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 μg 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.
Gene transfer by electroporation is gaining increasing momentum as a non-viral approach in gene therapy. Electro gene transfer (EGT) is obtained when an external electric field, which transiently destabilises the membrane, renders the cells accessible for cDNA entry.1 Recent developments in the EGT procedure have provided highly efficient pulses with minimal perturbation of cell physiology and membrane integrity, when using a combination of one high-voltage (HV) pulse followed by one low-voltage (LV) pulse.2, 3, 4
Electroporation has been used for several years in the clinic for enhanced drug delivery to tumours, showing that electroporation-based treatments are feasible and safe in the clinical setting.5, 6, 7 Moreover, the first clinical trials with EGT to tumours have been undertaken.8 EGT to skeletal muscle is particularly advantageous since the muscle is capable of producing large amounts of the transgenic product and can act as an artificial endocrine organ.9
There are several reports on successful in vivo EGT of erythropoietin (EPO) into skeletal muscles in both mice10, 11, 12 and rats,13, 14 and EPO expression has been detected more than a year after EGT in mice.15 The delivery of EPO was followed by a marked increase in haematocrit (Hct) levels both in normal and uraemic rats.16, 17, 18 In these studies, EPO expression was controlled by constitutive promoters, which induced high expression in the first months after transfection, followed by a reduction in the expression level to a lower steady-state phase.19
In mice, the half-life of erythrocytes is 12 days compared to 120 days in humans.20 Month-long transgenic EPO expression does, therefore, have great regulatory effect on the Hgb level. Thus the EPO–Hgb system is an excellent model for studying regulation of transgene expression in an inducible promoter system as the effect and control of EPO expression is easily measured from blood samples, which can be taken repeatedly.
In the clinical setting, control of gene expression is of utmost importance. Systems to induce expression of transgenes have previously been established,21 and the most utilised of these seems to be the Tet-On system, which uses doxycycline (dox) as inducer drug.22 The Tet-On system comprises of a chimeric transactivator, which activates transcription from a silent tetO promoter in the presence of dox in a dose-dependent manner.22 The system has been hampered by high basal activity; however, this has been solved by two approaches: the development of a transcription silencer23 and new generations transactivators.24, 25 The inducer molecule dox is an orally available antibiotic, which induces gene expression in the range of 0.01–0.2 mg/ml. This dosing regimen for induction of gene expression is comparable to levels used in antibiotic therapy.21 It has been shown that expression can be induced, turned-off and re-induced at a later time.24 Other variables affecting the level of gene expression could be the amount of DNA transferred, but very few dose-dependent studies have been carried out as far as DNA dose is concerned. Finally, electroporation makes it possible to delineate precisely the area to be transferred, as transfer beyond the electrodes is minimal.26 However, only few data on ‘dose’ with respect to amount of transferred tissue are available.
As long-term high-level gene expression can be obtained after EGT,15, 27 treatment of, for example, protein deficiency syndromes is envisaged. However, to proceed with such a strategy, precise regulation of expression with options for changing level of expression both upward and downward is paramount.
We therefore engaged in an experiment to explore the possibilities of regulating expression of electro gene transferred genes by (1) amount of DNA, (2) area of muscle transfected, and (3) induction of promoter. Before starting with the actual ‘prescription’ experiment the influence of the three different variables were examined.
Optimisation of plasmid usage for local gene expression
The initial studies for optimising the amount of pTetO-Luc (plasmid encoding luciferase under a tetO inducible promoter) and pTet-On (plasmid encoding the tetO transactivator) needed for maximal gene expression, were performed by evaluating luciferase expression in muscles transfected with pTetO-Luc and pTet-On in C57Black/C mice. We found that 10 μg of each plasmid per muscle was sufficient to ensure maximal inducible gene expression (Figure 1a); however, a significant reduction in the gene expression response (P<0.05, Student's t-test) was not observed until the plasmid amount was decreased to 2.5 μg. Using enhanced green fluorescent protein (EGFP) for qualitative description, the plasmid amount could be reduced to 5 μg, before the maximal gene expression starts to decrease (Figure 1a). Importantly, the luciferase plasmid (9.5 kb) is almost twice as big as the EGFP plasmid (5.1 kb), this means that in molar concentrations, similar amounts (1.6 pmol) of both plasmids were needed for maximal gene expression.
In the non-induced state, we found high basal activity in the Tet-On system as has been reported previously by others.11, 23, 28 Addition of pTetS (plasmid encoding a silencer) to the injected plasmid solution reduced the basal luciferase expression in a dose-dependent way. pTetS (2.5 μg) reduced the basal activity, limiting activity to a few muscle fibres (Figure 1b), whereas luciferase expression was completely eliminated using 5, 7.5 or 10 μg pTetS. In the dox-induced state, pTetS had no effect on luciferase expression at any concentration, where expression stayed high. Studies using EGFP confirmed these results (Figure 1b).
When using the Tet-On system, the level of gene expression can be regulated by dox in a dose-dependent manner.22 Maximal gene expression of EGFP was achieved using 0.2 mg/ml. Reducing dox to 0.1 mg/ml resulted in 50% of the maximal gene expression and 0.01 mg/ml dox in the drinking water resulted in 25% expression (data not shown).
Dose–response correlation between the electrotransferred plasmids and haemoglobin levels
To determine the minimal effective dose of EPO plasmid, C57Black/C mice (n=10) were electrotransferred with 1, 2.5 or 5 μg of pUHD-10.3–mEPO (plasmid encoding EPO under a tetO inducible promoter), pTet-On and pTetS in either one or both hind legs. Gene expression was maximally induced by 0.2 mg/ml dox (Figure 2). Electrotransfer of 1 and 2.5 μg of the plasmids in one leg led to significant increases in Hgb to 12.6±2.7 and 14.6±2.6 mmol/l, respectively. Interestingly, EGT of 5 μg of each plasmid did not increase Hgb. In fact, the electrotransfer resulted in an initial increase in Hgb to 10.3±2.4 mmol/l, but after day 28 the Hgb levels started to decline and returned to control levels at day 56.
Electrotransfer of 1, 2.5 or 5 μg of each plasmid to both legs showed same pattern as transfer to one legs. One and 2.5 μg both induced significant increases in Hgb to 15.9±2.9 and 15.6±2.1 mmol/l, respectively, while electrotransfer of 5 μg induced an initial increase in Hgb, followed by a decline to control level after day 28. The levels of Hgb were slightly higher than those after transfer to one leg, but not as high as might be expected from transfer of the double amount of plasmid.
Highly elevated Hgb levels with long-term dox induction
To determine the duration of increased Hgb levels, groups of 10 mice were electrotransferred with 1 μg of pUHD-10.3–mEPO, 1 μg pTet-On and 1 μg pTetS in both legs, or simply injected with the same amounts of plasmid. All mice received 0.2 mg/ml dox throughout the experiment for maximal expression. In the EGT-treated group, the Hgb increased to 14.2±2.5 mmol/l and this level was maintained for 85 days, hereafter the median Hgb slowly decreased (Figure 3). The decrease in median Hgb was caused by some mice with rapidly declining Hgb (initial responders), while others maintained the elevated Hgb (responders). The Hgb stabilised at 9.5±2.5 mmol/l, which is the mean of responders and initial-responders. The actual mean level of the responders was 13.3±2.4 mmol/l, and expression in this subgroup was detected for at least 257 days. The observed decreases in Hgb were correlated with low serum iron levels (see below). DNA injection alone induced an initial increase in Hgb, which was lost within the first weeks. This increase might be due to transient transfection of single muscle fibres following the direct DNA injection.
Tight correlation between serum EPO level and haemoglobin level during dox regulation
To characterise the responsiveness in gene expression towards dox administration, two groups of four Wistar rats were electrotransferred with 2.5 μg of pUHD-10.3-mEPO, pTet-On and pTetS. Group A was induced with 0.2 mg/ml dox for 8 weeks, followed by 7 weeks of induction with 0.01 mg/ml dox, while group B did not receive dox. From week 15, both groups (A+B) were induced with 0.05 mg/ml dox (Figure 4). A non-transfected control group (C) was also included, receiving dox throughout the experiment. During the first induction at 0.2 mg/ml in group A, the Hct levels increased significantly (P=0.017) in all four rats ranging from 49 to 74 (9.9–15.5 mmol/l) with a control level of 42.1±1.1 (9.1±0.44 mmol/l). Reducing the dox concentration to 0.01 mg/ml led to a significant drop in the Hgb with three rats returning to control level (Hct: 46±8.2, Hgb: 9.1±0.44 mmol/l), while the Hgb of one rat remained elevated. From week 15, when gene expression was induced with 0.05 mg/ml dox in all eight transfected rats, a secondary increase was observed in group A, which had already been induced, while a significant increase (P=0.0024) was observed in two of the four rats in group B, which were induced for the first time. Serum EPO levels were measured in all animals and the levels were closely correlated to the Hct levels (Figure 4). In fact the serum EPO level in the two non-responder rats was not detectable, explaining why no significant increases in Hct levels were observed.
Dox drinking water intake
Dox drinking water was administrated without sucrose to ensure normal levels of drinking;29 indeed the mice receiving dox drank between 4.0 and 5.7 ml/day, which was not significantly different from the control groups (4.1 ml/day). There was no correlation between the drinking water intake and the dox concentration in the drinking (R2=0.11); in fact the drinking water intake was more closely correlated to the median Hgb level in the groups (R2=0.61). This is not surprising as thirst is partly controlled by haemoconcentration.
Treatment strategy for obtaining preset haemoglobin levels
The experiment presented in Figure 5 was performed as a prescription experiment where the three authors were to prescribe an EGT treatment to obtain a level of 10, 12 or 14 mmol Hgb/l, respectively. The authors could control the following parameters: the transfected area (one or both tibialis cranialis (TC) muscles), the plasmid amount, the administered dox concentration and finally they could prescribe re-transfection.
For 10 mmol/l (Figure 5a), small amounts of plasmids, 1 μg/leg, 0.25 μg/leg and 0.25 μg/2 legs, respectively, were electrotransferred. In group 1, which was electrotransferred 1 μg/leg followed by dox induction at 0.1 mg/ml, a significant increase in Hgb to 13.9±1.17 mmol/l was observed after first prescription. Turning down the dox concentration to 0.05 mg/ml did not alter the Hgb level; however complete withdrawal of dox led to a decrease to control level. Thus 1 μg induced too high increases in Hgb, but importantly the Hgb could be reduced to control level by withdrawing the dox. In the other two groups with smaller amounts of DNA transferred, no significant increases in Hgb were observed after first prescription. Two strategies were employed to amend this. In group 2, the dox concentration was increased first to 0.2 mg/ml dox, and then 0.3 mg/ml dox; however this was not enough to reach 10 mmol/l at any time. In group 3, the mice were re-transfected at the third prescription with 0.5 μg plasmids/2 legs followed by dox induction at 0.2 mg/ml dox; this led to an increase in Hgb to 10.8±1.03 mmol/l.
For 12 mmol/l (Figure 5b), slightly higher concentrations of plasmid were used with submaximal dox concentration to regulate the gene expression through the dox administration. Group 1 was electrotransferred with 2 μg/leg followed by dox induction at 0.1 mg/ml. This led to an increase in Hgb to 14.2±0.95 mmol/l. Reducing the dox concentration to 0.05 mg/ml led to a decrease in median Hgb to 12.5±2.57 mmol/l, which was close to the predetermined value. Continuous induction at 0.05 mg/ml was not sufficient, however, to maintain the elevated Hgb level, as the Hgb dropped to control level (8.25±2.89 mmol/l). Group 2 was electrotransferred with 0.5 μg/leg with dox induction at 0.1 mg/ml, leading to an increase in Hgb of 11.0±1.60 mmol/l, increasing the dox concentration to 0.2 mg/ml dox at the second prescription resulted in exactly the target of 12.0±1.60 mmol/l. This level, however, could not be maintained with continuous induction with 0.2 mg/ml dox. Group 3 was electrotransferred with 1 μg/leg with dox induction at 0.05 mg/ml, resulting precisely in the 12.0 (±1.87) mmol/l. Despite continuous induction with 0.05 mg/ml dox a minor reduction in Hgb was observed.
The averages of the three treatment strategies were 12.4 mmol/l after the first prescription, and precise 12.0 mmol/l after the second prescription. However after the third prescription, the mean Hgb was reduced to 9.88 mmol/l as none of the groups were capable of maintaining the elevated Hgb.
For 14 mmol/l (Figure 5c), higher amounts of plasmids were electrotransferred but submaximal dox concentrations were still administered to give room for adjustments. Group 1 was electrotransferred with 3 μg/leg followed by dox induction at 0.1 mg/ml. This led to an increase in Hgb to 13.5±0.54 mmol/l. Increasing the dox concentration to 0.2 mg/ml led to an increase in Hgb to 15.2±3.26 mmol/l. This level was maintained throughout the experiment. Group 2 was electrotransferred with 1.5 μg/leg followed by dox induction at 0.1 mg/ml. This led to an increase in Hgb to 13.2±1.58 mmol/l. Increasing the dox concentration to 0.2 mg/ml did not increase the Hgb level. At 12 weeks, the Hgb dropped off sharply nearly to control level (9.0±2.29 mmol/l). This could, however, be explained by low serum iron levels (see below). Group 3 was electrotransferred with 1 μg/2 legs followed by dox induction at 0.2 mg/ml. This increased Hgb to 14.5±1.17 and this level was maintained throughout the experiment.
The averages for the three strategies were 13.7 and 14.5 mmol/l after the first two prescriptions, respectively, showing precise regulation within the range of the preset Hgb values.
Loss of EPO responsiveness after continuous stimulation
Most mice were capable of maintaining the elevated Hgb levels; however some mice lost this ability over time. Possible explanations of this loss of responsiveness towards EPO transfer could be serum iron deficiency or silencing of transgenic EPO transcription. In investigating these possible explanations, mice from the prescription experiment were divided into a group with decreased Hgb level (mean=8.4±0.48 mmol/l), a group with elevated Hgb (mean=12.8±1.38 mmol/l) and the control group (mean=8.3±0.32 mmol/l).
Serum iron levels
Serum iron contents were measured from terminal blood samples. The serum iron levels in mice losing the ability to maintain elevated Hgb levels were 15.97±2.23 mmol/l (n=14), which were significantly lower (P=0.0006, Student's t-test) than control level (21.03±1.19 mmol/l, n=4), indicating that the decrease in Hgb could be caused by low serum iron levels. The serum iron levels in mice maintaining the elevated Hgb levels were 25.03±3.72 mmol/l (n=6), which were not significantly different from the control group.
Transcription of transgenic EPO
To determine whether the transcription rate of EPO was altered between the mice with low or high Hgb, Q-PCR was performed on RNA isolated from the transfected muscles. There were no significant differences in the mRNA levels between the two groups (data not shown).
Electroporation is already in use in clinical trials to promote the uptake of chemotherapeutics in malignant tumours (reviewed in Gothelf et al., 200330) and several studies have documented the feasibility of this approach.5, 6, 31 It is likely that this efficient mean of non-viral gene transfer will expand into gene therapy of diseases of organs, as well as organisms. Of particular interest is gene transfer to muscle tissue, as long-term, high levels of expression of the transferred genes can be obtained.15, 32, 33 Gene transfer of, for example, anti-tumour agents to the muscle is a very promising field, while other examples of interesting pre-clinical experiments include gene transfer of EPO for correction of anaemia16, 17, 18 or β-thalassemia.12, 34
Being able to regulate expression of the transgene is of utmost importance for clinical use. The long-term expression obtainable is an excellent opportunity to offer treatments for protein deficiency syndromes – but is also a challenge, as the expression of the inserted gene needs to be controlled. In this study, we demonstrate a therapeutic approach with transfer of low amounts of DNA and determination of biological activity.
If the level of biological activity is satisfying, the gene expression can be sustained over a long period, for example, regulated through the inducible promoter.
If the level of biological activity is too low, new transfers can be undertaken until a satisfactory level is reached.
Too high levels of biological activity can be adjusted by deactivating or downregulating the inducible promoter.
Finally, the electroporation procedure itself selectively targets the area encompassed by the electrodes, and thus electrode placement can be used as an additional restrictor of the anatomical extent of the transfected host tissue, thus of the number of cells expressing the transgene.
Prescription of precise levels of Hgb
To our knowledge, no attempts have yet been made at devising treatment strategies for the use of EGT to obtain specific levels of the transgene product. We carried out a very simple experiment: by giving the three authors of this paper privileges to prescribe any combination of (1) DNA amount, (2) amount of tissue transfected (one or two muscles), and (3) dose of induction drug (dox), we explored the possibilities of prescribing drugs delivered by EGT with inducible control systems. Interestingly, the prescription experiment showed that several strategies could be employed, resulting in the same range of Hgb. Most interestingly, calculating the average Hgb of the three strategies in each subexperiment, demonstrated a close approximation to the preset Hgb level (Figure 5).
Another advantage of the system is the possibility to regulate stepwise transgene expression. Blood sampling following EGT offers the opportunity to adjust gene expression, while the transgenic expression is proceeding. Undershooting the therapeutic level can be corrected by turning up the dox concentration or re-transferring new DNA amounts, while, more importantly, overshooting the therapeutic level can be amended by withdrawing dox, leading to return of Hgb to control level (Figure 5a). For clinic use, it is of utmost importance that the transgenic expression can be turned on and off – indeed turning off the transgene expression is imperative.
The first parameters to determine in relation to EGT are the DNA amount and the area transfected.
Several factors affect the level of transcriptional activity from a plasmid including the promoter strength.35, 36 Previous studies using the Tet-On system to control EPO production have introduced 10 μg of plasmid.11 We found that it was feasible to reduce the DNA amount to 0.5–1 μg plasmid for obtaining therapeutic levels of Hgb. In fact, exceeding 5 μg led to decreased expression. The decreased efficacy of transferring higher amounts of DNA might be due to local toxicity of the DNA in the muscle. This is supported by our findings that electrotransfer of 2.5 μg in 2 legs caused significant increases in Hgb, while electrotransfer of 5 μg in 1 leg (same total amount of DNA) led to small Hgb increases, which declined to control level within 42 days. It must be noted that no decrease in gene expression was found when introducing higher levels of plasmids encoding GFP or luciferase.
Extrapolating these findings in mice to adult humans suggests that 1.4 mg DNA would be enough to stimulate a significant increase in Hgb in humans. Certainly, this proves the clinical relevance of the EGT, as this amount of DNA is indeed manageable in the clinical setting.
The particular characteristic of EGT is that transfection only occurs in the area injected with DNA and delineated by the electrodes,1 whereas other transfection systems, for example viral vectors and lipofection rely on systemic or intra-organ injections with little control of the tissue and area transfected. In this study, the transfected area was controlled by transfection to one or both TC muscles. However in larger animals defined areas in the muscle could be transfected independently of the rest of the muscle. Thus EGT to a new area within the same muscle will be possible. Another interesting aspect is the possibility of obtaining measurable systemic levels of the transgene product, as well as therapeutic effects, by transfecting a muscle as small as the mouse TC muscle, weighing approximately 50 mg (0.2% of total mouse body weight). This is extremely encouraging for future clinical use of the technology.
Post-transfectional, the level of gene expression may be controlled through dox induction and re-transfection.
The Tet-On system has had problems with high levels of basal activity; however in this study, we were capable of eliminating the basal activity by increasing the concentration of the TetS silencer. A very close correlation was found between dox concentration in the drinking water and transgene expression. In the prescription experiments, it was found that expression levels can be adjusted over a relatively wide dynamic range by changing dox concentrations and that expression can be turned-off completely by withdrawal of dox. Finally, it was shown in another experiment that gene expression can be induced long time after EGT has taken place.11
The potential to re-transfer new amounts of DNA, if efficient levels of gene expression are not obtained, is unlimited. Indeed several studies have shown significant EPO expression and increases in Hgb after re-transfection.11, 12, 13
Transgenic EPO expression
As the main regulator of erythropoiesis, EPO was expected to stimulate increases in Hgb after EGT. Indeed we found that there was a tight correlation between serum EPO level and Hgb level (Figure 4). This close correlation indicates that the transgenic EPO production can supersede the endogenous control of Hgb. In some mice, we observed decreases in the Hgb levels, which correlated with low levels of serum iron. Exhaustion of iron stores is a common side effect of recombinant EPO treatment;37 therefore in future experiments, iron supplementation may eliminate this problem.
Less is more: reducing DNA amounts
Our studies with both GFP, luciferase and EPO confirm that increasing the amount of electrotransferred plasmid does not correlate with increasing expression efficacy. We found that reducing the amount of plasmid to 5 μg ensures optimal GFP expression in the muscle, whereas 1 μg was enough to stimulate EPO production resulting in significant increases in the Hgb levels. Generally, other groups have used larger amounts of plasmids, ranging from 10 μg and onwards.39, 40, 41, 42 The rationale for reducing the amount of DNA is both for practical purposes including expenses and administration volumes, as well as the fact that DNA is cytotoxic when introduced in vivo in large amounts.43, 44 DNA-dependent muscle damage with necrotic myofibres and infiltration of inflammatory cells was observed after EGT of 50 μg supercoiled endotoxin-free plasmid DNA.43
Gene transfer to muscle: different requirements depending on aim
In this study, we found that the DNA requirement for obtaining optimal gene expression was different between locally expressed proteins (GFP and luciferase) and the systemically expressed EPO, even though the promoter was the same in the three plasmids. This suggests that for proteins with endocrine/paracrine action, the effect of transgene production is amplified in the body through the signalling cascades induced by the hormone. Therefore EGT of a small amount of DNA encoding a hormone may have significant effect on systemic scale, whereas larger amounts of DNA are required for proteins, whose action is not amplified by the innate signalling pathways.45, 46, 47, 48 For these proteins, the amount of DNA required to obtain a physiological response might be correlated with whether the effect is local or systemic (Figure 6).
In conclusion, this study shows that expression of a transfected gene can be tightly controlled, making it possible to reach and maintain a certain preset value. As control is an important prerequisite for bringing the method to the clinic, this study represents an important step towards clinical application of EGT for gene therapy.
Methods and materials
Animals and muscle preparation
All animal experiments were conducted in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimentation and after permission from the Danish Animal Experiments Inspectorate. Mice experiments were performed at University of Copenhagen at Herlev Hospital on 8 to 10 weeks old female C57Black/C mice from Taconic (Tornbjerggaard, Denmark), while rat experiments were carried out at University of Aarhus, Department of Physiology and Biophysics on 4 to 5 weeks old male or female Wistar rats (own breed). The animals were maintained in a thermo-stated environment under a 12-hour light/dark cycle and had free access to food (Altromin pellets, Spezialfutter-Werke, Germany) and drinking water. The animals were killed by cervical dislocation, and the rats were then decapitated. Intact TC without tendons were dissected out and quickly frozen on dry ice and absolute alcohol.
Plasmid constructs and in vivo EGT
The plasmids pTet-On, encoding the reverse tet-responsive transcriptional activator (rtTA) transactivator,49, 50 pTetS, encoding the tS silencer,24 pBI-EGFP, encoding the enhanced green fluorescent protein and pBI-Luc, encoding luciferase, the last two both under the control of an rtTA-dependent promoter, were all obtained from Clontech (Palo Alto, CA, USA). PUHD-10.3-mEPO encoding murine EPO under the control of an rtTA-dependent promoter was kindly provided by Dr Fattori.11 All DNA preparations were performed using Qiafilter Plasmid Maxiprep kits (Qiagen, Germany), and the concentration and quality of the plasmid preparations were controlled by spectrophotometry and gel electrophoresis.
The animals were anaesthetised 15 min before electroporation (EP) by injection of Hypnorm (0.4 ml/kg, Janssen Saunderton, Buckinghamshire, UK) and Dormicum (2 mg/kg, Roche, Basel, Switzerland). For mice, 20 μl plasmid solution was injected intramuscularly (i.m.) along the fibres into the TC muscle using a 29G insulin syringe. The rats were injected with 10 μl plasmid solution in the TC also using an insulin syringe. Plate electrodes with 5-mm gap were fitted around the hind legs. Good contact between electrode and skin was ensured by hair removal and use of electrode gel. The electric field was applied using the Cliniporator™ (IGEA, Italy), applying a combination of a HV (1000 V/cm (applied voltage=400 V), 100 μs) pulse followed by a LV (100 V/cm (applied voltage=40 V), 400 ms) pulse. Induction of gene expression was obtained by administering drinking water containing dox (doxycycline hyclate, Sigma-Aldrich, Denmark) at various concentrations (0.01–0.2 mg/ml) in distilled water.
Evaluation of EGFP expression
Forty-eight hours after treatment the mice were euthanised and muscles were removed and evaluated by fluorescence stereomicroscopy (Nikon SMZ1500, Japan) in blinded and randomised order. Gene expression was scored based on the area of muscle fibres expressing EGFP on a scale from 0 to 5. No EGFP expression was set at 0, less than 20% of the muscle expressing EGFP was given the mark 1, 20–40%=2, 40–60%=3, 60–80%=4, while more than 80% of the muscle surface expressing EGFP was rewarded 5.
Evaluation of luciferase expression
Forty-eight hours after treatment, the mice were euthanised and the muscles were removed and quickly frozen in dry ice and 100% alcohol. Muscle extracts were prepared by homogenising the muscles with a S8N-5G homogeniser (IKA-werke, Germany) in 1 ml cell lysis buffer (Roche, Germany). Cell debris was spun down, and luciferase activity was measured on the supernatant in a luminometer (LumiStar, Ramcom A/S, Denmark).
Protocol for experiment presented in Figure 5
The three authors of this paper acted as ‘doctors’, trying to prescribe an EGT treatment, which would result in 10, 12 or 14 mmol Hgb/l, respectively. Each author had three groups of eight mice at their disposal. The following parameters could be controlled during the prescription: EGT to one or both TC muscles, the amount of plasmid electrotransferred, the administered dox concentration and finally the mice could be re-transfected in one or both TC muscles. Blood samples were taken every 4 weeks, whereafter the ‘doctors’ had 3 days to make adjustments in their prescriptions.
Evaluation of EPO expression and determination of haemoglobin and serum iron levels
In mice, blood samples (20–30 μl) were drawn from the retro-orbital sinus and haemoglobin levels were measured from a drop of blood using the HemoCue Hb201+ (HemoCue AB, Sweden). The precision of the apparatus was ±0.1 mmol/l, controlled by repeated measurements from the same blood sample. At the termination of the experiments, 300 μl blood were drawn from the mice and free iron was measured using the Vitros 950 Chemistry System. In rats, blood samples (ca. 200 μl) were drawn from the tail vein and Hct level was determined by hairpin centrifugation. The Hct levels are reported, but were also converted to haemoglobin levels by multiplying the Hct value with 0.207, and expressed in mmol/l. The remaining blood sample was spun down, and serum was isolated for determination of EPO levels.
Detection of EPO expression after DNA electrotransfer
Total muscle RNA was isolated from frozen muscles by muscle homogenisation and RNA extraction using the TRIzol reagent (Invitrogen Life Technologies, Denmark). RT-PCR was performed using random p(dN)6 primers (Roche, Germany) and AMV reverse transcriptase (Roche, Germany). EPO was amplified from cDNA using the Brilliant SYBR Green Q-PCR kit 600548 (Stratagene, Cedar Creek, TX, USA). The sequence of the pUHD1-mEPO primers were: sense: 5′-IndexTermatgtcgcctccagataccac, and antisense: 5′-IndexTermcctctcccgtgtacagcttc and PCR and detection was performed by Q-PCR (MX3000P, Stratagene).
Detection of EPO in rat serum
The levels of EPO in serum samples were determined by ELISA (Quantikine Mouse Erythropoietin Kit, R&D systems). Shortly, 50 μl undiluted serum samples were incubated in monoclonal anti-EPO antibodies coated wells for 2 h. After washing, secondary anti-EPO peroxidase-conjugated antibodies were added for 2 h incubation. Following incubation and washing, a colour reaction was developed and EPO concentration was determined by optical density measurements and reported as pg EPO/ml. The sensitivity was determined by the minimum detectable dose, which was 12.5 pg/ml, while no cross-reactivity was observed with other cytokines or other factors.
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We thank Vibeke Uhre and Marianne Fregil for excellent technical assistance. The study was supported by the Danish Research Agency (22-03-0367) and (22-02-0523).
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Hojman, P., Gissel, H. & Gehl, J. Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation. Gene Ther 14, 950–959 (2007). https://doi.org/10.1038/sj.gt.3302951
- electro gene transfer
- skeletal muscle
- tet-on system
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