Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression. Our results show that the combination of electroporation and a plasmid vector carrying these DNA elements results in increased and more persistent gene expression of the luciferase reporter gene in BALB/c mouse muscle. At 14 days after gene delivery, the gene expression was 16-fold higher in muscles injected and electroporated with the plasmid carrying the SV40 enhancers than with control plasmid. We have also studied the effects of the vehicle in which the plasmid was delivered, and the DNase inhibitor aurintricarboxylic acid (ATA), on gene expression. By combining ATA with 150 mM sodium phosphate buffer we were able to obtain a 2-fold increase in gene expression compared to delivery of the plasmid in physiological saline. These results are of importance for the development of efficient delivery techniques for naked DNA.     

Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-microl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 microl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-microl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.     

Transduction of satellite cells after prenatal intramuscular administration of lentiviral vectors

BACKGROUND: We have previously reported long-term expression of lacZ in myocytes after in utero intramuscular injection of Mokola and Ebola pseudotyped lentiviral vectors. In further experiments, we have noted that these vectors also transduce small cells at the periphery of the muscle fibers that have the morphology of satellite cells, or muscle stem cells. In this study we performed experiments to further define the morphology and function of these cells. METHODS: Balb/c mice at 14-15 days gestation were injected intramuscularly with Ebola or Mokola pseudotyped lentiviral vectors carrying CMV-lacZ. Animals were harvested at various time points, muscles were stained with X-gal, and processed for electron microscopy (EM) and immunofluorescence. To determine whether transduced satellite cells were functionally capable of regenerating injured muscles, animals were injected with notexin in the same area 8 weeks after the in utero injection of viral vector. RESULTS: Transmission EM of transduced cells confirmed the ultrastructural appearance of satellite cells. Double immunofluorescence for beta-galactosidase and satellite cell markers demonstrated co-localization of these markers in transduced cells. In the notexin-injured animals, small blue cells were seen at the areas of regeneration that co-localized beta-galactosidase with markers of regenerating satellite cells. Central nucleated blue fibers were seen at late time points, indicating regenerated muscle fibers arising from a transduced satellite cell. CONCLUSIONS: This study demonstrates transduction of muscle satellite cells following prenatal viral vector mediated gene transfer. These findings may have important implications for gene therapy strategies directed toward muscular dystrophy.     

A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation

Efficient gene transfer is a key factor in gene therapy. Reducing the damage caused by gene transfer to muscle by electroporation is very important for its clinical application. Extensive investigation of optimal conditions for gene transfer by electroporation is required. The parameters used for electroporation, including plasmid concentration; injection volume; the plasmid dose of the injection; the concentration of saline media; the size of plasmid DNA; the age of the mice; the lag time between plasmid injection and electroporation; and the effect of repeated gene transfer by electroporation, were systematically investigated in the present study. The efficiencies of gene transfer by electroporation in normal and rodent models of diabetes were also evaluated. We found that electroporation used for non-viral gene transfer could be repeated in the same place in the muscle, but the expression efficiency was closely related to the muscle damage. Increasing pulse times could enhance the efficiency of gene transfer with a lower strength of electric field. It was better to use a higher plasmid concentration than to use a larger dose of plasmid and repeated injection to achieve a high level of transgene expression. Optimal conditions varied in different animal models, being milder for diabetic mice than for normal mice, and it was also shown that the conditions that worked well on these small rodents were not necessarily suitable for larger animals. Our results provide a comprehensive view of the factors that affect the efficiency of gene transfer into skeletal muscle by electroporation.     

In vivo electroporation restores the low effectiveness of DNA vaccination against HER-2/neu in aging

Experimental evidence has been provided that cancer vaccines are less effective at older age than in young adults. In this study, we evaluated the possibility to recover the low effectiveness of DNA immunization against HER-2/neu increasing plasmid uptake by cells from old mice through electroporation with the aim to enhance the activation of specific immune responses. Young and old Balb/c mice received two immunizations with a pCMV-ECDTM DNA plasmid using plasmid intramuscular injection followed by electroporation (IM + E) or plasmid intramuscular injection alone (IM), and successively, they were challenged with syngeneic HER-2/neu overexpressing TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice after IM immunization. IM + E immunization completely protected old mice against a TUBO cell challenge. The protection was associated with increased transgene expression in the site of immunization and with the induction of both humoral and cell-mediated immunity in old mice. We conclude that the effectiveness of anticancer DNA vaccination in old ages may be improved increasing plasmid uptake and transgene expression through electroporation, suggesting the relevant role of the first steps of the immunization process in the success of cancer vaccines at older age.     

Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.     

Skeletal muscle regeneration in mice is stimulated by local overexpression of V1a-vasopressin receptor

Skeletal muscle has a remarkable capacity to regenerate after mechanical or pathological injury. We show that the V1a receptor (V1aR) for vasopressin, a potent myogenic-promoting factor that stimulates differentiation and hypertrophy in vitro, is expressed in mouse skeletal muscle and modulated during regeneration after experimental injury. We used gene delivery by electroporation to overexpress the myc-tagged vasopressin V1aR in specific muscles, thus sensitizing them to circulating vasopressin. The correct localization on the surface of the fibers of the recombinant product was demonstrated by confocal immunofluorescence directed against the myc tag. V1aR overexpression dramatically enhanced regeneration. When compared with mock-transfected controls, V1aR overexpressing muscles exhibited significantly accelerated activation of satellite cells and increased expression of differentiation markers. Downstream of V1aR activation, calcineurin was strongly up-regulated and stimulated the expression of IL-4, a potent mediator of myogenic cell fusion. The central role of calcineurin in mediating V1aR-dependent myogenesis was also demonstrated by using its specific inhibitor, cyclosporine A. This study identifies skeletal muscle as a physiological target of hormones of the vasopressin family and reveals a novel in vivo role for vasopressin-dependent pathways. These findings unveil several steps, along a complex signaling pathway, that may be exploited as potential targets for the therapy of diseases characterized by altered muscle homeostasis and regeneration.     

Satellite cell activation on fibers: modeling events in vivo--an invited review

Knowledge of the events underlying satellite cell activation and the counterpart maintenance of quiescence is essential for planning therapies that will promote the growth and regeneration of skeletal muscle in healthy, disease and aging. By modeling those events of satellite cell activation in studies of single muscle fibers or muscles in culture, the roles of mechanical stretching and nitric oxide are becoming understood. Recent studies demonstrated that stretch-induced activation is very rapid and exhibits some features of satellite cell heterogeneity. As well, gene expression studies showed that expression of the c-met receptor gene rises rapidly after stretching muscles in culture compared to those without stretch. This change in gene expression during activation, and the maintenance of quiescence in both normal and dystrophic muscles are dependent on NO, as they are blocked by inhibition of nitric oxide synthase (NOS). Mechanical, contractile activity is the defining feature of muscle function. Therefore, ongoing studies of stretch effects in satellite cell activation and quiescence in quiescent fiber and muscle cultures provides appropriate models by which to explore the regulatory steps in muscle in vivo under many conditions related to disease, repair, rehabilitation, growth and the prevention or treatment of atrophy.     

In vivo DNA electrotransfer into muscle

Naked plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed. Among the non-viral techniques for gene transfer in vivo , this method is especially simple, inexpensive, and safe. However, the relatively low expression levels attained by this method have limited its applications for uses other than as a DNA vaccine. We and other groups investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA vector. The results demonstrated that gene transfer into muscle by in vivo electroporation is far more efficient than simple intramuscular DNA injection and provides a potential approach to systemically delivering cytokines, growth factors, and other serum proteins for basic research and human gene therapy.     

Enhanced delivery of naked DNA to the skin by non-invasive in vivo electroporation

DNA delivery to skin may be useful for the treatment of skin diseases, DNA vaccinations, and other gene therapy applications requiring local or systemic distribution of a transgene product. However, the effective, consistent and patient-friendly transfection of skin cells remains a challenge. In a mouse model, we evaluated the effectiveness of intradermal injection of plasmid DNA followed by noninvasive in vivo electroporation (EP) as a method to improve transfection in skin. We achieved a several hundred-fold stimulation of gene expression by EP, sufficient to produce clinically relevant amounts of transgene product. We studied the effect of DNA dose and time after treatment as well as various EP pulse parameters on the efficiency of gene expression. EP under conditions of constant charge transfer revealed that the applied voltage was the main determinant for transgene expression efficiency while other pulse parameters had lesser effects. Patient-friendly, noninvasive meander electrodes which we designed for clinical applications proved equally effective and safe as plate electrodes. We also showed for the first time that noninvasive EP is effective in stimulating transfection and gene expression in human skin, particularly in the epidermis. Our findings demonstrate the applicability of EP-enhanced DNA delivery to skin for gene therapy, DNA immunization and other areas.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

An early post‐traumatic reaction of lymph‐heart striated muscle fibers in adult frog Rana temporaria during the first postoperative week: An electron microscopic and autoradiographic study

According to the current opinion, lymph‐heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron‐microscopic autoradiography. We present evidence that both, the satellite cells and pre‐existing muscle fibers bordering the site of injury, contribute directly to the lymph‐heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron‐microscopic autoradiography showed that 4 h after single ³H‐thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re‐enter into the S‐phase. Our results indicate that the nuclei of lymph‐heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis. J. Morphol. 276:1525–1534, 2015. © 2015 Wiley Periodicals, Inc.     

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

BACKGROUND: Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. METHODS: Electroporation was applied to mouse quadriceps and rat tibialis anterior muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP), beta-galactosidase (beta-gal), luciferase or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. RESULTS: Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. CONCLUSIONS: Our findings suggest that the optimal electroporation parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.     

Functional in vivo gene transfer into the myofibers of adult skeletal muscle

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.     

Single-fiber isolation and maintenance of satellite cell quiescence.

The activity of satellite cells during myogenesis, development, or skeletal muscle regeneration is strongly modelled using cultures of single muscle fibers. However, there are variations in reported features of gene or protein expression as examined with single-fiber cultures. Here, we examined the potential differences in activation of satellite cells on normal mouse muscle fibers produced during a standard isolation protocol, with or without agitation during collagenase digestion. Activation was detected in satellite cells on fibers after 24 and 48 h of culture in basal growth medium using immunodetection of the incorporation of bromodeoxyuridine (BrdU) into DNA and quantification of the number of BrdU-positive cells per fiber. After 24 and 48 h in culture under nonactivating conditions, the number of activated (BrdU+) satellite cells was greater on fibers that had received gentle agitation during collagenase digestion than on those that were subject to digestion without agitation during isolation. The findings are interpreted to mean that at least some of the variation among published reports may derive from the application of various methods of fiber isolation. The information should be useful for maintaining satellite cell quiescence during studies of the regulatory steps that lead to satellite cell activation.     

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ～80% from pre-EP levels. Within 3 h, torque recovered to ～50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ～25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ～20% and ～70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.