Anita Gothelf

Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation

Over the last decade a new cancer treatment modality, electrochemotherapy, has emerged. By using short, intense electric pulses that surpass the capacitance of the cell membrane, permeabilization can occur (electroporation). Thus, molecules that are otherwise non-permeant can gain direct access to the cytosol of cells in the treated area.A highly toxic molecule that does not usually pass the membrane barrier is the hydrophilic drug bleomycin. Once inside the cell, bleomycin acts as an enzyme creating single- and double-strand DMA-breaks. The cytotoxicity of bleomycin can be augmented several 100-fold by electroporation. Drug delivery by electroporation has been in experimental use for cancer treatment since 1991. This article reviews 11 studies of electrochemotherapy of malignant cutaneous or subcutaneous lesions, e.g., metastases from melanoma, breast or head- and neck cancer. These studies encompass 96 patients with altogether 411 malignant tumours. Electroporation was performed using plate or needle electrodes under local or general anaesthesia. Bleomycin was administered intratumourally or intravenously prior to delivery of electric pulses. The rates of complete response (CR) after once-only treatments were between 9 and 100% depending on the technique used. The treatment was well tolerated and could be performed on an out-patient basis.

Gene electrotransfer to skin; review of existing literature and clinical perspectives.

Gene electrotransfer, which designates the combination of gene transfer and electroporation, is a non-viral means for transfecting genes into cells and tissues. It is a safe and efficient method and reports regarding the use of this technique in a variety of animal models and organs have been published in the literature. We find that gene electrotransfer to skin is of particular interest; not only due to the easy accessibility of this organ, which renders both treatment and evaluation feasible, but also the capability of the skin to produce transgenes and elicit immunological responses. Up to now more than 40 papers have been published in which gene electrotransfer was the technique used for gene transfection to skin in vivo. The aim of this review is to summarize which plasmids were injected and the electrical parameters applied. Furthermore an overview of the clinical perspectives of gene electrotransfer to skin will be presented.

What you always needed to know about electroporation based DNA vaccines

Vaccinations are increasingly used to fight infectious disease, and DNA vaccines offer considerable advantages, including broader possibilities for vaccination and lack of need for cold storage. It has been amply demonstrated, that electroporation augments uptake of DNA in both skin and muscle, and it is foreseen that future DNA vaccination may to a large extent be coupled with and dependent upon electroporation based delivery. Understanding the basic science of electroporation and exploiting knowledge obtained on optimization of DNA electrotransfer to muscle and skin, may greatly augment efforts on vaccine development. The purpose of this review is to give a succinct but comprehensive overview of electroporation as a delivery modality including electrotransfer to skin and muscle. As well, this review will speculate and discuss future uses for this powerful electrotransfer technology.

Duration and level of transgene expression after gene electrotransfer to skin in mice

In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations. Level and duration of transgene expression after gene electrotransfer to skin is essential and here we present data from two independent quantitative studies. Using in vivo bioimaging of a far-red fluorescent molecule, Katushka, allowing for continuous monitoring of local gene expression, compared with measurements of a systemic transgene, that is, serum erythropoietin (EPO) after gene electrotransfer with EPO to skin, we found a significant increase in transgene expression (P< 0.01) with a peak 9 days (Katushka) and 14 days (EPO) after transfection. Duration of expression could be 3–4 weeks, which is a suitable time frame for vaccinations and is applicable, for example, in gene therapy for wound healing or treatment of cancer.

Therapeutic levels of erythropoietin (EPO) achieved after gene electrotransfer to skin in mice

Gene electrotransfer refers to gene transfection by electroporation and is an effective non-viral method for delivering naked DNA into cells and tissues. This study presents data from gene electrotransfer with erythropoietin (EPO) to mouse skin. Nine-week-old female NMRI mice received one, two or three intradermal injections of 50 μg EPO plasmid and were subsequently electroporated. With plate electrodes and 100 μg of EPO, a significant increase in hemoglobin (P<0.01) was observed compared with controls. The level of hemoglobin peaked after 5 weeks but stayed significantly elevated for more than 3 months. Serum EPO was significantly increased (P<0.001) 24 h after the transfection and remained significantly different compared with controls until the maximum level of serum EPO was reached after 2 weeks. Eight weeks after the transfection serum EPO returned to baseline. In this study, we have established that gene electrotransfer to skin of even small amounts of DNA can lead to systemically therapeutic levels of protein. This means that in addition to DNA vaccinations, there is a potential utility for electroporation in alleviating systemic diseases such as cancer and protein deficiency disorders.

Gene Electrotransfer to Skin

Gene electrotransfer to skin is achieving increasing interest and is likely to gain considerable clinical application due to the ease with which it is performed and the safety of the procedure. There is a potential use of gene electrotransfer to skin in e.g., DNA vaccinations, local production of therapeutic molecules as well as production of molecules for systemic therapy. More than 30 preclinical studies concerning gene electrotransfer to skin have been reported in the literature and this chapter aims at creating an overview of plasmids injected, electrical parameters used, and duration and level of transgene expression.

Efficacy of transgene expression in porcine skin as a function of electrode choice

Gene electrotransfer is a non-viral technique using electroporation for gene transfection. The method is widely used in the preclinical setting and results from the first clinical study in tumours have been published. However, the preclinical studies, which form the basis for the clinical trials, have mainly been performed in rodents and the body of evidence on electrode choice and optimal pulsing conditions is limited. We therefore tested plate and needle electrodes in vivo in porcine skin, which resembles human skin in structure. The luciferase (pCMV-Luc) gene was injected intradermally and subsequently electroporated. Simultaneously, studies with gene electrotransfer to porcine skin using plasmids coding for green fluorescent protein (GFP) and betagalactosidase were performed. Interestingly, we found needle electrodes to be more efficient than plate electrodes (p<0.001) and electric field calculations showed that penetration of the stratum corneum led to much more homogenous field distribution at the DNA injection site. Furthermore, we have optimised the electric pulse regimens for both plate and needle electrodes using a range of high voltage and low voltage pulse combinations. In conclusion, our data support that needle electrodes should be used in human clinical studies of gene electrotransfer to skin for improved expression.

Paclitaxel, cisplatin and gemcitabine in treatment of carcinomas of unknown primary site, a phase II study

Abstract Background. The present study was conducted to evaluate the efficacy and toxicity of a combination of paclitaxel, cisplatin and gemcitabine in patients with carcinoma of unknown primary site (CUP). Patients and methods. Patients with CUP, ECOG performance status 0–1 and age between 18 and 65 years old were treated with paclitaxel 175 mg/m2 day 1, cisplatin 75 mg/m2 day 1 and gemcitabine 1000 mg/m2 day 1 and 8 in a three-week schedule. Results. Ninety-eight patients were enrolled between 1998 and 2008. Ninety-one patients had target lesions according to the RECIST guidelines. The overall response rate was 42.9% (39 patients), including five complete responses (5.5%) and 34 partial responses (37.4%). The median survival time was 10.7 months, and the survival rates at one and two years were 42% and 14%, respectively. The most frequent grade 3 or more adverse events were neutropenia and thrombocytopenia. There were 3 treatment-related deaths. Conclusions. Combination of paclitaxel, cisplatin and gemcitabine is an active regimen in patients with CUP with response and survival rates at least similar to other platinum- and taxane-containing regimens. The treatment was well tolerated by most patients although neutropenia and thrombocytopenia were relatively common. The present regimen represents an attractive regimen in younger CUP patients with a good performance status.

Change in Hemoglobin Levels due to Anesthesia in Mice: An Important Confounder in Studies on Hematopoietic Drugs

Analgesic and anesthetic drugs may have an impact on the results achieved from animal experiments. In the study presented here, we try to enlighten whether anesthesia with fentanyl/fluniasone and midazolam (Hypnorm and Dormicum) has an influence on measurements of hemoglobin in mice. In a cross-over study, we have compared hemoglobin levels in two groups of mice: anesthetized versus non-anesthetized and found significant decrease in hemoglobin levels in the anesthetized group (p < 0.05) unrelated to which group received the anesthesia. The mean hemoglobin levels after intraperitoneal administration of Hypnorm and Dormicum was 8.7 mmol/L compared to mean hemoglobin 9.9 mmol/L before anesthesia (p < 0.001), and the decrease lasted for more than 30 min. These results show that anesthesia can be an important confounder in studies involving measurements of hemoglobin, and this should be taken into account when planning studies and analyzing data.

Electroporation-Based DNA Delivery Technology: Methods for Gene Electrotransfer to Skin

DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses.