Neoadjuvant calcium electroporation for potentially curable colorectal cancer.

BACKGROUND: The development of new perioperative treatment modalities to activate the immune system in colorectal cancer might have a beneficial effect on reducing the risk of recurrence after surgery. Calcium electroporation is a promising treatment modality that potentially modulates the tumor microenvironment. The aim of this study was to evaluate the safety of the procedure in the neoadjuvant setting in localized left-sided colorectal cancer (CRC). METHODS: The study included patients with potentially curable sigmoid or rectal cancer with no indication for other neoadjuvant treatment. Patients were offered calcium electroporation as a neoadjuvant treatment before elective surgery. Follow-up visits were conducted on the preoperative day before elective surgery, POD2, POD14, and POD30, with an evaluation of adverse events, impact on elective surgery, clinical examination, and quality of recovery. RESULTS: Endoscopic calcium electroporation was performed as an outpatient procedure in all 21 cases, with no procedure-related complications reported. At follow-up, five adverse events were registered, two of which were classified as serious adverse events. Surgery was performed as planned in 19 patients (median time to surgery, 8 days), and the final two patients underwent surgery with a delay due to adverse events (14 and 33 days). No significant impact on the quality of recovery scores nor inflammatory markers were seen before and after calcium electroporation, nor baseline and POD30. CONCLUSIONS: Endoscopic calcium electroporation is a safe and feasible procedure in patients with potentially curable CRC. The study showed limited side effects and limited impact on the following elective surgical resection.

Electrochemotherapy in the treatment of cutaneous malignancy: Outcomes and subgroup analysis from the cumulative results from the pan-European International Network for Sharing Practice in Electrochemotherapy database for 2482 lesions in 987 patients (2008-2019).

BACKGROUND: Electrochemotherapy (ECT) is a treatment for both primary and secondary cutaneous tumours. The international Network for sharing practices on ECT group investigates treatment outcomes after ECT using a common database with defined parameters. METHODS: Twenty-eight centres across Europe prospectively uploaded data over an 11-year period. Response rates were investigated in relation to primary diagnosis, tumour size, choice of electrode type, route of bleomycin administration, electrical parameters recorded and previous irradiation in the treated field. RESULTS: Nine hundred eighty-seven patients, with 2482 tumour lesions were included in analysis. The overall response (OR) rate was 85% (complete response [CR]: 70%, partial response rate: 15%, stable disease: 11%, and progressive disease: 2%). For different histologies, OR and CR rates for metastases of malignant melanoma were 82% and 64%, basal cell carcinoma were 96% and 85%, breast cancer metastases were 77% and 62%, squamous cell carcinoma were 80% and 63% as well as Kaposi's sarcoma were 98% and 91%, respectively. Variance was demonstrated across histotypes (p < 0.0001) and in accordance with size of lesion treated (dichotomised at diameter of 3 cm (p < 0.0001). Hexagonal electrodes were generally used for larger tumours, but for tumours up to 3 cm, linear array electrodes provided better tumour control than hexagonal electrodes (80%:74%, p < 0.003). For tumours more than 2 cm, intravenous administration was superior to intratumoural (IT) administration (p < 0.05). Current recorded varied across tumour histologies and size but did not influence response rate. In previously irradiated areas, responses were selectively lower for IT administration. CONCLUSIONS: These cumulative data endorse efficiency of ECT across a broad range of histotypes. Analysis of 2482 lesions details subgroup analysis on treatment response informing future treatment choices.

Calcium electroporation and electrochemotherapy for cancer treatment: Importance of cell membrane composition investigated by lipidomics, calorimetry and in vitro efficacy.

Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy.

Calcium electroporation for treatment of cutaneous metastases; a randomized double-blinded phase II study, comparing the effect of calcium electroporation with electrochemotherapy.

BACKGROUND: Calcium electroporation is a novel anticancer treatment, which utilizes high voltage pulses to permeabilize cell membranes and expose the cell to supraphysiological doses of calcium. Preclinical studies on calcium electroporation have shown strikingly high tumor response with cell necrosis. Calcium electroporation builds on the treatment electrochemotherapy, where chemotherapeutic drugs, mostly bleomycin, are internalized by electroporation. This double-blinded randomized study compared calcium electroporation to electrochemotherapy in terms of objective response measured 6 months after treatment. METHODS: Seven patients with a total of 47 cutaneous metastases from breast cancer and malignant melanoma were included in the protocol. A total of 37 metastases were randomized and evaluated for response, another 10 metastases were used for biopsy. This was a non-inferiority trial and metastases were randomized individually in each patient to either intratumoral calcium or bleomycin followed by application of electric pulses to tumor site. All metastases were treated once, and after 6-months of follow-up, the randomization code was revealed. RESULTS: Objective response of calcium electroporation was 72% (13/18) with complete response in 66% (12/18). For electrochemotherapy, objective response was 84% (16/19) with complete response in 68% (13/19). There was no statistically significant difference between the two treatments (p = 0.5). After 1 year, only three out of 25 metastases had relapsed. Ulceration, itching and exudation were reported slightly more frequently in metastases treated with bleomycin, and hyperpigmentation was only seen in metastases treated with bleomycin. CONCLUSION: This study shows that calcium electroporation is feasible and effective in patients with cutaneous metastases.

Electrochemotherapy of unresectable cutaneous tumours with reduced dosages of intravenous bleomycin: analysis of 57 patients from the International Network for Sharing Practices of Electrochemotherapy registry.

BACKGROUND: Electrochemotherapy (ECT) is currently used to treat unresectable superficial tumours of different histotypes through the combination of cytotoxic chemotherapy and local application of electric pulses. In 2006, a collaborative project defined the ESOPE (European Standard Operating Procedures of Electrochemotherapy) guidelines to standardize the procedure. The International Network for Sharing Practices of Electrochemotherapy (InspECT) aims to refine the ESOPE and improve clinical practice. Limiting patient exposure to systemic chemotherapy would be advisable to ameliorate ECT safety profile. OBJECTIVE: The aim of this study was to evaluate the efficacy and toxicity of ECT with reduced chemotherapy dosages. METHODS: In a retrospective analysis of a prospectively maintained database (InspECT registry), we evaluated the outcome of patients who received ECT with reduced dosages of bleomycin (7500, 10 000 or 13 500 IU/m2 , instead of the standard dose of 15 000 IU/m2 ). Tumour response in melanoma patients was compared with melanoma patients of the InspECT registry who received the standard dose of bleomycin. RESULTS: We identified 57 patients with 147 tumours (melanoma, 38.6%; squamous cell carcinoma, 22.8%; basal cell carcinoma, 17.5%; breast cancer 7%; Kaposi sarcoma 7%; other histotypes, 7.1%). Per-tumour complete response (CR) rate at 60 days was 70.1% (partial, 16.3%); per-patient CR was 57.9% (partial, 21.1%). Local pain was the most frequently reported side-effect (n = 22 patients [39%]), mostly mild; two patients experienced flu-like symptoms, one patient nausea. We observed the same CR rate (55%) in patients with melanoma treated by reduced or conventional bleomycin dosages (P = 1.00). CONCLUSIONS: Electrochemotherapy performed with reduced bleomycin dosages could be as effective as with currently recommended dose. Patients with impaired renal function or candidate to multiple ECT cycles could benefit from a reduced dose protocol. Our findings need prospective confirmation before being adopted in clinical practice.

Electrochemotherapy in the treatment of metastatic malignant melanoma: a prospective cohort study by InspECT.

BACKGROUND: (ECT) is an effective local treatment for cutaneous metastasis. Treatment involves the administration of chemotherapeutic drugs followed by delivery of electrical pulses to the tumour. OBJECTIVES: To investigate the effectiveness of ECT in cutaneous metastases of melanoma and to identify factors that affect (beneficially or adversely) the outcome. METHODS: Thirteen cancer centres in the International Network for Sharing Practices on Electrochemotherapy consecutively and prospectively uploaded data to a common database. ECT consisted of intratumoral or intravenous injection of bleomycin, followed by application of electric pulses under local or general anaesthesia. RESULTS: In total, 151 patients with metastatic melanoma were identified from the database, 114 of whom had follow-up data of 60 days or more. Eighty-four of these patients (74%) experienced an overall response (OR = complete response + partial response). Overall, 394 lesions were treated, of which 306 (78%) showed OR, with 229 showing complete response (58%). In multivariate analysis, factors positively associated with overall response were coverage of deep margins, absence of visceral metastases, presence of lymphoedema and treatment of nonirradiated areas. Factors significantly associated with complete response to ECT treatment were coverage of deep margins, previous irradiation of the treated area and tumour size (< 3 cm). One-year overall survival in this cohort of patients was 67% (95% confidence interval 57-77%), while melanoma-specific survival was 74% (95% confidence interval 64-84%). No serious adverse events were reported, and the treatment was in general very well tolerated. CONCLUSIONS: ECT is a highly effective local treatment for melanoma metastases in the skin, with no severe adverse effects noted in this study. In the presence of certain clinical factors, ECT may be considered for local tumour control as an alternative to established local treatments, or as an adjunct to systemic treatments.

Electrochemotherapy: technological advancements for efficient electroporation-based treatment of internal tumors.

Electrochemotherapy, a combination of high voltage electric pulses and of an anticancer drug, has been demonstrated to be highly effective in treatment of cutaneous and subcutaneous tumors. Unique properties of electrochemotherapy (e.g., high specificity for targeting cancer cells, high degree of localization of treatment effect, capacity for preserving the innate immune response and the structure of the extracellular matrix) are facilitating its wide spread in the clinics. Due to high effectiveness of electrochemotherapy in treatment of cutaneous and subcutaneous tumors regardless of histological origin, there are now attempts to extend its use to treatment of internal tumors. To advance the applicability of electrochemotherapy to treatment of internal solid tumors, new technological developments are needed that will enable treatment of these tumors in daily clinical practice. New electrodes through which electric pulses are delivered to target tissue need to be designed with the aim to access target tissue anywhere in the body. To increase the probability of complete tumor eradication, the electrodes have to be accurately positioned, first to provide an adequate extent of electroporation of all tumor cells and second not to damage critical healthy tissue or organs in its vicinity. This can be achieved by image guided insertion of electrodes that will enable accurate positioning of the electrodes in combination with patient-specific numerical treatment planning or using a predefined geometry of electrodes. In order to be able to use electrochemotherapy safely for treatment of internal tumors located in relative proximity of the heart (e.g., in case of liver metastases), the treatment must be performed without interfering with the heart's electrical activity. We describe recent technological advances, which allow treatment of liver and bone metastases, soft tissue sarcomas, brain tumors, and colorectal and esophageal tumors. The first clinical experiences in these novel application areas of electrochemotherapy are also described.

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 microg EPO plasmid and were subsequently electroporated. With plate electrodes and 100 microg 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.

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.

Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation.

Electroporation-based gene transfer (electro gene transfer (EGT)) is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. Induction of expression using the Tet-On system was previously established; however, attempts to reach a predefined target dose - a prescription, have not been reported. We set three target haemoglobin levels (10, 12 and 14 mmol/l, base level was 8.2 mmol/l) and aimed at them by transferring the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle, and varying (1) DNA amount, (2) muscle mass transfected and (3) induction with the Tet-On system. Results showed that (a) using GFP, luciferase and EPO low DNA amounts were needed. In fact, 0.5 microg of DNA to one TC muscle led to significant Hgb elevation - this amount extrapolates to 1.4 mg of DNA in humans, (b) three prescribers hit the targets with average Hgb of 10.5, 12.0 and 13.7 mmol/l, (c) different approaches could be used, (d) undershooting could be corrected by retransferring, and (e) overshooting could be alleviated by reducing dose of inducer (doxycycline (dox)). In conclusion, this study shows that using EGT to muscle, a preset level of protein expression can be reached. This is of great interest for future clinical use.

Evaluation of cytotoxic effect of photodynamic therapy in combination with electroporation in vitro.

Under the influence of electric pulses cells undergo membrane electroporation (EP), which results in increased permeability of the membrane to exogenous compounds. EP is applied in oncology as a method to enhance delivery of anticancer drugs. For that reason it was essential to combine photodynamic tumor therapy (PDT)--the cancer treatment method based on the use of photosensitizers that localize selectively in malignant tumors and become cytotoxic when exposed to light, and EP, with the aim to enhance the delivery of photosensitizers into the tumor and therefore to increase the efficacy of PDT. Thus, the aim of study was to evaluate the cytotoxic effect of PDT in combination with EP. A Chinese hamster lung fibroblast cell line (DC-3F) was used. The cells were affected by photosensitizers chlorin e(6) (C e(6)) at the dose of 10 mug/ml and aluminium phthalocyanine tetrasulfonate (AlPcS4) at the dose of 50 microg/ml. Immediately after adding of photosensitizers the cells were electroporated with 8 electric pulses at 1200 V/cm intensity, 0.1 ms duration, 1 Hz frequency. Then, after 20 min of incubation the cells were irradiated using a light source--a visible light passing through a filter (KC 14, emitted light from 660 nm). The fluence rate at the level of the cells was 3 mW/m(2). Cytotoxic effect on cells viability was evaluated using MTT assay. Our in vitro data showed that the cytotoxicity of PDT in combination with EP increases fourfold on the average. Based on the results we suggest that EP could enhance the effect of PDT.

Elevated neutrophil and monocyte counts in peripheral blood are associated with poor survival in patients with metastatic melanoma: a prognostic model.

We aimed to create a prognostic model in metastatic melanoma based on independent prognostic factors in 321 patients receiving interleukin-2 (IL-2)-based immunotherapy with a median follow-up time for patients currently alive of 52 months (range 15-189 months). The patients were treated as part of several phase II protocols and the majority received treatment with intermediate dose subcutaneous IL-2 and interferon-alpha. Neutrophil and monocyte counts, lactate dehydrogenase (LDH), number of metastatic sites, location of metastases and performance status were all statistically significant prognostic factors in univariate analyses. Subsequently, a multivariate Cox's regression analysis identified elevated LDH (P<0.001, hazard ratio 2.8), elevated neutrophil counts (P=0.02, hazard ratio 1.4) and a performance status of 2 (P=0.008, hazard ratio 1.6) as independent prognostic factors for poor survival. An elevated monocyte count could replace an elevated neutrophil count. Patients were assigned to one of three risk groups according to the cumulative risk defined as the sum of simplified risk scores of the three independent prognostic factors. Low-, intermediate- and high-risk patients achieved a median survival of 12.6 months (95% confidence interval (CI), 11.4-13.8), 6.0 months (95% CI, 4.8-7.2) and 3.4 months (95% CI, 1.2-5.6), respectively. The low-risk group encompassed the majority of long-term survivors, whereas the patients in the high-risk group with a very poor prognosis should probably not be offered IL-2-based immunotherapy.

Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research.

Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. This transient, permeabilized state can be used to load cells with a variety of different molecules, either through simple diffusion in the case of small molecules, or through electrophoretically driven processes allowing passage through the destabilized membrane--as is the case for DNA transfer. Initially developed for gene transfer, electroporation is now in use for delivery of a large variety of molecules: From ions to drugs, dyes, tracers, antibodies, and oligonucleotides to RNA and DNA. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumours has been performed. Whereas initial electroporation procedures caused considerable cell damage, developments over the past decades have led to sophistication of equipment and optimization of protocols. The electroporation procedures used in many laboratories could be optimized with limited effort. This review (i) outlines the theory of electroporation, (ii) discusses factors of importance for optimization of electroporation protocols for mammalian cells, (iii) addresses particular concerns when using electroporation in vivo, e.g. effects on blood flow and considerations regarding choice of electrodes, (iv) describes DNA electrotransfer with emphasis on use in the in vivo setting, and (v) sums up data on safety and efficacy of electroporation used to enhance delivery of chemotherapy to tumours in cancer patients.

Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer.

Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. We have evaluated the effects of various combinations of square wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulse) or a series of four low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the (51)Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by four long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may represents a clear improvement in view of further clinical development.

Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy.

Electric pulses can cause transient permeabilization of cell membranes (electroporation) and this can be utilized to increase the uptake of chemotherapy (electrochemotherapy). Preclinical studies have shown that in vivo electroporation causes transient shut down of blood flow both in normal and, in particular, malignant tissues. We report the successful palliation of a malignant melanoma patient with bleeding skin metastases using electrochemotherapy. In an on-going study of combined electrochemotherapy and low dose interleukin-2, one patient with bleeding skin metastases was included. Nine skin metastases, of which seven were ulcerated, were treated. After intratumoral bleomycin injection, needle electrodes with two arrays 4 mm apart were inserted into the tumours. Eight square wave electric pulses each 99 micros in duration and with an applied voltage to electrode distance ratio of 1.2 kV/cm were administered. In all the treated lesions, bleeding immediately stopped on administration of the electric pulses and did not recur. The treated metastases developed crusts and the lesions healed in a matter of weeks. Treatments were given under local anaesthesia, lasted a few minutes, and patient discomfort was brief and modest. In conclusion, we propose that electrochemotherapy should be considered for the palliation of haemorrhaging metastases as it is an efficient, tolerable, brief, outpatient, once-only treatment.

Electroporation of muscle tissue in vivo.

Electroporation (also termed electropermeabilization) of muscle tissue has been studied in several contexts. It has been shown that electroporation plays an important role in muscle damage as a result of electrical injury (1,2) and that electroporation of cardiac muscle occurs during defibrillation or cardioversion (3,4). As electroporation has been shown to greatly enhance the cytotoxic effect of certain chemotherapeutic agents, and as clinical Phase I-II studies (5-8) have shown that the combination of electroporation and chemotherapy (electrochemotherapy) is highly efficient against various localized cancers, the question of normal tissue sensitivity to electroporation needs to be investigated. Finally, efficient in vivo gene transfection to muscle tissue by electroporation has recently been reported (9,10), warranting increased knowledge on in vivo electroporation of muscle tissue.

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.

Determination of optimal parameters for in vivo gene transfer by electroporation, using a rapid in vivo test for cell permeabilization.

In vivo gene transfer to muscle tissue by electroporation can produce long-term, high-level gene expression. In the present study, we report quantitative results on muscle fiber permeabilization using Cr(51)-EDTA as a marker, and we analyze the influence of electric field strength, pulse duration, and pulse number. The comparison of these results to recently published data on gene transfer (Mir et al., P.N.A.S. (USA), 1999), using an identical experimental setup provides the basis for discussing the importance of the level of permeabilization for gene transfer. The threshold for permeabilization was determined by measuring uptake of Cr(51)-EDTA, and DNA transfer was optimal for field strengths just above the threshold for permeabilization. This means that when designing in vivo electric-field-mediated gene transfer protocols for various tissues, determining the threshold for permeabilization using a rapid test such as incorporation of Cr(51)-EDTA, can be used to predict the optimal window for gene transfer.

High-efficiency gene transfer into skeletal muscle mediated by electric pulses.

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.