system for TP53, based on the CRISPR/Cas9 genome editing technology. This system will restore the normal p53 phenotype in tumor cells by replacing the mutant TP53 gene with a functional copy, leading to sustained expression of p53 protein and tumor regression.

CRISPR/Cas9: Transcending the Reality of Genome Editing.

With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

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.