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

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

Walther W, Schmeer M, Kobelt D, et al. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis. Human gene therapy. Clinical development. 2013;24(4):147-153.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

Additional file 4: Figure S3. of Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

Influence of optCPE in vivo gene transfer on body weight. Body weight of Co7515* PDX bearing mice was measured during tumor growth inhibition. In all animals no systemic toxicities, such as body weight loss, were observed, which strongly indicates the safety of this gene therapeutic approach. (JPG 283 kb

Additional file 2: Figure S1. of Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

Knockdown of claudin-3 and -4 leads to reduced CPE activity in human colon cancer cells. a Sequences of used short interfering RNA (siRNA) targeting claudin-3 and -4. b Western blot analysis for claudin-3 and claudin-4 gene expression in human colon cancer cell lines SW480 (left panel) and HCT116 (right panel) 72 h after siRNA treatment, showing an efficient down-regulation of both with two independent siRNA compared to control (siCo). c Specific toxin responsiveness of claudin-3 and -4 down-regulated colon cancer cells. 72 h after siRNA transfection tumor cells were treated with recCPE at indicated concentrations for another 72 h. The cytotoxicity was determined by MTT assay and compared to siCo treated cells. A significantly reduced responsiveness (*** P < 0.0001) was demonstrated in both colon cancer cell lines, SW480 (left panel) and HCT116 (right panel). All assays were performed in two independent experiments and are expressed as mean percent of untreated control. Bars: SD. Level of significance was calculated by 2way-ANOVA (Bonferroni posttest). b Cytotoxicity of optCPE gene transfer in siRNA treated colon cancer cells and proof of claudin specificity. The siCldn3 + siCldn4 treated SW480 and HCT116 cells were transfected with optCPE construct 72 h after siRNA treatment. MTT assay was performed 72 h after CPE treatment and a significantly reduced CPE mediated cytotoxicity was observed in down-regulated SW480 (left panel) and also in HCT116 (right panel) cells compared to siCo treated cells. All assays were performed in two independent experiments and expressed as survival in optical density [OD]. Bars: SD. Level of significance was calculated by nonparametric, unpaired students t-test, *** P < 0.0001. Both assays demonstrate high selectivity of CPE on claudin-3 and -4 as down-regulated cells remain unaffected. (JPG 600 kb

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