Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy.

BACKGROUND: Bacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector. METHODS: In this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice. RESULTS: Claudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression. CONCLUSIONS: This novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo.

Effective Oncoleaking Treatment of Pancreatic Cancer by Claudin-Targeted Suicide Gene Therapy with Clostridium perfringens Enterotoxin (CPE).

Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

Novel jet-injection technology for nonviral intratumoral gene transfer in patients with melanoma and breast cancer.

PURPOSE: This phase I clinical trial evaluated safety, feasibility, and efficiency of nonviral intratumoral jet-injection gene transfer in patients with skin metastases from melanoma and breast cancer. EXPERIMENTAL DESIGN: Seventeen patients were enrolled. The patients received five jet injections with a total dose of 0.05 mg beta-galactosidase (LacZ)-expressing plasmid DNA (pCMVbeta) into a single cutaneous lesion. Clinical and laboratory safety monitoring were done. Systemic plasmid clearance was monitored by quantitative real-time PCR of blood samples throughout the study. All lesions were resected after 2 to 6 days. Intratumoral plasmid DNA load, DNA distribution, and LacZ expression was analyzed by quantitative real-time PCR, quantitative reverse transcription-PCR, Western blot, immunohistochemistry, and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining. RESULTS: Jet injection of plasmid DNA was safely done in all patients. No serious side effects were observed. Thirty minutes after jet injection, peak plasmid DNA levels were detected in the blood followed by rapid decline and clearance. Plasmid DNA and LacZ mRNA and protein expression were detected in all treated lesions. Quantitative analysis revealed a correlation of plasmid DNA load and LacZ-mRNA expression confirmed by Western blot. Immunohistochemistry and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside staining showed LacZ-protein throughout the tumor. Transfected tumor areas were found close and distant to the jet-injection site with varying levels of DNA load and transgene expression. CONCLUSION: Intratumoral jet injection of plasmid DNA led to efficient LacZ reporter gene expression in all patients. No side effects were experienced, supporting safety and applicability of this novel nonviral approach. A next step with a therapeutic gene product should determine antitumor efficacy of jet-injection gene transfer.

Heat-inducible in vivo gene therapy of colon carcinoma by human mdr1 promoter-regulated tumor necrosis factor-alpha expression.

The promoter of the human multidrug resistance gene (mdr1) harbors defined heat-responsive elements, which could be exploited for construction of heat-inducible expression vectors. To analyze the hyperthermia inducibility of the mdr1 promoter in vitro and in vivo, we used the pcDNA3-mdrp-hTNF vector construct for heat-induced tumor necrosis factor alpha (TNF-alpha) expression in transfected HCT116 human colon carcinoma cells at mRNA level by quantitative real-time reverse transcription-PCR and at protein level by TNF-alpha ELISA. For the in vitro studies, the pcDNA3-mdrp-hTNF-transfected tumor cells were treated with hyperthermia at 43 degrees C for 2 h. In the animal studies, stably transfected or in vivo jet-injected tumor-bearing Ncr:nu/nu mice were treated for 60 min at 42 degrees C to induce TNF-alpha expression. Both the in vitro and in vivo experiments show that hyperthermia activates the mdr1 promoter in a temperature- and time-dependent manner, leading to an up to 4-fold increase in mdr1 promoter-driven TNF-alpha expression at mRNA and an up to 3-fold increase at protein level. The in vivo heat-induced TNF-alpha expression combined with Adriamycin (8 mg/kg) treatment leads to the inhibition of tumor growth in the animals. These experiments support the idea that heat-induced mdr1 promoter-driven expression of therapeutic genes is efficient and feasible for combined cancer gene therapy approaches.

Oncoleaking: Use of the Pore-Forming Clostridium perfringens Enterotoxin (CPE) for Suicide Gene Therapy.

Suicide gene therapy has been shown to be very efficient in tumor eradication. Numerous suicide genes were tested in vitro and in vivo demonstrating their therapeutic potential in clinical trials. Apart from this, still growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard bacterial toxins are an alternative, which add to the broad spectrum of different suicide strategies. In this context, the claudin-targeted bacterial Clostridium perfringens enterotoxin (CPE) is an attractive new type of suicide oncoleaking gene, which as pore-forming protein exerts specific and rapid toxicity towards claudin-3- and -4-overexpressing cancers. In this chapter we describe the generation and use of CPE-expressing vectors for the effective tumor cell killing as novel suicide gene approach particularly for treatment of therapy refractory tumors.

Chemosensitization by diverging modulation by short-term and long-term TNF-α action on ABCB1 expression and NF-κB signaling in colon cancer.

Multidrug resistance (MDR) is a major cause for cancer chemotherapy failure. Among the numerous strategies to overcome persistent action of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) permits downregulation of MDR-associated genes, including ATP-binding cassette, subfamily B 1 gene (ABCB1). A key regulator of ABCB1 expression is the transcription factor nuclear factor κ light chain enhancer (NF-κB)/p65. We analyzed diverging short- and long-term effects of TNF-α regarding modulation of NF-κB/p65 signaling and ABCB1 expression in colon cancer cells. Highly resistant ABCB1 overexpressing human HCT15 colorectal carcinoma cells were subjected to short- (30-120 min) or long-term (24-96 h) TNF-α treatment. TNF-α mediated modulation of ABCB1 expression was analyzed by real-time RT-PCR and western blot analysis. The TNF-mediated chemosensitization was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. The involvement of TNF receptors and of NF-κB/p65 signaling was analyzed by western blot analysis, ABCB1 promoter analysis and electrophoretic mobility shift assay (EMSA). The study revealed, that long-term, but not short-term TNF-α treatment leads to TNF-receptor 1 (TNFR1) mediated downregulation of ABCB1 resulting in sensitization towards drug treatment. It dampens NF-κB/p65 activation and nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α (IκBα) resynthesis, associated with reduced nuclear accumulation of NF-κB/p65 and reduced binding to its consensus sequence in the ABCB1 promoter. The study reveals the diverging effects of short- or long-term TNF-α action and provides novel insights on downregulation of ABCB1 expression by TNF-mediated repression of NF-κB signaling.

Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector.

Nonviral gene therapy represents a realistic option for clinical application in cancer treatment. This preclinical study demonstrates the advantage of using the small-size MIDGE(®) DNA vector for improved transgene expression and therapeutic application. This is caused by significant increase in transcription efficiency, but not by increased intracellular vector copy numbers or gene transfer efficiency. We used the MIDGE-hTNF-alpha vector for high-level expression of hTNF-alpha in vitro and in vivo for a combined gene therapy and vindesine treatment in human melanoma models. The MIDGE vector mediated high-level hTNF-alpha expression leads to sensitization of melanoma cells towards vindesine. The increased efficacy of this combination is mediated by remarkable acceleration and increase of initiator caspase 8 and 9 and effector caspase 3 and 7 activation. In the therapeutic approach, the nonviral intratumoral in vivo jet-injection gene transfer of MIDGE-hTNF-alpha in combination with vindesine causes melanoma growth inhibition in association with increased apoptosis in A375 cell line or patient derived human melanoma xenotransplant (PDX) models. This study represents a proof-of-concept for an anticipated phase I clinical gene therapy trial, in which the MIDGE-hTNF-alpha vector will be used for efficient combined chemo- and nonviral gene therapy of malignant melanoma.

Performance of high quality minicircle DNA for in vitro and in vivo gene transfer.

Plasmid DNA is frequently used particularly for nonviral gene therapy. Conventional plasmid DNA contains bacterial backbone and resistance gene sequences, as well as immunogenic CpG motifs. These components are not required for transgene expression. They represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. To overcome these drawbacks, the minicircle technology is removing such sequences, to improve performance and also to reduce DNA size. Here, we show the effective production of luciferase, GFP, or lacZ-carrying minicircle DNA with high yield and reproducible high quality. They are used for lipofection or electroporation gene transfer into human melanoma and colon carcinoma cell lines. Comparison of respective parental plasmid and minicircle-mediated luciferase gene transfer shows improved luciferase expression by minicircle in all cell lines. This is not associated with increase in intracellular minicircle copy numbers after lipofection or electroporation. The minicircles rather mediate enhanced transgene mRNA transcription compared to their parental plasmids. In addition, FACS analysis revealed increase in counts of GFP positive cells after minicircle gene transfer, indicating higher gene transfer rates. Furthermore, minicircle showed also improved performance in vivo after jet-injection gene transfer. Therefore, availability of minicircles with reproducible high quality and sufficient amount makes them an applicable and effective alternative to conventional plasmid gene vectors.

A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVß reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

Intratumoral dispersion, retention, systemic biodistribution, and clearance of a small-size tumor necrosis factor-α-expressing MIDGE vector after nonviral in vivo jet-injection gene transfer.

For nonviral applications of therapeutic DNA, highly efficient and safe vector systems are of crucial importance. In the majority of nonviral approaches plasmid vectors are in use. A novel minimalistic gene expression vector (MIDGE) has been developed to overcome the limitations of plasmid vectors. This small-size double-stranded linear DNA vector has shown improved transgene expression. However, only limited knowledge on uptake, biodistribution, and clearance of this vector exists. In this study we investigated the intratumoral and systemic biodistribution, clearance, and expression kinetics of the tumor necrosis factor (TNF)-α-carrying MIDGE-CMVhTNF vector in NMRI-nu/nu mice with subcutaneously xenotransplanted human A375 melanoma. Biodistribution was analyzed by quantitative real-time PCR in tumors, blood, and organs 0 to 60 min and 3 to 48 hr after intratumoral jet-injection of 50 µg of MIDGE-CMVhTNF. We examined TNF mRNA expression in tumor tissue and organs, using real-time RT-PCR and TNF-specific ELISA. High levels of MIDGE DNA in the tumor tissue demonstrated efficient gene transfer of the small-size vector, resulting in inhomogeneous DNA dispersion and efficient transgene expression. Intratumoral jet-injection of the vector DNA was accompanied by leakage into the blood circuit and appearance in peripheral organs within 5 min to 6 hr. However, this did not lead to TNF-α expression and was followed by rapid vector clearance resulting in the disappearance of MIDGE DNA 24 hr after gene transfer. These data provide important new information for the kinetics of intratumoral and systemic biodistribution and rapid clearance of the jet-injected small-size MIDGE vector.

S100A4-induced cell motility and metastasis is restricted by the Wnt/ß-catenin pathway inhibitor calcimycin in colon cancer cells.

The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/ß-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced ß-catenin mRNA and protein levels despite the expression of Δ45-mutated ß-catenin. Consequently, calcimycin inhibited Wnt/ß-catenin pathway activity and the expression of prominent ß-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.

Activation of the CMV-IE promoter by hyperthermia in vitro and in vivo: biphasic heat induction of cytosine deaminase suicide gene expression.

The cytomegalovirus-immediate early (CMV-IE) promoter is widely used as a strong and constitutively active promoter. Although the CMV-IE promoter does not harbor heat-responsive sequences, we determined its heat inducibility. We analyzed in vitro and in vivo heat responsiveness and possible mechanisms of heat induction of the CMV-IE promoter. We used transfected SW480 human colon carcinoma cells (SW480/CMVCD), expressing CMV-IE promoter-driven bacterial cytosine deaminase (CD) gene. These cells were heated at 42 degrees C. The SW480/CMVCD cells were also used for in vivo studies, in which tumor-bearing animals were treated with hyperthermia at 41.5 degrees C. As controls, SW480 (SW480/HSPCD) cells were used, in which CD expression is driven by the HSP70-promoter. In vitro, we observed a biphasic, up to 25-fold heat induction of CMV-IE-driven CD expression after hyperthermia in SW480/CMVCD cells. In vivo, we found a 2.5-fold induction of CD expression after hyperthermia in SW480/CMVCD tumor-bearing animals. The analysis of the CMV-IE promoter sequence revealed several transcription factor-binding sites, which mediate stress responsiveness. YB-1 and C/EBP-beta might mediate heat responsiveness of the CMV-IE promoter. These data point to limitations in heat-induction gene therapy studies, in which the CMV-IE promoter is used as control system. In addition, the CMV-IE promoter itself could well be used for construction of heat-inducible vectors.

Nonviral jet-injection gene transfer for efficient in vivo cytosine deaminase suicide gene therapy of colon carcinoma.

Jet-injection technology has developed into an efficient gene delivery system for nonviral in vivo gene transfer. In this study the jet-injector system was used for the intratumoral gene transfer of small volumes of naked DNA encoding the Escherichia coli cytosine deaminase (CD) suicide gene. In our in vivo studies human colon carcinoma (patient-derived tumor model Colo5734 and SW480 colon carcinoma)-bearing NMRI-nu/nu male mice received four jet injections (10 microl per injection) of the CD-gene-carrying plasmid, representing 40 microg plasmid DNA per animal. Forty-eight hours after jet-injection, treatment of tumors with 5-fluorocytosine (5-FC; 500 mg/kg ip) was started and during treatment tumor volumes were measured. Starting from day 5 of 5-FC treatment inhibition of tumor growth was seen in the CD-gene-transduced tumors compared to the respective control groups, which lasted for the entire observation time. Expression analysis at the mRNA and protein levels revealed efficient expression of the CD gene in the jet-injected tumors. Therefore, in this in vivo study jet-injection gene transfer of 40 microg CD-expressing naked plasmid DNA leads to a significant tumor growth inhibition. This study demonstrates the applicability of the jet-injection technology for in vivo gene transfer into tumors to achieve efficient tumor gene therapy.

Interleukin-3 promotes proliferation and differentiation of human hematopoietic stem cells but reduces their repopulation potential in NOD/SCID mice.

In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34(+) cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.