Introduction

Gene therapy is a novel approach for treatment or prevention of different genetic and non-genetic diseases. The most straightforward approach to therapeutic gene delivery is direct injection of plasmid DNA into the tissue of interest (1). However, this is a relatively inefficient method of gene transfer with highly variable interindividual transfection efficiency. Preclinical studies show that transfection efficiency of plasmid DNA application alone can be significantly enhanced when it is followed by electroporation (EP) of target tissue (2, 3).

EP is a method for delivery of various molecules into the cells by transiently increasing permeability of cell membrane using application of controlled external electric field to the cells (4). It is already well established as an in vitro method for increasing delivery of various molecules (e.g., RNA, DNA, oligonucleotides, dyes, ions, chemotherapeutic drugs, etc.) into different types of cells. In vivo, it is gaining much interest as a tool for two prospective therapeutic modalities in cancer treatment: electrochemotherapy, which is already used in clinical practice (i.e., application of controlled electric pulses to tumor cells in order to increase uptake and cytotoxicity of chemotherapeutic drugs bleomycin and cisplatin) (5, 6), and electrogene therapy (i.e., combining injection of plasmid DNA, encoding therapeutic genes into target tissue with application of electric pulses) (2, 3), which is already being tested in a number of clinical trials (7). A number of different types of tissues have been successfully transfected in vivo using this approach, including tumors (8, 9), skeletal muscle (10, 11), skin (12, 13), and liver (14).

In skeletal muscle EP enhances expression of plasmid DNA up to 2000-times and significantly reduces variability of gene expression, compared to application of plasmid DNA alone, resulting in long-term expression of exogenous DNA, which can last up to 1 year (11). Electrotransfer of plasmid DNA into skeletal muscle has been successfully achieved in different experimental animals: mice, rats and rabbits (10, 11), cattle (15, 16), goats (15), sheep (17), pigs (18, 19), dogs (20, 21), and monkeys (22). There have been two different types of EP protocols developed for this purpose. At first, only low voltage (LV) electric pulses with long duration (100-200 V/cm, 20-50 ms) were successfully used. Lately, it has been shown that a better transfection efficiency can be achieved using combination of one high voltage (HV) electric pulse (600-800 V/cm, 100 μs) followed by different numbers of LV electric pulses (80-100 V/cm, duration in time range of tens to hundreds of milliseconds) (23, 24). It has been hypothesized that the HV pulse first causes permeabilization of cell membrane, followed by electrophoresis of DNA across destabilized cell membrane during the LV pulses (23, 25). However, recent published paper presented evidence that does not support this hypothesis (26); therefore, further research is clearly needed to elucidate this aspect of electrotransfer of plasmid DNA into the cells.

To date there have been only a few reports on electrotransfection of canine skeletal muscle (20, 21, 27, 28). They account of two different plasmids that were efficiently transfected: plasmid encoding growth hormone-releasing hormone and plasmid encoding human coagulation factor IX. Three similar EP protocols were used for transfection of these two plasmids, all employing LV pulses only. None of the published experiments on dogs has yet utilized the combination of high and low voltage pulses.

Proinflammatory cytokine interleukin-12 (IL-12) has been extensively studied as an antitumor agent, since it exhibits a number of activities, which are potentially important in immunotherapy of cancer, including induction of T-cells and triggering of interferon-γ response. Despite having potential in anticancer therapy (29, 30), systemic application of recombinant IL-12 can result in severe toxicity (31). Therefore, a new approach to IL-12 immunotherapy, utilizing gene therapy, has been investigated and successfully employed on preclinical level on different types of tumors (32, 33, 34, 35, 36).

Canine and human IL-12 share approximately 90% genetic identity based on amino acid sequence analysis (37). Human IL-12 activates proliferation of canine peripheral blood mononuclear cells (PBMC) in in vitro setting and triggers a number of immune responses in canine PBMC (38), which leads to speculation that it could have therapeutic potential in dogs.

The aim of this study was to compare the efficiency of established low voltage EP protocols with EP protocols utilizing combination of HV and LV pulses, which to date have never been used in dogs. For this purpose we used plasmid encoding green fluorescent protein (GFP), injected into m. semitendinosus of beagle dogs, followed by delivery of five different EP protocols. Furthermore, the best two of these protocols were evaluated for transfection efficiency of a single intramuscular application of therapeutic gene, encoding human IL-12. Additionally, local and systemic side effects of electrotransfection of both plasmids were assessed.

Materials and Methods

Experimental Animals

In our experiments, one female and five male beagle dogs, aged from 8-10 years and weighing 13-23 kg, were used. The study was approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia with the limitation on the number of animals used in the study. All procedures on animals were performed under general anesthesia. Prior to all experimental procedures, complete blood count with differential white blood cell count was performed in all animals, using automated laser haematology analyser (Technicon H^*1, Bayer, Germany) with species-specific software (H^*1 Multi-Species V30 Software). Automated chemistry analyser Technicon RA-XT (Bayer, Germany) was used for determination of the following biochemical parameters: blood urea, creatinine, serum alkaline phosphatase (SAP), and creatinine kinase (CK). In addition, alanin aminotransferase was determined only in the second part of experiment, featuring plasmid encoding human IL-12.

Plasmids

In our experiments, two different plasmids were used: pEGFP-N1, encoding GFP (Clontech Laboratories, Inc.; Mountain View, CA, USA) and pORF-hIL-12 (InvivoGen, San Diego, CA, USA), encoding human IL-12. They were both prepared using the Qiagen Maxi Endo-Free kit (Qiagen, Hilden, Germany), according to manufacturer?s instructions and diluted to concentration of 1 mg/ml. Purified plasmid DNA was subjected to quality control and quantity determinations, performed by agarose gel electrophoresis and by means of spectrophotometry. Quality control included the ratio 260/280 between 1.7-1.9 and minimum presence of genomic DNA and RNA determined on gel electrophoresis. The purified DNA contained less that 0.1 EU of bacterial endotoxin per μg of DNA according to the manufacture declaration.

Plasmid Transfection Protocol

In the first part of the study, plasmid encoding GFP was electrotransfected into skeletal muscle of experimental animals. Dogs were premedicated with combination of acepromazine (Promace, Fort Dodge Animal Health, Iowa, USA; 0.02 mg/kg of bodyweight) and metadone (Heptanon, Pliva Zagreb, Croatia; 2 mg/kg of bodyweight). Thirty minutes later, general anesthesia was induced using thiopental (Nesdonal, Merial, Lyon, France; 5 mg/kg of bodyweight) and maintained with isoflurane (Forane, Abbott Laboratories LTD, Queensborough, United Kingdom). During the anesthesia the animals were receiving Hartmann?s solution (B. Braun Melsungen AG, Melsungen, Germany) at rate 10 ml/kg of bodyweight/hour.

In animals under general anesthesia hair on femoral regions of both legs was clipped and regions were surgically prepared. Incision of the skin and fascia was made in order to expose m. semitendinosus, followed by infiltrative intramuscular injection of 150 μg of plasmid encoding GFP, using 1 ml syringe with 22 G needle. Infiltrative injection is an injection where part of tissue is infiltrated with single application. Position of the needle is slightly changed during emptying of syringe, allowing the content of syringe to infiltrate the target tissue more uniformly. A surgical suture was placed on muscle at the exact site of plasmid injection using nonresorbable polifilament material (Sofsilk® 3-0, USSC, Norwalk CT, USA) to allow future identification of exact plasmid application site for correct placement of electrodes and performing biopsies. Electric pulses were applied to muscles 20 minutes after plasmid injection, according to data available in the literature (39, 40). Electric pulses generator Cliniporator™ (IGEA s.r.l., Carpi, Italy) was employed, using needle electrodes N-18-4B (IGEA s.r.l., Carpi, Italy), which consist of two arrays, each composed of four electrodes with 4 mm distance between them. Skin incisions were closed with standard surgical procedure immediately after application of electric pulses and animals were let to recover from anesthesia spontaneously. Postsurgically, analgesia was provided to all dogs with single intravenous application of carprofen (Rimadyl, Pfizer Animal Health, Dundee, United Kingdom; 4mg/kg of bodyweight).

In the first part of the study using GFP, altogether five different EP protocols were used, each applied to two muscles in such order that each dog received two different EP protocols. Three of these protocols (EP 1 - EP 3) utilized combination of 1 HV pulse, followed by different number of LV pulses. Lag between the HV pulse and the first LV pulse in all three protocols was 1 s. The duration of LV pulses was kept in all protocols constant in order to avoid tissue heating due to the longer duration of pulses delivery in the case of protocols EP2 and EP3. Another two protocols were performed by application of LV pulses only: one of them (EP 4) was the standard train of eight identical pulses of 200 V/cm, 20 ms duration, which was long considered as an optimal EP protocol for electrotransfer of DNA into skeletal muscle. The second low voltage EP protocol (EP 5), utilizing six pulses of 100 V/cm, 60 ms, was chosen due to the fact that this is a protocol which has been employed in majority of published experiments using muscle targeted electrotransfer on dogs (24). The control group received only plasmid application without electric pulses. Details of each protocol are provided in Table I.

Approximately one month after the completion of the first part of the study, electrotransfer of plasmid encoding human IL-12 was performed on the same six animals under the same conditions. The animals were divided into two groups, each comprising three dogs. Two different EP protocols were used, one on each experimental group (Table II). Different doses of plasmid DNA were injected infiltratively into the right m. semitendinosus muscle of each dog, using part of the muscle, as far as possible from the site, where electrotransfer of plasmid encoding GFP was performed. Plasmid was injected through intact skin, followed by transcutaneous positioning of needle electrodes and delivery of EP protocol 20 minutes after the plasmid injection (Table II). Postprocedure recovery and analgesia was provided in the same manner as in the first experiment.

Assessment of Transfection Efficiency

In case of experiment, featuring plasmid encoding GFP, transfection efficiency was assessed on day 2 and day 7 after the electrotransfection procedure. Dogs were anaesthetized using the same protocol as described above and skin sutures were removed. At both time points incision biopsies of transfected muscles were performed, each time removing approximately 0.5 cm × 0.5 cm × 0.5 cm of muscle tissue. Skin incisions were closed with standard surgical procedure immediately after collection of muscle samples and animals received single intravenous application of carprofen.

Samples were embedded in Tissue-Tek O.C.T. Compound (Miles Inc., Elkhart, IN, USA) at -20 °C. Frozen samples were cut into 20 μm thick sections. Transfection efficiency was determined by assessment of green fluorescence using fluorescence microscope (Olympus BX51, Olympus, Hamburg, Germany). Green fluorescence of each sample was evaluated by two independent observers in a blind fashion, with estimation of fluorescence intensity ranging from zero (no visible fluorescence) up to maximum of 5 points.

In case electrotransfection of plasmid encoding human IL-12, transfection efficiency was evaluated with determination of serum concentrations of human IL-12 and canine IFN-γ. Blood samples were collected before and on day 2, 7, 14, and 28 after the procedure. Human IL-12 was determined using Interleukin-12 (p40) (human) ELISA (DRG Instruments GmbH, Marburg, Germany). Canine IFN-γ was determined using Quantikine® Canine IFN-γ Immunoassay (R&D Systems, Inc., Minneapolis, USA).

Assessment of Side Effects

In both parts of our study, animals were under close observation in the first month after the experimental procedure in order to assess possible local and systemic side effects of performed electrotransfection. Clinical examination of each animal was conducted on daily basis as well as assessment of appearance of the area on the leg, where electric pulses were delivered, for any clinical signs, including erythema, edema, pain, secretions, necrosis, et cetera. On day 2, 7, 14, and 28 after both electrotransfection procedures, the same hematological and biochemical analyses were performed, as prior to the procedures (described above), in order to assess possible systemic effects of the electrotransfection procedure on the experimental animals.

Results

Electrotransfection of Plasmid Encoding GFP

Transfection Efficiency: The highest level of GFP fluorescence in the muscle was observed in two sets of muscle samples (Table I): samples, taken from the group, where EP 2 (1 HV pulse, followed by 4 LV pulses) was applied and from the group, where EP 4 (8 LV pulses) was applied. In both protocols significant GFP fluorescence was detectable both 2 and 7 days after the transfection (Fig. 1).

Figure 1: Images of frozen tissue sections of muscle samples, in which GFP fluorescence was detected. Similar level of GFP fluorescence was observed in two sets of muscle samples: in group, where EP 2 (1 HV + 4 LV) was applied (A and B, which represent samples collected on 2nd and 7th day, respectively) and in group, where EP 4 (8 x LV) was applied (E and F, which represent samples collected on 2nd and 7th day, respectively). EP 3 (1 HV + 8 LV) yielded in markedly lower degree of transfection, observed only on 2nd day (C). In this group, only autofluorescence could be observed in muscle samples, collected seven days after electrotransfection (D).

Markedly lower degree of transfection was achieved using EP 3 (1 HV pulse, followed by 8 LV pulses) (Fig. 1). In this group, GFP fluorescence was less pronounced compared to EP 2 or EP 4. Furthermore, fluorescence was detectable only in samples, taken at day 2 after electrotransfection. In this group, no GFP fluorescence was observed in muscle samples, taken one week after the procedure.

No GFP fluorescence was detectable either at day 2 or 7 days after electrotransfection on muscle samples, taken from control group and from groups, where EP 1 (1 HV pulse, followed by 1 LV pulse) or EP 5 (6 LV pulses) were used (Table I).

Assessment of Side Effects of the Procedure: Throughout the whole electrotransfer procedure dogs? heart and respiratory rate, ECG, end tide CO₂, noninvasive blood pressure and temperature were routinely monitored for purposes of safe conduct of general anesthesia. During delivery of electric pulses to dogs, increase of dog?s heart and respiratory rate were noted for up to 60% (data not shown). This response correlated with delivery of low voltage electric pulses, during which significant muscle contractions of hind legs were also noticed. Heart rate returned to baseline values immediately after intravenous application of analgesic (ketamine hydrochloride, Bioketan, Vetoquinol, Paris, France; 1 mg/kg), additionally demonstrating that the heart rate acceleration was consequence of painful stimulus.

In order to record any possible local side effects, caused by electrotransfection procedure, clinical examination of each experimental animal was performed on daily basis. It was established that the procedure was very well tolerated by the animals. The only observed side-effect was tissue swelling at the site of electroporation, which spontaneously resolved within two days after the procedure, without any signs of impaired locomotory function. Clinically, we couldn?t detect any marked difference in severity of observed side effects with regard to different EP protocols used.

To determine possible systemic side effects, complete blood count with differential white blood cell count and selected biochemistry parameters were analyzed in blood samples, collected before the procedure and 2, 7, 14, and 28 days afterwards. All hematological parameters measured in samples, collected before the procedure, were within reference limits, except one dog with mild thrombocytosis (platelets 564 × 10⁹/L; reference values 200-500 × 10⁹/L) and three cases of mild relative neutropenia (neutrophils ranging from 57%-59.7%; reference values 60-80%). After the procedure, only mild alterations from reference values were observed, including relative neutropenia on day 28 in one dog that received EP 2 and EP 4 protocols (neutrophils 50%), relative lymphocytosis in two dogs treated with EP 1, EP 4 and untreated (lymphocytes 35.9% in one dog on day 7 and 42% in one dog on day 28; reference values 12-35%), and thrombocytosis in two dogs treated with EP 1, EP 3, EP 4, and untreated (platelet count 544 × 10⁹/L in one dog on day 2 and 684 × 10⁹/L in one dog on day 28). These alterations were not associated with any clinical signs in animals.

Selected biochemistry parameters of dogs, measured before electrogene transfer demonstrated values of blood urea and CK to be within reference limits (reference values for dogs are presented in Table III), low concentration of creatinine (mean value 50.05 μmol/L), and slightly elevated activity of SAP (mean value 138.08 U/L). Similar biochemistry profile could be seen in dogs after the procedure. Urea and CK remained within reference limits, concentration of creatinine was low at all three post-procedure measures (mean value 49.6 μmol/L at day 2, 77.5 μmol/L at day 7, and 71.4 μmol/L at day 28) and activity of SAP was slightly elevated at day 7 and 28 (mean value 129.5 U/L and 139.4 U/L, respectively). In animals none of these alterations in biochemistry parameters could be detected clinically.

Electrotransfection of Plasmid Encoding Human IL-12

Serum Concentrations of Human IL-12 and Canine IFN-γ: In order to determine serum concentrations of human IL-12 in dogs, blood samples were collected in different time points after the single electrically-assisted intramuscular delivery of plasmid encoding human IL-12. Human IL-12 was detected with ELISA in only one serum sample, which was collected seven days after the electrotransfection procedure, from dog No. 3 (Table II).

Canine IFN-γ was detected at different time points after the electrotransfer procedure in serum samples of three animals (Table II), including dog No. 3, the only animal with detectable human IL-12 levels. In this dog, induction of interferone response did not correlate with production of human IL-12, since IFN-γ was detected in serum sample, taken on day 2 and human IL-12 was detected on day 7 after the electrotransfer procedure.

Assessment of Side Effects of the Procedure: In the first month after electrotransfection of therapeutic plasmid, animals didn?t show any abnormalities in their clinical status or behavior. Tissue swelling at the site of electroporation (Fig. 2), which was observed, was slightly less pronounced as in the first part of experiment, where we used plasmid encoding GFP.


Figure 2: Local side effects of electrotransfection procedure seen as a tissue swelling at the site of injection of plasmid encoding human IL-12 and subsequent transcutaneous application of electric pulses (arrow).

All hematological parameters measured in samples, collected before the procedure, were within reference limits. The only exception was a dog with clinically nonsignificant thrombocytosis (platelets 684 × 10⁹/L). Hematological parameters measured in samples, collected after the procedure, were mainly within reference limits with only two exceptions: mild haemoconcentration on day 28 in dog No. 2 (hemoglobin 187 g/L; reference values 115-180 g/L) and No. 5 (hemoglobin 185 g/L). The only animal, in which human IL-12 was detected (dog No. 3), had all hematology parameters within reference limits at all four measurements after the procedure.

There were some minor abnormalities in biochemistry parameters in blood samples, collected before the procedure, as well as in samples, taken at selected time points after the procedure. However, altered postprocedure values did not have any clinical value, and could not be directly linked to effects of electrotransfection procedure. Typical values of biochemistry parameters are presented in Table III, which shows biochemistry profile of all six dogs on day 7 after electrotransfection of therapeutic plasmid. Measurements on all other time points were similarly uncharacteristic (data not shown).

Discussion

Results of our study suggest, that combination of high and low voltage pulses is more suitable for use in muscle targeted DNA electrotransfer in dogs, than use of standard established trains of low voltage pulses only. Although the best transfection of plasmid encoding GFP was achieved with either combination of one HV pulse (600 V/cm, 100 μs), followed by four LV pulses (80 V/cm, 100 ms, 1Hz) or eight identical LV pulses (each 200 V/cm, 20 ms, 1Hz), the first protocol was shown to be more suitable for potential clinical use. Electrogene therapy, using either of these two plasmids was associated with only mild and transient local side effects and did not result in any detectable systemic toxicity.

In the first part of the study, reporter gene encoding GFP was used to evaluate transfection efficiency of five different electroporation protocols, selected according to published reports and recommendations for successful transfection of skeletal muscle using EP in different animal species. Historically, first successful in vivo attempts have used only low voltage pulses, according to results of comprehensive study by Mir and colleagues on a number of different animals (11). In that study it was determined that the optimal protocol for electrotransfection of skeletal muscle consists of delivery of eight identical pulses with electric field strength 200 V/cm, duration of 20 ms, and repetition frequency 1 Hz, which became standard optimal EP protocol for electrotransfection of skeletal muscle. In dogs, three different EP protocols have been successfully used for efficient muscle targeted electrotransfection. The highest number of dogs in published experiments received plasmid encoding growth-hormone releasing hormone, either using EP protocol consisting of six pulses of 100 V/cm and 60 ms duration (20, 27), or five pulses of 100 V/cm and 52 ms duration (28). Another plasmid encoding human clotting factor IX, was delivered using six pulses of 200 V/cm, 60 ms duration, and reversed polarity after each pulse (21).

In vitro studies regarding the role of electric pulses in DNA electrotransfer demonstrated that electric pulses could have two roles in successful DNA electrotransfer: electropermeabilization of target cell membrane and electrophoretic effect on DNA molecules. This hypothesis has been investigated by in vivo experiments in mice featuring use of two different types of square wave electric pulses for electrotransfection of skeletal muscle (23, 25). It was proposed that the HV pulse (600-800 V/cm) of very short duration (100 μs) causes permeabilization of muscle cells. This field strength was shown to be the highest not to cause irreversible electropermeabilization of muscle cells (41). Subsequent LV pulses (usually less than 100 V/cm, with duration in time range from tens to hundreds of ms) act on DNA molecules, enabling electrophoretic movement of DNA molecules toward and across the cell membrane. However, a recently published study (26) indicates that electrophoretic force may not play as important role in movement of DNA molecules across cell membrane as proposed in these reports. Nevertheless, different combinations of HV and LV pulses were evaluated, and combination of one HV pulse, followed by 4 LV pulses was as efficient as the standard electroporation protocol of 8 identical LV pulses of 200 V/cm, 20 ms, 1 Hz (25).

Our results on dogs confirm and extend the results of previous studies on mice. In addition, to date, the combination of HV and LV pulses has not yet been used in any of the published studies employing electrotransfection of canine skeletal muscle. In our study, the highest transfection efficiency was achieved using either EP 2, i.e., combination of one HV pulse (600 V/cm, 100 μs), followed by four LV pulses (each 80 V/cm, 100 ms, repetition frequency 1 Hz), or using EP 4, which was standard train of eight identical pulses of 200 V/cm, 20 ms duration with repetition frequency of 1 Hz. We didn?t detect any marked difference in intensity of fluorescence with both EP protocols. Despite the fact, that both mentioned protocols yielded the same transfection efficiency, there are two major advantages for the use of combination of 1 HV and 4 LV pulses compared to the use of 8 LV pulses only. The first one is that the combination is less harmful to target cells, since the intensity of LV pulses is lower compared to standard train of eight pulses (80 V/cm versus 200 V/cm, respectively) (25). The goal of gene therapy targeted to muscle cells is to produce sufficient levels of therapeutic proteins, which can be achieved only, if target cells remain viable after gene delivery to be able to express transgene products. Therefore, the least possible stress to target cells should be attempted. The second advantage could be important for eventual clinical use of this procedure. In combination, where 1 HV pulse, followed by 4 LV pulses were used, the delivery of electric pulses was concluded faster compared to delivery of eight identical LV pulses (frequency of electric pulse delivery in both protocols was the same, 1 Hz) and caused only four instead of eight painful muscle contractions.

In both protocols, which yielded the highest intensity of GFP fluorescence, expression remained the same on the 2nd and 7th day after gene delivery. According to other reports, transgene expression in skeletal muscle of dogs lasts longer; sufficient levels of transgene products to achieve biological effect after single injection of plasmid in healthy dogs were detected for at least 180 days (28). Therefore, we can speculate that transgene expression of GFP in canine muscle is actually longer than one week, but due to ethical considerations, we designed our experiment in such way as to perform only two muscle biopsies and could not monitor transgene expression beyond that time period.

Based on results of the first part of our study using reporter gene, encoding GFP, we used the best two EP protocols, to achieve electrotransfection of plasmid encoding human IL-12 into canine skeletal muscle. In the second part of the study, injection of therapeutic plasmid as well as placement of electrodes was performed transcutaneously without skin and fascia incisions as in the first experiment. This modification was performed with the intention to develop and evaluate a protocol, which could be in the future used in clinical work for muscle targeted gene delivery in dogs. Transcutaneous application is faster and especially less invasive, which is an important advantage for clinical pattern.

Although low number of samples was used, due to ethical considerations and legal restrictions in designing the experiment, some additional conclusions can be drawn from results of this part of the study, which confirm results, obtained utilizing plasmid encoding GFP. Human IL-12 was detected in serum of a dog, in which the highest dose of plasmid (1 mg diluted in 1 ml) was applied and EP protocol utilizing combination of 1 HV and 4 LV pulses (EP 2) was delivered. The concentration of human IL-12 was low (19 pg/ml), and was detected only in serum sample, collected at seven days after the procedure. To establish whether systemically secreted human IL-12 manifests immunostimulatory effect in vivo in dogs, serum levels of canine IFN-γ in blood samples were determined. Canine IFN-γ was detected in low levels (2.15-36.6 pg/ml) in three dogs, but interferon response did not correlate with detection of human IL-12. Although hematology and biochemistry analysis did not show any significant alterations, which could explain induction of IFN-γ production in these three animals, we conclude, that concentration of human IL-12 was probably too low to exert in vivo biological effect in the dog and induce unequivocal IFN-γ response. In contrast to our results, in the study of Fewell and colleagues, therapeutic concentrations of human coagulation factor IX in dogs after a single muscle-targeted electrotransfection of therapeutic plasmid were achieved (21). It should be noted that in that study extremely high doses of plasmid were needed to achieve therapeutic response; even up to 3 mg of plasmid per kg of bodyweight. Therefore, it is possible that 1 mg of plasmid encoding human IL-12 per dog, is not a sufficient dose to achieve detectable levels of human IL-12 in beagle dogs and that higher dosage of plasmid should be transfected. Another possible improvement of transgene expression could lie in multiple consecutive repetitions of procedure in order to achieve higher concentrations of human IL-12 in blood, resulting in successful induction of IFN-γ response.

In both parts of our study, all animals were monitored for possible side effects of electrotransfection procedure. Local side effects after delivery of both plasmids included a mild swelling of subcutaneous tissue at the site of electroporation, which didn?t cause any impairment of locomotory function. The swelling was slightly more pronounced in experiment with plasmid encoding GFP, which was probably due to tissue damage, caused by incision of skin and fascia. These observations are in agreement with other studies, featuring electrotransfection of muscle in large animals, where only transient local effects were observed (e.g., transitory erythema at the injection point), without any permanent damage to skin or muscle (27, 42). Furthermore, lack of any clinically significant adverse effects on muscle was confirmed by measuring serum activity of CK, which is a very sensitive indicator of muscle damage (43). In the previous studies, featuring electrotransfection of canine skeletal muscle, the activity of CK was measured in only one published paper (21), where transient increase in CK activity was detected, which returned to normal levels by day 7. The increase was clearly dose-dependent, with animals receiving plasmid in more injection sites and, thus, more sites to which electric pulses were delivered, having significantly higher CK activity.

The only negative effect of the electrotransfection procedure in dogs was considerable muscle contractions observed during delivery of electric pulses, which dissipated immediately thereafter. Muscle contractions are reported to be the major cause of pain in human patients, undergoing electrochemotherapy (44, 45). Routine monitoring of animals? vital signs during general anaesthesia revealed increased heart rate without any alterations in ECG and increased respiratory rate during delivery of LV electric pulses, which is the usual response to painful stimulus in an anaesthetised dog, and returned to baseline values immediately after intravenous application of analgesic. During electrochemotherapy pain can be significantly alleviated by raising the treated tumor nodule if possible away from subcutis prior to electric pulses delivery, to avoid painful contractions of subcutaneous muscle. In muscle targeted electrogene therapy this is of course not possible. Therefore, it is mandatory to perform the procedure in animals under adequately deep general anesthesia and to provide sufficient analgesia during delivery of electric pulses.

Our study asserted that electrotransfection of either plasmid did not cause any important systemic side effects. Hematological and biochemical parameters in collected blood samples remained within reference limits throughout the whole observation period with only few clinically nonsignificant and nonspecific alterations. This is a particularly important finding in gene therapy utilizing IL-12. Namely, the systemic administration of recombinant IL-12 is associated with multiple serious adverse effects, including renal and systemic toxicity (46); and high-dose levels were linked to temporary immune suppression, which would not be favorable for effective immunotherapy. Toxicity of electrically-mediated intratumoral delivery of plasmid encoding IL-12 was evaluated in a mouse melanoma model (47). Significant delay in tumor growth was demonstrated without any detectable serum concentrations of IL-12. The only histopathological abnormality, specific to animals, which received plasmid encoding IL-12, was inflammation, associated with the kidney by day 30 after the gene delivery, but without any hematological or biochemistry alterations, contributable to diminished kidney function. In our study, hematological (complete blood count, differential white cell count) and biochemistry parameters (urea, creatinine, SAP, and alanin aminotransferase) in blood samples, collected in different time intervals after delivery of plasmid encoding human IL-12, remained within reference limits, with individual alterations, which were clinically not significant and could not be linked to the performed procedure. Clinical status of all animals remained unaltered and they didn?t show any changes in appetency, water intake, and general behavior.

In conclusion, our study shows that electrotransfection is a feasible, effective, and safe method for muscle targeted gene therapy in dogs, which yields in gene expression lasting at least one week. Based on results of our study, we conclude that EP protocol, utilizing one HV pulse (600 V/cm, 100 ?s), followed by four LV pulses (80 V/cm, 100 ms, 1 Hz) is more suitable for electrotransfection of canine skeletal muscle than use of EP protocols employing LV pulses only. This EP protocol proved in our study to be more suitable than use of established low voltage protocols, which is an important aspect for eventual clinical use of this gene delivery method, and resulted in comparable level of transfection of muscle cells with plasmid encoding GFP. Our results suggest that this novel approach to gene therapy could have potential for clinical applications in veterinary medicine of small animals.

Acknowledgements

The authors acknowledge the financial support of the state budget by Slovenian Research Agency (Projects No. P3-0003, J3-7044 and P4-0053). All the authors declare that they have no conflict of interest.

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