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The intentional use of viruses for cancer therapy dates back over a century. As viruses are inherently immunogenic and naturally optimized delivery vehicles, repurposing viruses for drug delivery, tumor antigen presentation, or selective replication in cancer cells represents a simple and elegant approach to cancer treatment. While early virotherapy was fraught with harsh side effects and low response rates, virus-based therapies have recently seen a resurgence due to newfound abilities to engineer and tune oncolytic viruses, virus-like particles, and virus-mimicking nanoparticles for improved safety and efficacy. However, despite their great potential, very few virus-based therapies have made it through clinical trials. In this review, we present an overview of virus-inspired approaches for cancer therapy, discuss engineering strategies to enhance their mechanisms of action, and highlight their application for overcoming the challenges of traditional cancer therapies. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a unique treatment approach used to decrease a disease's excessive gene expression, including cancer. SiRNAs may find and destroy homologous mRNA sequences within the cell thanks to RNAi processes. However, difficulties such poor cellular uptake, off-target effects, and susceptibility to destruction by serum nucleases in the bloodstream restrict the therapeutic potential of siRNAs. Since some years ago, siRNA-based therapies have been in the process of being translated into the clinic. Therefore, the primary emphasis of this work is on sophisticated nanocarriers that aid in the transport of siRNA payloads, their administration in combination with anticancer medications, and their use in the treatment of cancer. The research looks into molecular manifestations, difficulties with siRNA transport, the design and development of siRNA-based delivery methods, and the benefits and drawbacks of various nanocarriers. The trapping of siRNA in endosomes is a challenge for the majority of delivery methods, which affects the therapeutic effectiveness. Numerous techniques for siRNA release, including as pH-responsive release, membrane fusion, the proton sponge effect, and photochemical disruption, have been studied to overcome this problem. The present state of siRNA treatments in clinical trials is also looked at in order to give a thorough and systematic evaluation of siRNA-based medicines for efficient cancer therapy. [ABSTRACT FROM AUTHOR]

The latest advancements in oncology involves the creation of multifunctional nanostructures. The integration of nanoparticles into the realm of cancer therapy has brought about a transformative shift, revolutionizing the approach to addressing existing challenges and limitations in tumor elimination. This is particularly crucial in combating the emergence of resistance, which has significantly undermined the effectiveness of treatments like chemotherapy and radiotherapy. GO stands as a carbon-derived nanoparticle that is increasingly finding utility across diverse domains, notably in the realm of biomedicine. The utilization of GO nanostructures holds promise in the arena of oncology, enabling precise transportation of drugs and genetic material to targeted sites. GO nanomaterials offer the opportunity to enhance the pharmacokinetic behavior and bioavailability of drugs, with documented instances of these nanocarriers elevating drug accumulation at the tumor location. The GO nanostructures encapsulate genes, shielding them from degradation and facilitating their uptake within cancer cells, thereby promoting efficient gene silencing. The capability of GO to facilitate phototherapy has led to notable advancements in reducing tumor progression. By PDT and PTT combination, GO nanomaterials hold the capacity to diminish tumorigenesis. GO nanomaterials have the potential to trigger both cellular and innate immunity, making them promising contenders for vaccine development. Additionally, types of GO nanoparticles that respond to specific stimuli have been applied in cancer eradication, as well as for the purpose of cancer detection and biomarker diagnosis. Endocytosis serves as the mechanism through which GO nanomaterials are internalized. Given these advantages, the utilization of GO nanomaterials for tumor elimination comes highly recommended. [Display omitted] • Development and introduction of nanostructures for cancer therapy can evolutionalize the treatment prospect of patients, • Graphene oxide (GO) nanoarchitectures are considered as novel platforms for targeted gene and drug delivery, • GO induce phototherapy in increasing tumor ablation and promoting response to therapy, • GO nanostructures provide immunotherapy and are beneficial for vaccine development, • GO nanoparticles can be utilized for diagnosis of cancer biomarkers and cells. [ABSTRACT FROM AUTHOR]

Breast carcinoma is a molecularly diverse illness, and it is among the most prominent and often reported malignancies in female across the globe. Surgical intervention, chemotherapy, immunotherapy, gene therapy, and endocrine treatment are among the currently viable treatment options for the carcinoma of breast. Chemotherapy is among the most prevalent cancer management strategy. Doxorubicin (DOX) widely employed as a cytostatic medication for the treatment of a variety of malignancies. Despite its widespread acceptance and excellent efficacy against an extensive line up of neoplasia, it has a variety of shortcomings that limit its therapeutic potential in the previously mentioned indications. Employment of nanoparticulate systems has come up as a unique chemo medication delivery strategy and are being considerably explored for the amelioration of breast carcinoma. Polylactic- co -glycolic acid (PLGA)-based nano systems are being utilized in a number of areas within the medical research and medication delivery constitutes one of the primary functions for PLGA given their inherent physiochemical attributes, including their aqueous solubility, biocompatibility, biodegradability, versatility in formulation, and limited toxicity. Herein along with the different application of PLGA-based nano formulations in cancer therapy, the present review intends to describe the various research investigations that have been conducted to enumerate the effectiveness of DOX-encapsulated PLGA nanoparticles (DOX-PLGA NPs) as a feasible treatment option for breast cancer. [Display omitted] [ABSTRACT FROM AUTHOR]

To explore new therapeutic options for cervical cancer, the inhibitory effect on cervical cancer of targeted CD133-loaded sPD1 gene microbubbles (MBs) combined with low-frequency ultrasound was studied and its mechanism was explored. We prepared microbubbles conjugated with anti-CD133 antibody to deliver the sPD1 gene and determined concentration, particle size, and potentials of MBs. In addition, we verified that CD133 targeted-MBs could specifically bind to U14 cervical cancer cells in vitro. A mouse model of subcutaneous xenograft cervical cancer was established and mice were divided into a control group, an non-targeted microbubble group, a CD133-MBs group, an sPD1-MBs group and a CD133/sPD1-MBs group. Compared with the control group, tumor growth was inhibited in each group, with the CD133/sPD1 group showing the strongest inhibitory effect after treatment. The tumor volume and weight inhibition rates in the CD133/sPD1-MBs group were 78.01% and 72.25% respectively, which were statistically different from the other groups (P < 0.05), and HE staining and TUNEL immunofluorescence showed necrosis and apoptosis in tumor tissue. Flow cytometry, lactate dehydrogenase, and indirect immunofluorescence experiments showed that T lymphocytes were activated and a large number of CD8-positive T cells infiltrated the tumor tissue after treatment, with the CD133/sPD1-MBs group showing the most prominent effects (P < 0.05). The combination of ultrasound with anti- CD133 antibody-conjugated microbubbles loaded with the sPD1 gene can inhibit the growth of cervical cancer, suggesting that the immunosuppressive microenvironment of the tumor is improved after treatment. • We rationally designed a type of multi-functional microbubbles. • We successfully down-regulate expression of PD-L1 and CD133. • sPD1 / Anti-CD133 Ab -MBs could activate anti-tumor immune response. • sPD1 / Anti-CD133 Ab -MBs could inhibit cervical cancer stemness. • sPD1 / Anti-CD133 Ab -MBs could inhibit the growth of cervical cancer. [ABSTRACT FROM AUTHOR]

Abstract The gene therapy of cancer, due to the limit of its efficiency and safety, has not been widely used in clinical. Recently, bacterial magnetic particles (BMPs), which are membrane-bound nanocrystals found in magnetotactic bacteria, have been exploited as a new gene delivery system. However, its application on gene therapy remains to be explored. In our previous study, we found that a combination of cecropin B (ABPs) and apoptin (VP3) could serve as an effective gene therapeutic agent. Thus, in this study, we used BMPs to deliver the co-expression plasmid of these two gene, namely pVAX1-VA, and evaluated its therapeutic effect on human hepatocellular carcinoma (HepG2). Our results showed that BMPs significantly improved the efficiency of gene transfection (almost 3-fold than Lipofectamine 2000 at 48 h, P <.001), which led to stronger apoptosis (in a peak almost 2-fold than Lipofectamine 2000-pVAX1-VA, P <.01) and growth inhibition of HepG2 cells. More importantly, compared with Lipofectamine 2000-pVAX1-VA group, BMP-pVAX1-VA strikingly inhibited tumor growth (0.60 ± 0.09 g vs. 0.88 ± 0.11 g, P <.05) in nude mouse tumor models and increased the tumor-infiltrating lymphocytes considerably without apparent cytotoxicity. These findings suggest that BMPs could be an attractive gene delivery system for gene therapy and provide a potential available treatment for human hepatocellular carcinoma and maybe some other kinds of tumors. Highlights • This is the first time to use BMPs as a gene delivery system for gene therapy. • BMPs significantly improved the transfection efficacy in HepG2 cells. • BMPs enhanced the effect of pVAX1-VA on apoptosis and growth inhibition of HepG2 cells. • BMP-pVAX1-VA strikingly inhibited tumor growth without apparent cytotoxicity in a nude mice tumor model. [ABSTRACT FROM AUTHOR]

Combination of gene therapy and photothermal therapy (PTT) has drawn much attention in cancer therapy in recent years. However, this joint treatment process lacks fluorescence imaging visualization guidance that limits its clinical applications in oncotherapy. Herein, we report the use of gene therapy and tungsten oxide (W 18 O 49 , WO) synthetized with template method for combined PTT of cancer. In this system, a novel nanoplatform, with Bax gene, WO and indocyanine green (ICG) loaded in mesoporous silica nanoparticle had been successfully constructed, which was used as the near-infrared imaging-guided gene/optothermal multi-modal oncotherapy. These nanoparticles could achieve a synergistic therapy effect of gene therapy and PTT for tumor under 808 nm near-infrared (NIR) laser excitation. In vivo animal experiments showed that they could cause solid tumor regression under 808 nm NIR light irradiation, revealing the potential of these nanocomposites as a fluorescence imaging-guided multi-modal therapeutic nanosystem for tumor visual synergistic treatment. [ABSTRACT FROM AUTHOR]

Compared to conventional treatments, gene therapy offers a variety of advantages for cancer treatment including high potency and specificity, low off-target toxicity, and delivery of multiple genes that concurrently target cancer tumorigenesis, recurrence, and drug resistance. In the past decades, gene therapy has undergone remarkable progress, and is now poised to become a first line therapy for cancer. Among various gene delivery systems, nanoparticles have attracted much attention because of their desirable characteristics including low toxicity profiles, well-controlled and high gene delivery efficiency, and multi-functionalities. This review provides an overview on gene therapeutics and gene delivery technologies, and highlight recent advances, challenges and insights into the design and the utility of nanoparticles in gene therapy for cancer treatment. [ABSTRACT FROM AUTHOR]

Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo , this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy. [ABSTRACT FROM AUTHOR]

Objective To assess the cost-effectiveness of a strategy employing genomic-based tumor testing to guide therapy for platinum-resistant ovarian cancer. Methods A decision model was created to compare standard of care (SOC) cytotoxic chemotherapy to a genomic-based treatment strategy. The genomic arm included tumor testing with treatment directed at targets identified. Overall survival was assumed to be similar between strategies; quality of life (QOL) was assumed superior during targeted therapy compared to chemotherapy. Pertinent uncertainties (cost of targeted therapy and genomic testing, response to targeted therapy, probability of a tumor having a targetable alteration, and impact on QOL) were evaluated in a series of one-and two-way sensitivity analyses. Results The genomic testing strategy was more expensive ($90,271 vs. $74,926) per patient than SOC. The incremental cost-effectiveness ratio (ICER) of the genomic strategy was $479,303 per quality-adjusted life year saved (QALY). Model results were insensitive to the cost of genomic testing, differences in QOL, and the probability of identifying a targetable alteration. However, the model was sensitive to the cost of targeted therapy. For example, when the cost of targeted therapy was reduced to 56% of its current cost (or $6400/cycle), the genomic strategy became more cost-effective with an ICER of $96,612/QALY. Conclusions Genomic-based tumor testing and targeted therapy in patients with platinum-resistant ovarian cancer is not cost-effective compared with SOC. However, reducing the cost of targeted therapy (independently, or in combination with reducing the cost of the genomic test) provides opportunities for improved value in cancer care. [ABSTRACT FROM AUTHOR]

T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRβ is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and β pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases. [ABSTRACT FROM AUTHOR]

Cancers of the brain remain one of the greatest medical challenges. Traditional surgery and chemo-radiation therapy are unable to eradicate diffuse cancer cells and tumor recurrence is nearly inevitable. In contrast to traditional regenerative medicine applications, engineered neural stem cells (NSCs) are emerging as a promising new therapeutic strategy for cancer therapy. The tumor-homing properties allow NSCs to access both primary and invasive tumor foci, creating a novel delivery platform. NSCs engineered with a wide array of cytotoxic agents have been found to significantly reduce tumor volumes and markedly extend survival in preclinical models. With the recent launch of new clinical trials, the potential to successfully manage cancer in human patients with cytotoxic NSC therapy is moving closer to becoming a reality. [ABSTRACT FROM AUTHOR]

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β- d -ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β- d -allofuranosyl)-6-methylpurine ( 3 ) and 9-(6-deoxy-5- C -methyl-β- d -ribo-hexofuranosyl)-6-methylpurine ( 4 ), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di- O -acetyl-3,5-di- O -benzyl-α- d -allofuranose ( 10 ) and 1- O -acetyl-3- O -benzyl-2,5-di- O -benzoyl-6-deoxy-5- C -methyl-β- d -ribohexofuran-ose ( 15 ) were prepared from 1,2:5,6-di- O -isopropylidine-α- d -glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5′- C -methylated-6-methylpurine nucleosides 3 and 4 , respectively. Unlike 9-(6-deoxy-α- l - talo -furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β- d - allo -furanosyl derivative 3 and the 5′-di- C -methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes. [ABSTRACT FROM AUTHOR]

Adoptive transfer of T cells gene-engineered with T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to accurately select TCRs that demonstrate antigen-selective responses that are restricted to tumor cells and, at the same time, include strategies that restore or enhance the entry, migration and local accumulation of T cells in tumor tissues. Here, we present the current standing of TCR-engineered T cell therapy, discuss and propose procedures to select TCRs as well as strategies to sensitize the tumor to T cell trafficking, and provide a rationale for combination therapies with TCR-engineered T cells. [ABSTRACT FROM AUTHOR]

Background Peritoneal dissemination of cancer is a terminal condition with limited therapeutic options. Because the peritoneal cavity is a single enclosed space, regional treatment approaches for isolated peritoneal cancrinomatosis are appealing. There is a potential role for gene therapy in the management of peritoneal cancrinomatosis. Materials and methods An adenoviral construct of the human p14ARF gene (a tumor suppressor) and a 22 amino acid sequence of the ARF gene product, which has cell membrane penetrating properties, were assayed for proapoptotic properties in a human colorectal cancer cell line (Clone A) cells in vitro. Peritoneal carcinomatosis derived from Clone A cells was also established in nude mice and then treated with intraperitoneal administration of an adenoviral construct of the human p14ARF gene. Results Treatment of ARF-negative Clone A cells with Ad-hARF in vitro reestablished ARF function. However, the cell penetrating ARF-related peptide did not restore ARF function in Clone A cells. Treatment of Clone A peritoneal xenografts with a single intraperitoneal dose of Ad-hARF (9 x 106 viral particles) suppressed the progression of peritoneal disease. Weekly (six times) administration of the Ad-hARF at a lower dose (3 x 106 viral particles) also suppressed tumor progression. Conclusions Treatment of peritoneal carcinomatosis by intraperitoneal administration of adenoviral constructs of inactivated tumor suppressor genes may be a feasible clinical approach, and ARF may represent a suitable molecular target for tumors where the ARF gene is inactivated. [ABSTRACT FROM AUTHOR]

Replication deficient human adenovirus type 5 (hAd5) is an important gene delivery vehicle and has been used in various fields of biomedical sciences such as gene therapy, cancer therapy and vaccination. Inspite of its various useful features, emergence of replication competent adenovirus (RCA) in recombinant virus stocks is a great concern. In the present study, recombinant adenovirus expressing foot-and-mouth disease virus serotype-O capsid proteins was propagated in HEK-293 cells and purified by CsCl density gradient ultracentrifugation. The virus was serially passaged in HEK-293 cells and at passage level (P) 2–4, 6, 8 and 12, tested for the presence of RCA. A vector dose of 2 × 10 8 and 1 × 10 10 TCID 50 of the virus was used in cell line bioassay and PCR, respectively. Our results demonstrated that the PCR is more sensitive and rapid technique for the detection of RCA in recombinant adenovirus stocks as early as at P3, whereas the bioassay detected the RCA at P8. [ABSTRACT FROM AUTHOR]

Targeting the human epidermal growth factor receptor 2 (HER2) has yielded major advances in breast cancer treatment. Accordingly, it has generated interest in targeting HER2 to treat gynecologic malignancies. Multiple studies have evaluated the rates of HER2 overexpression and/or amplification in ovarian and uterine cancers. HER2 has also been studied as a prognostic factor but resulting data has been contradictory. Moreover, clinical trials of HER2-directed therapies, including trastuzumab, pertuzumab, and lapatinib in ovarian and uterine cancers have been largely disappointing. Current research on HER2 in gynecologic malignancies has focused on identifying mechanisms of resistance and looking further into how HER2 signaling in gynecologic cancers differs from breast cancer. In this review, we highlight the existing data of targeting HER2 in ovarian and uterine carcinomas, many dating back more than a decade, and discuss future directions in pursuing HER2 as a potential target in these diseases. [ABSTRACT FROM AUTHOR]

Abstract: SNPs in human AFP promoter are associated with serum AFP levels in hepatocellular carcinoma (HCC), suggesting that AFP promoter variants may generate better transcriptional activities while retaining high specificity to AFP-producing cells. We sequenced human AFP promoters, cloned 15 different genotype promoters and tested their reporter activities in AFP-producing and non-producing cells. Among various AFP variant fragments tested, EA4D exhibited the highest reporter activity and thus was selected for the further study. EA4D was fused with tBid and coupled with nano-particle vector (H1) to form pGL3-EA4D-tBid/H1. pGL3-EA4D-tBid/H1 could express a high level of tBid while retain the specificity to AFP-producing cells. In a HCC tumor model, application of pGL3-EA4D-tBid/H1 significantly inhibited the growth of AFP-producing-implanted tumors with minimal side-effects, but had no effect on non-AFP-producing tumors. Furthermore, pGL3-EA4D-tBid/H1 could significantly sensitize HCC cells to sorafenib, an approved anti-HCC agent. Collectively, pGL3-EA4D-tBid/H1, a construct with the AFP promoter EA4D and the novel H1 delivery system, can specifically target and effectively suppress the AFP-producing HCC. This new therapeutic tool shows little toxicity in vitro and in vivo and it should thus be safe for further clinical tests. [Copyright &y& Elsevier]

Abstract: Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently. Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin–endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies. [Copyright &y& Elsevier]

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1×108, 1×109, or 1×1010 viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1×109 vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. [ABSTRACT FROM AUTHOR]

Abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers specific apoptosis in tumor cells and is one of the most promising candidates for cancer gene therapy. However, resistance to TRAIL is one of the main impediments to use of TRAIL in cancer treatment. We showed previously that the lipoxygenase inhibitor MK886 in combination with TRAIL exhibits enhanced antitumor activities compared with each agent alone in human glioma cells. In this study, we elucidated the molecular mechanisms responsible for MK886-mediated sensitization to TRAIL-induced apoptosis. We found that MK886 sensitized glioma cells to TRAIL-induced apoptosis by upregulating the death receptor 5 (DR5) and that specific knockdown of DR5 attenuated cell death. The mechanisms underlying this sensitization involved activation of the MK886-induced p38 mitogen-activated protein kinase (MAPK) pathway and subsequent DR5 overexpression. However, treatment with a specific inhibitor or gene silencing of p38 MAPK abolished both the DR5 induction and the increase in apoptosis caused by TRAIL. Taken together, our findings indicate that the increased expression of DR5 in a p38 MAPK-dependent manner plays an important role in the sensitization of MK886 to TRAIL-induced apoptosis. [Copyright &y& Elsevier]

Abstract: Recent advances have highlighted the central role of DNA methylation in leukemogenesis and have led to clinical trials of epigenetic therapy, notably hypomethylating agents, in myelodysplasia and acute myeloid leukemia. However, despite these advances, our understanding of the dynamic regulation of the methylome remains poor. We have attempted to address this shortcoming by producing a dynamic, six-compartmental model of DNA methylation levels based on the activity of the Dnmt methyltransferase proteins. In addition, the model incorporates the recently discovered Tet family proteins which enzymatically convert methylcytosine to hydroxymethylcytosine. A set of first order, partial differential equations comprise the model and were solved via numerical integration. The model is able to predict the relative abundances of unmethylated, hemimethylated, fully methylated, and hydroxymethylated CpG dyads in the DNA of cells with fully functional Dnmt and Tet proteins. In addition, the model accurately predicts the experimentally measured changes in these abundances with disruption of Dnmt function. Furthermore, the model reveals the mechanism whereby CpG islands are maintained in a hypomethylated state via local modulation of Dnmt and Tet activities without any requirement for active demethylation. We conclude that this model provides an accurate depiction of the major epigenetic processes involving modification of DNA. [Copyright &y& Elsevier]

Abstract: Cancer gene therapy is a promising direction for the treatment of cancer patients. A primary goal of all cancer therapies is to selectively target and kill tumour cells. Such therapies are administered via different approaches, including both viral and non-viral delivery; however, both methods have advantages and disadvantages. Transcriptional targeting enables genes encoding toxic proteins to be expressed directly in cancer cells. Numerous vectors have been created with the purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Data concerning the function of vectors bearing genes that encode cytotoxic proteins under the control of different promoters, including tissue/tumour specific and constitutive promoters, is summarised here. This review focuses on vectors that bear genes encoding diphtheria toxin, Pseudomonas exotoxin A, caspases, gef, streptolysin, and melittin. Data describing the efficacy of such vectors have been summarised. Notably, there are vectors that killed cancer cell lines originating from the same type of cancer with differential efficiency. Thus, there is differential inhibition of cancer cell growth dependent on the cell line. In this review, the constructs employing genes whose expression induces cell death and the efficiency with which they suppress cancer cell growth will be summarised. [Copyright &y& Elsevier]

Abstract: Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC. [Copyright &y& Elsevier]

Abstract: The retroviral-vector-targeted CD59 gene (pSUPER-siCD59) was constructed and transfected into breast cells (MCF-7). The results demonstrated that the retroviral vector-mediated RNAi successfully suppressed human CD59 gene. The expression of CD59 decreased at both mRNA and protein levels. Knockdown of CD59 abrogated its protective effect on complement-mediated cytolysis. Fas and caspase-3 were remarkably upregulated, which induced apoptosis and tumor growth suppression in MCF-7 cells. In addition, overexpression of CD59 promoted the proliferation of MCF-7 cells and inhibited anti-apoptotic Bcl-2 expression. In conclusion, CD59 may be a promising target in the gene therapy of breast cancer. [Copyright &y& Elsevier]

Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer. [Copyright &y& Elsevier]

Abstract: Glioblastoma multiforme (grade IV astrocytoma) is a highly malignant brain tumor with poor treatment options and an average lifespan of 15months after diagnosis. Previous work has demonstrated that BST2 (bone marrow stromal cell antigen 2; also known as PDCA-1, CD137 and HM1.24) is expressed by multiple myeloma, endometrial cancer and primary lung cancer cells. BST2 is expressed on the plasma membrane, which makes it an ideal target for immunotherapy. Accordingly, several groups have shown BST2 mAb to be effective for targeting tumor cells. In this report, we hypothesized that BST2 is expressed in human and mouse brain tumors and plays a critical role in brain tumor progression. We show that BST2 expression is upregulated at both the mRNA and protein level in high grade when compared to low grade human astrocytoma (p <0.05). To test the relevance of BST2, we utilized the intracranially (IC)-injected GL261 cell-based malignant brain tumor mouse model. We show that BST2 mRNA expression is increased in mouse brain IC-injected with GL261 cells, when compared to mouse brain IC-injected with saline at 3weeks post-operative (p <0.05). Furthermore, BST2 immunofluorescence predominantly localized to mouse brain tumor cells. Finally, mice IC-injected with GL261 cells transduced with shRNA for BST2±preincubated with BST2 mAb show no difference in overall lifespan when compared to mice IC-injected with GL261 cells transduced with a scrambled shRNA±preincubated with BST2 mAb. Collectively, these data show that while BST2 expression increases during brain tumor progression in both human and mouse brain tumors, it has no apparent consequences to overall lifespan in an orthotopic mouse brain tumor model. [Copyright &y& Elsevier]

Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAFV600E, as a result of the BRAF T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAFV600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC50 values (0.115–1.156μM) in BRAFV600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC50 values (56.674–1349.788μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF T1799A mutation-selective therapeutic agent for thyroid cancer. [Copyright &y& Elsevier]

Abstract: Delivery vectors having targeting ligands provide an impending impact on cancer gene therapy. Our work focuses on folate mediated targeting induced by conjugating poly(ethylene glycol)–folate (PEG–FA) with arginine modified chitosan polymer having low molecular weight of 15kDa and high degree of quaternization (ATFP15H). The ATFP15H derivative on condensation with plasmid DNA formed nanoparticles with core shell nanostructure. It also affirmed good buffering capacity. The derivative was found to protect DNA from DNase I degradation and also from disassembly in presence of negatively charged plasma proteins. It exhibited blood compatibility in terms of percentage hemolysis, erythrocyte aggregation and also by platelet activation. At a concentration of 10μg, the capability of the derivative to enhance cell growth at normal cell growing conditions was observed. The transfection efficiency was also found to be comparable to PEI when transfected in KB cell line, which over expressed the folate receptor (FR) in presence of 10% fetal bovine serum (FBS). On comparison with native chitosan and trimethylated chitosan, ATFP15H derivative exhibited high cellular uptake and nuclear localization due to the superior colloidal stability attained on conjugation with polyethylene glycol. This has been ascertained by flow cytometry and YOYO labeling of plasmid DNA. [Copyright &y& Elsevier]

Abstract: Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation. [Copyright &y& Elsevier]

Abstract: Epigenetic drugs have been shown to enhance gene expression and drug sensitivity in ovarian cancer cell lines and animal models. Based on promising preclinical studies, DNA methylation inhibitors in combination with existing chemotherapeutic agents have the potential for overcoming acquired drug resistance, laying the foundation for this specific class of epigenetic drug in ovarian cancer clinical trials. The recent completion of phase I trials of decitabine has yielded important information on dosing schedules and biological endpoints for evaluating patient responses. In addition, epigenetic drug effects on pharmacodyamic targets are beginning to emerge, and predictive epigenetic biomarkers and next generation epigenome therapeutics are being developed for application in clinical settings for ovarian cancer patients. [Copyright &y& Elsevier]

Abstract: MicroRNAs are involved in cancer-related processes. The microRNA-21(miR-21) has been identified as the only miRNA over-expressed in a wide variety of cancers, including cervical cancer. However, the function of miR-21 is unknown in cervical carcinomas. In this study, we found that the inhibition of miR-21 in HeLa cervical cancer cells caused profound suppression of cell proliferation, and up-regulated the expression of the tumor suppressor gene PDCD4. We also provide direct evidence that PDCD4-3′UTR is a functional target of miR-21 and that the 18bp putative target site can function as the sole regulatory element in HeLa cells. These results suggest that miR-21 may play an oncogenic role in the cellular processes of cervical cancer and may serve as a target for effective therapies. [Copyright &y& Elsevier]

Abstract: Tumor angiogenesis is the process by which new blood vessels are formed and enhance the oxygenation and growth of tumors. As angiogenesis is recognized as being a critical event in cancer development, considerable efforts have been made to identify inhibitors of this process. Cytostatic treatments that target the molecular events of the angiogenesis process have been developed, and have met with some success. However, it is usually difficult to preclinically assess the effectiveness of targeted therapies, and apparently promising compounds sometimes fail in clinical trials. We have developed a multiscale mathematical model of angiogenesis and tumor growth. At the molecular level, the model focuses on molecular competition between pro- and anti-angiogenic substances modeled on the basis of pharmacological laws. At the tissue scale, the model uses partial differential equations to describe the spatio-temporal changes in cancer cells during three stages of the cell cycle, as well as those of the endothelial cells that constitute the blood vessel walls. This model is used to qualitatively assess how efficient endostatin gene therapy is. Endostatin is an anti-angiogenic endogenous substance. The gene therapy entails overexpressing endostatin in the tumor and in the surrounding tissue. Simulations show that there is a critical treatment dose below which increasing the duration of treatment leads to a loss of efficacy. This theoretical model may be useful to evaluate the efficacy of therapies targeting angiogenesis, and could therefore contribute to designing prospective clinical trials. [Copyright &y& Elsevier]

Abstract: Background: Interleukin (IL)-23, composed of p19 and p40 subunits, has diverse functions in regulating immune systems, enhancing cell-mediated immunity. In the present study, we investigated whether forced expression of the p19-linked p40 gene in murine mammary cancer cells (MA891) produced antitumor effects in vivo. Tumor growth of MA-891 cells expressing IL-23 (IL-23/MA891) in mice was retarded compared with parental and vector DNA-transduced tumors and survival of the mice inoculated with IL-23/MA-891 cells was prolonged. Expressions of the CD4+ T cells and CD8+ T cells were up-regulated not only in IL-23/MA-891 tumor specimens but also in spleen cells of mice inoculated with IL-23/MA-891 as compared with those of mice inoculated with parental or vector DNA-transduced tumors. Cytotoxic CD8+ T lymphocyte (CTL) activity of spleen cells from mice inoculated with IL-23/MA-891 was also significantly higher than the other two groups. Th1-type cytokines such as interferon-γ, TNF-α and IL-12p70 secreted from spleen cells of mice bearing IL-23/MA-891 tumors were increased while Th2-type cytokine IL-4 was negative regulated. Moreover, we have identified that the quantity of DC in spleen cells of mice bearing IL-23/MA-891 tumors was increased as compared with those mice bearing parental or vector DNA-transfected tumors. [Copyright &y& Elsevier]

Abstract: Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2Å resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK. [Copyright &y& Elsevier]

Abstract: Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 (1–78REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated 1–78REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy. [Copyright &y& Elsevier]

Abstract: Much research over the past two decades has focussed on understanding the complex interactions of nitric oxide (NO ) in both physiological and pathological processes. As with many other aspects of NO biology, its precise role in tumour pathophysiology has been the cause of intense debate and we now know that it participates in numerous signalling pathways that are crucial to the malignant character of cancer. The available experimental evidence highlights contrasting pro- and anti-tumour effects of NO expression, which appear to be reconciled by consideration of the concentrations involved. This review addresses the complexities of the role of NO in cancer, whilst evaluating various experimental approaches to NO -based cancer therapies, including both inhibition of nitric oxide synthases, and overexpression of NO using donor drugs or nitric oxide synthase gene transfer. The evidence provided strongly supports a role for manipulation of tumour NO either as a stand-alone therapy or in combination with conventional treatments to achieve a significant therapeutic gain. [Copyright &y& Elsevier]

Abstract: Objective.: Previous studies have demonstrated the pivotal roles of matrix metalloproteinases (MMPs) in cervical cancer progression. Tissue inhibitor of metalloproteinases-3 (TIMP-3) can inhibit all of MMPs, and its overexpression can retard many kinds of tumor growth. Especially, a potent bystander effect would make it to be more advisable for treating cervical cancer whose primary growth pattern is to invade adjacent structures. These characters prompted the authors to explore its further applicability in human cervical cancer. Methods.: We constructed a replication deficient adenoviral vector carrying TIMP-3 (Ad-TIMP-3). It was transferred into different cervical cancer cell lines in vitro and was injected into the tumor xenografts of nude mice in vivo. Results.: Overexpression of TIMP-3 by adenovirus vector arrested cervical cancer cells in G2/M phase. It also promoted growth inhibition of cancer cells by bystander effects. Ad-TIMP-3 infection produced a more potent cell killing effect than Ad-p53 (P <0.05). A synergic effect was observed when Ad-TIMP-3 and cisplatin (cDDP) were used in combination (D <1). In vivo, Ad-TIMP-3 could inhibit cervical cancer xenografts growth. Compared with Ad-TIMP-3 and cDDP groups, tumor growth in Ad-TIMP-3 combined with cDDP group was inhibited more significantly (P <0.05). Conclusion.: These findings indicated that Ad-TIMP-3 bear a therapeutic potential for cervical cancer. [Copyright &y& Elsevier]

Abstract: Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies. [Copyright &y& Elsevier]

Abstract: The use of stem cells as a vehicle of therapeutic genes is an attractive approach for the development of new antitumoral strategies based on gene therapy. The aim of our study was to assess the potential of bone marrow-derived Multipotent Adult Progenitor Cells (rMAPCs) to differentiate in vitro and in vivo into endothelial cells and to be recruited to areas of tumor vasculogenesis. In vitro, rMAPCs obtained from Buffalo rats differentiated into cells expressing endothelial markers and demonstrated functional endothelial capacity. Intravenous injection of undifferentiated rMAPC transduced with a lentivirus expressing GFP in an orthotopic rat model of hepatocellular carcinoma, resulted in tumor recruitment of the injected cells and in vivo differentiation into endothelial cells in the tumor area with contribution to vasculogenesis. In summary, our results suggest that rMAPCs can be efficiently recruited by vascularized tumors and differentiate to endothelium and thus may represent a useful vehicle for delivery of therapeutic genes to sites of active tumor neovascularization. [Copyright &y& Elsevier]

Abstract: Adenovirus vectors (Adv) are used widely in cancer gene therapy research. However, the clinical application of Adv currently is limited to local, intratumoral administration; systemic administration leads to redundant transgene expression in the liver and subsequent hepatotoxicity. Here we replaced the conventional cytomegalovirus (CMV) promoter of Adv with a tumor-specific telomere reverse transcriptase (TERT) promoter, to restrict expression of the Adv-transduced transgene to tumor tissue alone. We evaluated the therapeutic and side effects after systemic administration of Adv expressing herpes simplex virus thymidine kinase (Ad-HSVtk) in mice bearing Meth-A tumors. Although systemically injected CMV promoter-driven Ad-HSVtk lacked therapeutic effect, mice injected with 2×1011 viral particles containing TERT promoter-driven Ad-HSVtk showed inhibited tumor growth and prolonged survival with minimal side effects. Our results suggest that Adv in which transgene expression is driven by the TERT promoter are a promising prototype of tumor-targeting vectors for effective and safe cancer gene therapy. [Copyright &y& Elsevier]

Purpose: Adenoviral gene therapy could potentially play a role in the treatment of esophageal cancer and Barrett’s esophagus. The adenoviruses can be categorized in different serotypes. The goal of the present study was to investigate the transduction efficacy of different adenoviral serotypes in different models of esophageal cancer and Barrett’s esophagus. Methods: Chimeras of the adenoviral serotype 5 backbone and fibers of serotypes 5, 16, 35, 40, and 50 were constructed with PCR technology. For esophageal cancer, cell lines were used originating from with adenocarcinoma and squamous cell carcinoma, respectively. Differentiating Caco-2 cells were used as an in vitro model for Barrett’s esophagus. GFP was used as a reporter gene and transduction efficacy was assessed by flow cytometry. Results: Overall transduction was rather efficient in the cancer cell lines. Especially serotype 16 and 50 exhibited an improved transduction compared with the other serotypes. In the Caco-2 cell lines, we observed a decreased transduction upon differentiation of the cells. All serotypes had a very limited transduction and no serotype had an additional value in this setting. Conclusions: Some serotypes could have an additional value in the development of gene therapy for esophageal cancer. Especially serotype 16 and 50 exhibited an improved transduction in esophageal cancer cells compared with the native serotype 5. In the setting of Barrett’s esophagus, none of the serotypes had an improved potency as in differentiated intestinal cells all serotypes had a very limited transduction. [Copyright &y& Elsevier]

Abstract: Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5′-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5′ deletion analysis identified the −1579bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; −4939bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between −345 and −359bp of the PC-1 5′-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy. [Copyright &y& Elsevier]

Abstract: Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer. [Copyright &y& Elsevier]

Abstract: Objectives. : Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). Methods. : Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (Δ24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. Results. : Compared to RGD-capsid CRAds (Δ24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p =0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p =0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over Δ24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p =0.005) compared to the clinical grade Δ24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p =0.001). Conclusions. : These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer. [Copyright &y& Elsevier]

Background: Using in vivo mouse models, we have demonstrated that the insulin promoter-driven suicidal gene therapy (RIPTK) could be used in the treatment of mouse insulinoma and human pancreatic cancer cells. However, limitations of this therapy include tumor cells lack of sufficient PDX-1 protein and low levels of transgene expression mediated by liposome delivery system. The purpose of this study was to determine 1) whether transient transfection of PDX-1 into selected pancreatic cancer cells would lead to increased RIPTK cytotoxicity, and 2) whether an adenoviral delivery system would increase the overall RIPTK gene expression in vitro. Material and methods: RIPlacZ and RSVlacZ plasmid DNA as well as AdCMVlacZ and AdRIPlacZ were used in transfection assays in human pancreatic cancer cell lines PANC-1 and MIA PaCa2 (n = 8). An expression plasmid DNA containing the mouse PDX-1 cDNA was also used. LacZ reporter assays were performed. RIPTK genes constructed either in plasmid or in adenoviral vectors were used in cytotoxic assays. RT-PCR assays were used to determine PDX-1 expression levels. Results: PDX-1 protein was detected in the human pancreatic ductal carcinoma cell line PANC-1, a little in MIA PaCa2 cells. Liposome mediated (L) RSVlacZ and RIPlacZ transfection in PANC-1 cells resulted in 10.1% and 9.3% transgene expression, respectively. Co-delivery of PDX-1 had no significant effect on RSVlacZ expression (9.3%, P = NS) but significantly increased RIPlacZ gene expression (14.9% P < 0.05). Adenoviral mediated (Ad) RIPlacZ transgene was highly expressed in PANC-1 cells (66.1%) and the reporter activity was further enhanced when PDX-1 was co-delivered (70.2%, P < 0.05). Liposomal transfection of MIA PaCa2 cells using RSVlacZ and RIPlacZ reporter genes resulted in 9.3% and 1.0% gene expression, respectively. Co-transfection of PDX-1 in these cells resulted in a significant activation of RIPlacZ gene expression (14.5%, P < 0.05) with no effects on RSVlacZ treated cells (9.8%). AdCMVlacZ and AdRIPlacZ significantly increased reporter activities in MIA PaCa2 cells (63.0% and 9.8%, respectively). Transfection of PDX-1 also significantly enhanced the AdRIPlacZ activities (46.0%, P < 0.05), with no significant effect in AdCMVlacZ treated cells (68.2%). The cytotoxic effect of liposome-RIPTK/ganciclovir (GCV) in PANC-1 cells was 18.6% and increased to 22.8% when PDX-1 was co-transfected into the cells (P = NS). MIA PaCa2 cells treated with RIPTK alone resulted in 4.9% cell death and increased to 18.2% when exogenous PDX-1 was co-delivered (P < 0.05). The AdRIPTK gene delivery with GCV treatment caused significant cytotoxic effect in PANC-1 (29.3%) and MIA PaCa2 (12.4%) compared with untreated cells. The cytotoxic effects were further increased to 43.4% and 29.4% in PANC-1 and MIA PaCa2 cells, respectively, when PDX-1 was co-transfected (P < 0.05 for both). Conclusions: These data demonstrated that adenoviral mediated gene delivery resulted in a significant increase of transgene expression compared with liposomal delivery systems. RIPTK mediated cytotoxicity was also significantly enhanced via co-delivery of exogenous PDX-1 in these cells. Thus, these results also indicated that PDX-1 plays critical roles in insulin promoter activation and demonstrated that PDX-1 production is essential for insulin promoter-directed gene therapy. [Copyright &y& Elsevier]

Abstract: Objectives. : Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). Methods. : We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. Results. : All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (αv integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. Conclusions. : The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa. [Copyright &y& Elsevier]

Abstract: Objective. : Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. Methods. : Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Δ24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Δ24-RGD, or icodextrin+Δ24-RGD. Survival data were plotted on Kaplan–Meier curve and statistical calculations performed using the log-rank test. Results. : There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Δ24-RGD group, and 105 days for icodextrin+Δ24-RGD (p =0.023). Of note, 5 of the 10 mice in the icodextrin+Δ24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). Conclusions. : These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are currently in development. [Copyright &y& Elsevier]

Background: Transcription factor PDX-1 is expressed by human pancreatic and breast cancers. Although cytotoxicity of PDX-1-directed RIP-TK/GCV gene therapy to pancreatic cancer cells has been demonstrated, the efficacy of this treatment in breast cancer cells is unknown. The purpose of this study was to determine the expression of PDX-1 and its effect on RIP activation in two human breast cancer cell lines, AU565 and T47D. We also investigated the efficacy of RIP-TK/GCV gene therapy and examined whether exogenous PDX-1 to would enhance its cytotoxic effect. Materials and methods: RT-PCR was used to determine PDX-1 expression. Gene constructs RSVLacZ and RIPLacZ were used for transient transfection and LacZ expression was determined using reporter assays. T47D cells were also transfected with adenoviral vectors. Cells were transfected with RIP-TK and the suboptimal level of GCV was determined for each cell line. Following GCV treatment, cytotoxicity was measured using MTS assays. The effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity was determined. Results: PDX-1 mRNA was expressed in human breast cancer cells and activated the RIP. Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not in T47D cells. Adenoviral transfection was more efficient in T47D cells than non-viral transfection. RIP-TK treatment was cytotoxic to AU565 and T47D cells and this effect was enhanced by exogenous PDX-1 with both transfection methods. Conclusions: RIP-TK/GCV therapy is cytotoxic to human breast cancer cells and exogenous PDX-1 enhances cytotoxicity. In vivo studies are necessary to determine the tumor specificity and efficacy of this treatment. [Copyright &y& Elsevier]

Abstract: The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors. [Copyright &y& Elsevier]