Jutta Aumann

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

Calcimycin restricts Wnt/β-catenin–regulated S100A4 expression, leading to reduced S100A4-mediated cell migration and invasion in colon cancer cells, as well as to inhibition of metastasis formation in xenografted mice.

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.

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 β-galactosidase (LacZ)-expressing plasmid DNA (pCMVβ) 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-β-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-β-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.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

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.

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 α (TNF-α) expression in transfected HCT116 human colon carcinoma cells at mRNA level by quantitative real-time reverse transcription-PCR and at protein level by TNF-α ELISA. For the in vitro studies, the pcDNA3-mdrp-hTNF–transfected tumor cells were treated with hyperthermia at 43°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°C to induce TNF-α 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-α expression at mRNA and an up to 3-fold increase at protein level. The in vivo heat-induced TNF-α 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. [Mol Cancer Ther 2007;6(1):236–43]

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.

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 pCMVβ 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.

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.

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.