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Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation  [PDF]
Kathleen A. Cashman,Kate E. Broderick,Eric R. Wilkinson,Carl I. Shaia,Todd M. Bell,Amy C. Shurtleff,Kristin W. Spik,Catherine V. Badger,Mary C. Guttieri,Niranjan Y. Sardesai,Connie S. Schmaljohn
Vaccines , 2013, DOI: 10.3390/vaccines1030262
Abstract: Lassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development.
A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer
Pierre Lefesvre, Joline Attema, Dirk van Bekkum
BMC Molecular Biology , 2002, DOI: 10.1186/1471-2199-3-12
Abstract: Intra-muscular DNA transfer of pLuciferase was increased by 2 logs after electroporation, confirming data described by others. However, the blood levels of the encoded protein were still lower than those obtained after injection of first generation adenoviral vectors. Also, the electroporation procedure, on its own, caused severe muscle damage consisting of rhabdomyolysis and infiltration, whereas the adenoviral vectors caused only a slight infiltration. As damage of targeted tissue may be an advantage in the case of tumour transfection, we also compared the two transfection methods in tumour tissue. In case of poorly permissive tumours, adenoviral vectors cannot transfect more than 2% of the tumour tissue without inducing significant liver damage. In contrast, the electroporation seems to offer a wider therapeutic window since it does not cause any systemic toxicity and still induce's significant transfection.Plasmid electroporation of the muscle induce severe local damage and is of no advantage over adenoviral vectors for obtaining high blood levels of a vector encoded protein. In contrast, electroporation of tumours might be safer than adenoviral gene transfer.Numerous diseases require treatment by systemic delivery of a therapeutic protein. Repetitive or continuous injections are the only delivery method used in daily practice. As a mean of reducing the inconvenience of multiple injections or implantation of mini pumps, gene transfer technology may offer certain advantages. In order to achieve a high plasma concentration, whatever the vector used, the transfection of a large tissue mass is required (e.g. liver or muscle). In rodents, intravenous injection of a non-targeted vector – viruses or plasmid preparations – induces mainly transfection in the liver and the spleen [1,2]. As of today, transfection of other large organs by viral vectors has not been accomplished in by way of systemic delivery [3]. The main inconveniences of intravenous administration of firs
Polydeoxyribonucleotide Dermal Infiltration in Male Genital Lichen Sclerosus: Adjuvant Effects during Topical Therapy  [PDF]
Luigi Laino,Silvia Suetti,Isabella Sperduti
Dermatology Research and Practice , 2013, DOI: 10.1155/2013/654079
Abstract: Background. Lichen sclerosus (LS) is an autoimmune inflammatory skin disease that leads to tissue sclerosis. Actually, the first-line treatment consists of local steroid as clobetasol propionate (CP). Polydeoxyribonucleotide (PDRN) has demonstrated anti-inflammatory effects through the reduction of cytokine production and growth stimulation of fibroblast. Objective. To evaluate the efficacy of intradermal administration of PDRN in male patients suffering from genital lichen sclerosus in addition to topical 0.05% CP, as compared to administering 0.05% CP without PDRN injection. Patients/Methods. A group of male patients ( = 28; aged 25 to 65) suffering from LS were observed during topical therapy or subdermal in addition to topical therapy. Disease activity at baseline was evaluated on Investigator’s Global Assessment (IGA) and the Dermatology Life Quality Index (DLQI). We used polydeoxyribonucleotide in a commercial preparation for human use and a topical CP emulsion. Results. After therapy, in all group A patients there has been a regression of most of clinical pathological signs, while there has been a moderate improvement in all group B patients. Conclusions. On site intradermal administration of PDRN, associated with CP 0.05% cream, seemed to be associated with a clinical improvement of lichen sclerosus better than CP used in single therapy. 1. Introduction Several researchers have shed new light on the importance of the action of extracellular nucleotides and nucleosides in increasing cell proliferation and reducing inflammation. PDRN, an A2A adenosine receptor, acts as mitogen for fibroblasts, endothelial cells, and preadipocytes [1, 2] working with different growth factors (VEGF, PGF, and FGF). PDRN is used in plastic and dermatologic surgery, and recently in urology, for its regenerative properties, restorative effects in ischemic skin flaps [3], and being used to improve intratesticular vascularisation [4]. Recently, the effects of PDRN have been analysed in a number of tissues, such as corneal epithelium [5], human bone [6], and skin [3]. PDRN is involved in protective and regenerative effects on UV-irradiated mouse cell cultures [7] and UV-irradiated dermal fibroblast [8]. PDRN has shown proliferation effects in human preadipocytes, which represent the richest reservoir of human adult stem cells [9]. In the light of these preliminary results, and because of these specific properties, we decided to perform the clinical observation, before and after therapy, of a local subdermal administration of PDRN in lichen sclerosus genital lesions,
Efficient expression of transgenes in adult zebrafish by electroporation
K Murali Rambabu, S Hari Rao, N Madhusudhana Rao
BMC Biotechnology , 2005, DOI: 10.1186/1472-6750-5-29
Abstract: Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V·cm-1 at 15 μg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita). To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy.Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.In vivo gene delivery into skeletal or cardiac muscle by direct injection of naked DNA is a convenient method to express proteins [1,2]. The efficiency of this procedure was improved substantially by applying electrical pulses at the site of injection [3,4]. Electroporation enhances transgene expression in tissues by causing electrical breakdown of membranes combined with electrophoresis of DNA into cells [5]. Since its discovery, electrotransfer of DNA into muscle has become a very popular method of gene delivery due to easy access of the muscle tissue, long life span of the muscle cell, abundant blood supply and its suitability for the production of proteins as systemic therapeutic agents [4]. Electroporation is routinely applied to a portion of the muscle, leading to transfection of cell layers around the site of injection. Recently, in ovo or in utero electroporation into the neural tubes and electroporation in tissue explants or solid
Electroporation-Induced Electrosensitization  [PDF]
Olga N. Pakhomova,Betsy W. Gregory,Vera A. Khorokhorina,Angela M. Bowman,Shu Xiao,Andrei G. Pakhomov
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017100
Abstract: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs.
In Vivo Electroporation Mediated Gene Delivery to the Beating Heart  [PDF]
Erick L. Ayuni,Amiq Gazdhar,Marie Noelle Giraud,Alexander Kadner,Mathias Gugger,Marco Cecchini,Thierry Caus,Thierry P. Carrel,Ralph A. Schmid,Hendrik T. Tevaearai
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014467
Abstract: Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.
Transformation of Xanthomonas axonopodis pv. citri by electroporation
Amaral, Alexandre M. do;Toledo, Cristiane P.;Baptista, Juliana C.;Machado, Marcos A.;
Fitopatologia Brasileira , 2005, DOI: 10.1590/S0100-41582005000300013
Abstract: this study describes the use of electroporation for transforming xanthomonas axonopodis pv. citri (xac), the causal agent of citrus (citrus spp.) canker. it also evaluates the methodology used for this species under different electrical parameters. the bacterium used in the study (xac 306) was the same strain used for recent complete sequencing of the organism. the use of a plasmid (pufr047, gentamycinr) is reported here to be able to replicate in cells of xac. following the preparation and resuspension of competent cells of xac at a density of ~4 x 1010 cfu/ml, in 10% glycerol, and the addition of the replicative plasmid, an electrical pulse was applied to each treatment. selection of transformants showed a high efficiency of transformation (1.1 x 106 transformants/mg dna), which indicates an effective, and inverse, combination between electrical resistance (50 w) and capacitance (50 μf) for this species, with an electrical field strength of 12.5 kv.cm-1 and 2.7-ms pulse duration. besides the description of a method for electroporation of xac 306, this study provides additional information for the use of the technique on studies for production of mutants of this species.
Single neuron electroporation in manipulating and measuring the central nervous system
Ti-Fei Yuan, Manuel Menéndez-González, Oscar Arias-Carrión
International Archives of Medicine , 2010, DOI: 10.1186/1755-7682-3-28
Abstract: Many strategies have been developed to introduce charged or non-charged compounds into cell cytoplasm, including chemically modified dyes or peptides to cross the cell membrane (e.g. penetrating peptides), viral vectors or lipocomplexes for gene introduction [1,2]. A main defect of these techniques is the lacking of targeting accuracy that precludes specificity to given groups of cells. Electroporation is a suited method that allows the cellular introduction of small as well as large molecules [3,4]. The principle of electroporation is based on the assumption that the cell membrane, which was non-permeable to charged molecules in normal conditions, could form nano-sized pores under transmembrane electrical fields, thus permitting charged macromolecules to cross through the cell membrane (Figure 1) [5]. The transient pores then disappeared and the cells would return to a normal (i.e. not destabilized) condition within minutes as the electrical pulses ceased [6]. Following the first success on plasmid introduction to mammalian cells in 1982 [7,8], similar successes have been achieved on plant cells, bacteria cells, and yeasts [9].Electroporation has, since the 1990 s, become a common laboratory technique for non viral transfection and molecule submission, however focused on bacteria or batch of cells in culture until the invention of in vivo electroporation - "modern electroporation". The most outstanding feature of this "modern electroporation" is the adoption of repeated charging with low voltage rectangular pulses, through which this approach could avoid the destroying currents to embryos or painful experiences to animals/human beings when high-voltage pulses were used. Developed on chick embryo [10,11], in vivo electroporation has now been successfully applied on liver, muscle, tumor, and skin for genetic material deliveries [12]. With stereotaxic apparatus facilitated microinjection, in vivo electroporation could target a defined small area in the adult brain [13
PENGARUH TEGANGAN ELEKTROPORASI TERHADAP EFISIENSI TRANSFORMASI PLAMID pND968 BAKTERI ASAM LAKTAT KE E. coli HB101 [The Effect of Electroporation Voltage on Transformation Efficiency of Plasmid pND968 of Lactic Acid Bacteria Into E. coli HB101]  [cached]
Jurnal Teknologi dan Industri Pangan , 2002,
Abstract: The research was conducted to examine the effect of different electroporation voltage on transformation efficiency of plasmid pND968. This was carried out by exposing the mixture of plasmid pND968 and competent cells of E. coli HB101 on a difference level of electroporation voltage at 1500, 2000 and 2500 Volts. After electroporation, the cells were resuspended in a recovery medium and then poured into ampicilin-containing LB agar. Transformants appeared after 48-72 hours were then recorded. The results showed that electroporation voltage of 2500 Volts with resistance 200 Ohm and capacitor 25 uF resulted in the highest electroporation efficiency at 7.72 x 104 CFU / ug DNA. Transformants confirmation by means of plasmid pND968 isolation showed that all of the ampicillin-resistant transformants harbored the intended pND968 with the expected size.
Skin Electroporation: Effects on Transgene Expression, DNA Persistence and Local Tissue Environment  [PDF]
Anna-Karin Roos, Fredrik Eriksson, James A. Timmons, Josefine Gerhardt, Ulrika Nyman, Lindvi Gudmundsdotter, Andreas Br?ve, Britta Wahren, Pavel Pisa
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007226
Abstract: Background Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. Methodology/Principal Findings This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. Conclusions/Significance This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.
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