We report the use of plasmid DNA-mediated combination gene therapy for tumor-bearing mice using in vivo electroporation, also called electro-gene therapy (EGT), that resulted in uncomplicated and complete cures in more than 90% of the mice. Subcutaneously inoculated CT26 tumors in syngeneic BALB/c mice were subjected to repeated EGT treatments consisting of intratumoral coinjection of naked plasmids encoding the cytokine interleukin-12 (IL-12) (p35 and p40 subunits) and the suicide gene herpes simplex virus thymidine kinase (HSV-tk), followed by in vivo electroporation. The early anti-tumor effect was always stronger, and the rate of cure, as seen in the long-term follow-up, was always greater in the groups treated with combination EGT than in those treated with IL-12 or HSV-tk EGT alone. Systemic levels of IL-12 and IFN-gamma increased in both combination and IL-12-alone EGT-treated groups. Moreover, combination EGT for established subcutaneous tumors strongly reduced hematogenous lung metastases and increased survival time when live CT26 tumor cells were injected through the tail vein. Limited experiments on C57/B16 mice with murine melanoma also showed very similar trends. These results suggest that this simple and safe method of plasm id-mediated combination EGT may provide a potentially effective gene therapy for cancer.

Interest continues to grow rapidly in clinical gene therapy. Although many protocols approved by the US NIH Recombinant Advisory Committee involve the genetic marking of cells to determine, for example, the source of relapse after autologous bone marrow transplantation for certain malignancies (1), increasing numbers are concerned with the correction of inherited single-gene disorders.

We report on an antitumor treatment involving electrogene therapy (EGT), a newly developed in vivo gene transfer method using electroporation. We carried out in vivo EGT in a subcutaneous model of CT26 colon carcinoma cells, using plasmid DNAs encoding interleukin 12 (IL-12) subunits. For this purpose, we developed two IL-12 expression systems: a cotransfer system using a plasmid encoding the IL-12 p40 subunit and a plasmid encoding the IL-12 p35 subunit, and a single-vector system using a plasmid expressing a p40-p35 fusion protein. Both transfer systems significantly inhibited the growth of CT26 tumor. Immunohistochemical analysis of IL-12 EGT-treated tumors revealed enhanced infiltration of CD8(+) cells into the tumor tissue, while reverse transcriptase-polymerase chain reaction confirmed the increased expression of interferon gamma within treated tumors. The same IL-12 EGT applied to the nude mouse model was not effective, suggesting the critical role of T cell infiltration in this treatment. The inhibitory effects revealed in experiments in which previously treated mice were rechallenged with a second inoculation of CT26 tumor cells suggested that IL-12 EGT may also establish partial systemic antitumor immunity. The growth of IL-12 EGT-treated Renca tumors, a renal cell carcinoma, was also significantly inhibited. These findings suggest that EGT of the IL-12 gene has the potential to be an effective anticancer gene therapy.

Cancer of the pancreas is currently the fifth leading cause of cancer related deaths with a five year survival of less than 1% In the United States (1). It is one of the most difficult cancers to treat, since it is hard to detect in the early stages. The patients remain asymptomatic until late in the course of the disease. An excellent review of pancreatic carcinoma has appeared (2). Despite the progress made in our understanding of the biology of this cancer (3), the final outcome for this disease has remained extremely poor. Conventional chemotherapeutic agents have not been very effective for human pancreatic adenocarcinoma (4). Use of intratumoral chemotherapy in combination with monoclonal antibodies have been reported to produce better response rate and also reduced toxicity (5,6). Smith and colleagues (7) have recently shown that an injectable gel with a sustained release profile can inhibit tumor growth in vivo in human pancreatic cancer xenografts. This was demonstrated in nude mice with BxPC-3 xenografts using fluorouracil, cisplatin, and doxorubicin with a consequent size reduction of the tumors between 72% and 79%, compared to the controls at day 28 after the first treatment. Although these figures are impressive, by any standard, no cure was reported.

In vivo electroporation. first reported in 1987, makes it possible to render cell membranes temporarily permeable to substances that otherwise would not be able to effectively enter the cell interior. Micro- or millisecond pulses of electrical field strengths exceeding the natural cellular transmembrane potential difference of approximately 1 V results in permeabilization ("poration") of cell membranes. This phenomenon opens up numerous applications in the medical field. Electroporative delivery of chemotherapeutic drugs into tumor cells has proven successful in clinical studies to treat malignant tumors and is nearing market introduction in Europe. For gene therapy applications, delivery of DNA by electroporation into a variety of tissues has been shown to consistently result in a 100-1000-fold enhancement of gene expression. Other applications of electroporation discussed in this paper include intravascular delivery of drugs and genes with electroporation catheters, electroinsertion of molecules into membranes, intraocular delivery of drugs and genes, and transdermal drug delivery. The use of electroporation for drug and gene delivery in vivo is clearly gaining momentum, and new medical applications are emerging at an increasing rate.

Electroporation, a standard laboratory method of introducing exogenous molecules into cells, has been gaining importance as a very effective non-viral physical technique of gene delivery. In this study, we have used subcutaneous model of the C6 rat glioma cells and established an optimal condition to obtain very high gene expression in tumor tissues using both reporter and functional genes. Tumors grown on the flanks of Wistar rats are exposed and directly injected with plasmid DNA having the constructs of luciferase, green fluorescent protein and, the fragment of the diphtheria toxin, DT-A. The tumors are then subjected to square wave pulses from an electroporator. Gene expression is found to be several orders of magnitude higher when the tumors are pulsed with the optimized electrical parameters compared to the controls. For luciferase, the enhancement is approximately 135-fold, for the green fluorescent protein, gene expression is seen over a wide area within the sections examined, as contrast to a few punctate dots in the control specimens, and finally, DT-A shows massive death in the tumor tissue. A special circular array of six needles through which pulses are delivered with rotating electric field is found to be highly efficient in transferring genes inside the tumor. Direct injection of plasmid DNA followed by electroporation allows very high in vivo gene transfer and its subsequent expression into tumor tissues. This method may be applicable to any solid tumor.

Hemophilia B is an X-linked genetic disorder that typically results from chronic circulating deficiency of blood coagulation factor IX (FIX) (1). While the occurrence of hemophilia B is significantly less frequent than hemophilia A (factor VIII, deficiency) it has received special attention as a model for gene therapy. This is because hemophilia B is one of the least complicated genetic diseases from the point of view of demonstrating the proof of principle of a gene therapy protocol. Specifically, hemophilia B is a single gene recessive disorder and a wide range of tissues can be targeted for FIX gene delivery and strict regulation of FIX expression is not required. In addition, the 2.8 kb FIX cDNA is much smaller than the 9 kb FVIII cDNA, and FIX expression in transfected mammalian cells has been less problematic than FVIII expression (2). Since clinical severity of bleeding episodes closely corresponds to a patient's FIX activity, achieving even partial restoration of normal FIX levels in the bloodstream can alleviate internal bleeding. Individuals with FIX levels less than 1% of normal experience severe symptomatic episodes but providing roughly 5% of normal levels (i.e., 250 ng/mL plasma) can significantly reduce the frequency and severity of bleeding episodes and reduce long term complications (3). Treatment of hemophilia B primarily relies on intravenous injections of FIX protein purified from pooled human plasma, or very recently, on newly developed recombinant FIX. Treatment is applied typically only when bleeding episodes have occurred or are expected, for example, in case of a trauma or surgery. Although the risk of viral transmission of HIV and hepatitis viruses has been largely eliminated the absolute safety of any product derived from blood cannot be guaranteed. Furthermore, supplies of factor concentrates are limited and costs (especially if prophylactic treatment is being considered) are high. Thus, the application of gene therapy to hemophilia, whereby long-term correction of factor IX deficiency might be achieved, would be extremely useful.

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

The curative effects of some chemotherapeutic drugs are impeded by their poor permeation through the cell membrane. This limitation can be overcome by a novel approach called electroporation therapy (EPT), electrochemotherapy (ECT), or electrical impulse chemotherapy (EIC). The method involves application of brief electrical pulses, which destabilize the cell membrane barrier, allowing intracellular access of chemotherapeutic drugs that otherwise would not be able to penetrate the cell membrane effectively. EPT makes it possible to lower the drug dose, thereby relieving the patient of adverse side effects associated with conventional chemotherapy. Even with the lower drug dose, EPT has shown significantly higher efficacy than has conventional chemotherapy. The method is currently being evaluated clinically for treating various cancer indications using the anticancer drugs bleomycin or cisplatin. This article provides a historical perspective and current insights into this new modality of cancer treatment, including basic physical, biological, and medical facts about EPT; computer-assisted development of electrical pulse generators and electrodes necessary to create effective electrical fields in the treatment area; results of cancer cell and tumor treatments in vitro, in animals, and in humans; safety aspects of EPT; potential combined delivery of chemotherapeutic drugs and biological agents to reduce or eliminate metastatic disease; and intracellular delivery of DNA by electroporation for cancer gene therapy.

Electroporation can deliver exogenous molecules like drugs and genes into cells by pulsed electric fields through a temporary increase in cell membrane permeability. This effect is being used for the treatment of cancer by intratumoral injection of low dosage of an otherwise marginally effective chemotherapeutic drug, bleomycin. Application of a pulsed electric field results in substantially higher uptake of the drug and enhanced killing of the cancer cells than is possible by conventional methods. The MedPulser(R), a new treatment system for local electroporation therapy (EPT) of head and neck tumors was developed and is described in this paper. EPT with bleomycin has been found to be very effective in killing cancer cells in vitro, in mouse tumor xenografts in vivo, and in tumors in humans. Ten head and neck cancer patients with recurring or unresponsive tumors were enrolled in a Phase I/II clinical trial. Treatment of the entire turner mass in each of eight patients resulted in five complete responses confirmed by biopsy and MRI, and three partial responses (/spl ges/50% shrinkage). Two additional patients who received partial treatment of their tumor mass had local response where treated, but no overall lesion remission. Duration of the complete responses ranges from 2-10 months to date. All patients tolerated the treatment well with no significant local or systemic adverse effects.

BACKGROUND:A new method of cancer treatment called electroporation therapy (EPT) which uses pulsed electric fields in combination with a chemotherapeutic agent is being developed to treat human pancreatic tumors. Such a combination has been found to increase the cytotoxic effect of the drug to tumor cells.METHODS:Human pancreatic tumors (Pan-4-JCK) were implanted subcutaneously onto nude mice. The animals were treated with EPT using bleomycin, mitomycin C or carboplatin as a single agent, and their effect on tumor growth was monitored over a period of 89 days.RESULTS:The tumors treated with either the drug or pulse alone showed increased tumor growth. However, tumors treated with EPT using any one of the three drugs showed significant to complete regression of tumors. Among the three drugs used, the order of efficacy was: bleomycin >> mitomycin C > carboplatin.CONCLUSIONS:These results are sufficient to warrant limited clinical trials of EPT for pancreatic cancers.

BACKGROUND:Electroporation therapy (EPT) uses reversible membrane permeabilization of cells by electrical pulses for intracellular delivery of poorly permeating drugs like bleomycin. This treatment modality has been found to significantly increase the cytotoxic effect of the drug to tumor cells.METHODS:Tumors of human epidermoid carcinoma of larynx (HEp-2) were xenografted subcutaneously in nude mice. EPT consisted of intratumoral injection of belomycin followed by 6 x 100 microseconds square wave electrical pulses of 1130 Volts. The effect of treatment on tumor growth was monitored over a period of 67 days.RESULTS:Complete regression of the tumors was observed in 83% of the treated mice 67 days after treatment. These findings were confirmed by histopathological analysis of tumor samples from the treated sites, which showed complete absence of tumor cells.CONCLUSIONS:The results indicate that electroporation therapy is very effective and has potential for treating laryngeal tumors clinically.

Cytotoxicity of anticancer drugs such as bleomycin, to tumor cells can be significantly enhanced by combining it with pulsed electric fields. This new modality of cancer treatment called electrochemotherapy has been very effective for localized tumors with tremendous potential for treatment of internal tumors. The present work deals with ECT experiments on human pancreatic and nonsmall cell lung cancers grown subcutaneously on the flank of nude mice. Results of pancreatic cancer indicate complete turner regressions in nearly 88% of the cases 28 days after a single treatment with 6 pulses of 1370 V and 99 mu s duration. About 64% of these mice showed no palpable tumor for 120 days after treatment, a complete cure by WHO definition. Histopathological studies sh owed absence of tumor cells in the excisional biopsy of tissue from tumor region. The ultrasonic guidance of acupuncture needles in internal tumors was found feasible indicating treatment possibilities in situ.

Electroporation therapy (EPT), also known as electrochemo-therapy (ECT), of a poorly differentiated human pancreatic carcinoma (Panc-3) implanted subcutaneously in nude mice significantly enhanced the cytotoxicity of bleomycin to tumor cells. A single treatment of intratumoral injection of bleomycin followed by 6 × 99 μs square wave electrical pulses of 1370 V resulted in complete tumor regression in 68% and partial regression (>80%) in 20% of the treated mice on day 28 following treatment. No palpable tumor was observed in 64% of the mice even 120 days after treatment. Histological studies of tissue samples taken from tumor sites 120 days after treatment in the D+E+ group showed complete absence of tumor cells.

Electroporation is a standard laboratory technique originally developed for in vitro transfer of molecules into cells. It involves application of electrical pulses ranging from micro- to milliseconds that create transient pores in the cell membrane allowing intracellular access of exogenous molecules. This technique has been successfully applied to regress tumors in animal models by combining electroporation with chemotherapeutic agents--a process known as electrochemotherapy (ECT) which substantially enhance cytotoxicity of some antineoplastic agents. Recently ECT has moved into clinical arena and patients with cutaneous tumors and head and neck cancers have been treated very effectively with ECT. Parallel to ECT, a technique has also been developed which makes it possible to inject plasmid DNA and combine it with in vivo electroporation--electro--genetherapy (EGT)--to deliver in a highly efficient manner both marker and functional genes into target tissue and achieve gene expression. Thus, in vivo electroporation is contributing to the development of a new strategy for cancer treatment with both drugs and genes.

This article is a compilation of the author's own observations on how biology is changing and is likely to change in the next decade or two, with views from leading biologists on current and future developments, particularly in terms of solving some of the most fundamental problems. Starting from the message of 'universality' which, in the author's view, is coded into the DNA structure, the possible consequences of the completion of the human genome project, namely the sequencing of all of the 50 000-100 000 human genes, the impact of new technology, especially in the field of medicine, and the relentless march of molecular biology in deciphering the secret of life are described. The role of bacteria in new drug development, routine use of gene therapy to tackle single gene disease, problems of evolution, behaviour, and development, and prediction of three-dimensional protein structure from amino acid sequences are discussed. It is argued that biology and technology have to go hand in hand to maintain the current momentum in the life sciences.