This work demonstrates that electrical muscle stimulation in the muscle. When dextran was injected together with the markedly increases the transfection efficiency of an intraplasmid DNA, it was also taken up by the transfected fibmuscular injection of plasmid DNA. In soleus or extensor ers. Stimulation-induced membrane permeabilization and digitorum longus muscles of adult rats the percentage of increased DNA uptake were therefore probably respontransfected fibers increased from about 1 to more than 10.sible for the improved transfection. The stimulation caused The number of transfected fibers and the amount of foreign some muscle damage but the fibers regenerated rapidly. protein produced could be graded by varying the numberThe described method, which is simple, efficient, and or duration of the electrical pulses applied to the muscle.reproducible, should become valuable for basic research, The stimulation had to be applied when DNA was present gene therapy and DNA vaccination.
We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 g of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.Genes can be transferred into skeletal muscle cells of rodents and primates by intramuscular injection of plasmid DNA, and the resulting gene expression has been reported to last as long as several months (1, 2). Similarly, various viral vectors such as adenoviral, retroviral, and AAV-based vectors (3), have been used to transduce myofibers in vivo. The i.m. injection of plasmid DNA, however, has several advantages over viral vectors. First, plasmid DNA vectors are easier to construct and can be prepared as pharmaceutical-grade solutions (4) without the risk of contamination with wild-type infectious particles. Second, previous infection by wild-type adenovirus or AAV may induce a neutralizing antibody response that could preclude administration of the recombinant virus. In contrast, anti-DNA antibodies have never been detected in experiments of muscle DNA injection (2), therefore it is possible to readminister plasmid DNA by i.m. injection if repeated therapy or escalation is required.Despite the promise of i.m. injection of plasmid vectors for treating serum protein deficiencies, several important issues remain to be addressed before this approach becomes feasible for human gene therapy. The potential clinical usefulness of direct gene transfer to muscle of plasmid DNA is in fact limited by the low and highly variable level of gene expression (1, 2, 5, 6). Therefore, although DNA injection is potentially very powerful as a vaccination method because a low level of gene expression is sufficient to trigger immunoresponses, it is necessary to increase the efficiency of DNA uptake after i.m. injection of plasmid vectors before using this technique as a standard gene correction procedure.One of the most efficient methods implemented to achieve gene transfer and expression in mammalian cells is based on electric pulses (7). Electroporation has been used to introduce foreign DNA in different cell types (7), but it has also recently met with some success in in vivo applications. Gene transfer by electrical permeabilization has been obtained in skin (8, 9), corneal endothelium (10), melanoma (11), brain (12), liver, (13) and muscle (14) of experimental animals.We have shown previously that electropermeabilization can increase severalfold the uptake by rat muscle of a plasmid encoding the Escherichia coli lacZ gene (15). In this study...
Attempts to correlate behavioral learning with cellular changes, such as increased synaptic efficacy, have often relied on increased extracellular potentials as an index of enhanced synaptic strength. A recent example is the enlarged excitatory field potentials in the dentate gyrus of rats that are learning spatial relations by exploration. The altered hippocampal field potentials do not reflect learning-specific cellular changes but result from a concomitant rise in brain temperature that is caused by the associated muscular effort. Enhanced dentate field excitatory potentials followed both passive and active heating and were linearly related to the brain temperature. These temperature-related effects may mask any learning-induced changes in field potential.
Since human immunodeficiency virus (HIV)-
The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.
In vivo electroporation (EP) has been shown to augment the immunogenicity of plasmid DNA vaccines, but its mechanism of action has not been fully characterized. In this study, we show that in vivo EP augmented cellular and humoral immune responses to a human immunodeficiency virus type 1 Env DNA vaccine in mice and allowed a 10-fold reduction in vaccine dose. This enhancement was durable for over 6 months, and re-exposure to antigen resulted in anamnestic effector and central memory CD8؉ T-lymphocyte responses. Interestingly, in vivo EP also recruited large mixed cellular inflammatory infiltrates to the site of inoculation. These infiltrates contained 45-fold-increased numbers of macrophages and 77-fold-increased numbers of dendritic cells as well as 2-to 6-fold-increased numbers of B and T lymphocytes compared to infiltrates following DNA vaccination alone. These data suggest that recruiting inflammatory cells, including antigen-presenting cells (APCs), to the site of antigen production substantially improves the immunogenicity of DNA vaccines. Combining in vivo EP with plasmid chemokine adjuvants that similarly recruited APCs to the injection site, however, did not result in synergy.Plasmid DNA vaccines have proven considerably less immunogenic in clinical studies than in preclinical studies (3,9,13,24,33), demonstrating the need to improve their potency. Various strategies are currently being pursued, including the use of plasmid cytokine and chemokine adjuvants (5,6,11,19,26,30), polymer adjuvants (29), novel transcriptional regulatory elements (7), and improved delivery techniques such as in vivo electroporation (EP) (2,23,25). In vivo EP involves the administration of electrical pulses to muscle tissue following intramuscular (i.m.) injection of DNA vaccines and has been shown to enhance the immunogenicity of DNA vaccines in a wide variety of small and large animal models (1,8,10,12,17,18,20,22,27,32). It has been suggested that in vivo EP functions in part by increasing myocyte permeability and thereby facilitating plasmid uptake and antigen expression by host cells (2, 14-16, 25, 28, 34).We have previously reported that there are very few professional antigen-presenting cells (APCs) in muscles after DNA vaccination (6), and we therefore hypothesized that DNA vaccines may be limited by insufficient APCs at the site of antigen production. Consistent with this hypothesis, we observed that plasmid chemokines and growth factors such as plasmid MIP-1␣ and Flt3L were able to recruit dendritic cells (DCs) and macrophages to the site of inoculation and to enhance DNA vaccine-elicited immune responses (26, 30). Whether APCs are similarly recruited by in vivo EP, however, has not previously been investigated. In addition, the phenotype of cellular immune responses elicited by DNA vaccination with in vivo EP has not been assessed in detail. In the present study, we investigated the magnitude, phenotype, and durability of cellular immune responses elicited in mice by human immunodeficiency virus type 1 (HIV-1) Env DN...
We report on the immunogenicity and clinical effects in a phase I/II dose escalation trial of a DNA fusion vaccine in patients with prostate cancer. The vaccine encodes a domain (DOM) from fragment C of tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA), PSMA27–35. We evaluated the effect of intramuscular vaccination without or with electroporation (EP) on vaccine potency. Thirty-two HLA-A2+ patients were vaccinated and monitored for immune and clinical responses for a follow-up period of 72 weeks. At week 24, cross-over to the immunologically more effective delivery modality was permitted; this was shown to be with EP based on early antibody data, and subsequently, 13/15 patients crossed to the +EP arm. Thirty-two HLA-A2− control patients were assessed for time to next treatment and overall survival. Vaccination was safe and well tolerated. The vaccine induced DOM-specific CD4+ and PSMA27-specific CD8+ T cells, which were detectable at significant levels above baseline at the end of the study (p = 0.0223 and p = 0.00248, respectively). Of 30 patients, 29 had a measurable CD4+ T-cell response and PSMA27-specific CD8+ T cells were detected in 16/30 patients, with or without EP. At week 24, before cross-over, both delivery methods led to increased CD4+ and CD8+ vaccine-specific T cells with a trend to a greater effect with EP. PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP. The high frequency of immunological responses to DOM-PSMA27 vaccination and the clinical effects are sufficiently promising to warrant further, randomized testing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1270-0) contains supplementary material, which is available to authorized users.
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