Comparison of Electrically Mediated and Liposome-Complexed Plasmid DNA Delivery to the Skin

BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery. METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model. RESULTS: Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery. CONCLUSION: Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired

Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells

In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFN β mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16. F10 cells in culture, IFN beta mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16. F10 cells, inducing effects in vitro and potentially in vivo

Plasma-Activated Air Mediates Plasmid DNA Delivery In Vivo

Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P \u3c 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P \u3c 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets

Effect of Electrically Mediated Intratumor and Intramuscular Delivery of a Plasmid Encoding IFN α on Visible B16 Mouse Melanomas

Interferon α may be used as a single agent therapy for metastatic malignant melanoma or as an adjuvant to chemotherapy. Delivery of interferon α by gene therapy offers an alternative to recombinant protein therapy. Electrically mediated delivery enhances plasmid expression in a number of tissues, for instance skin, liver, muscle and tumors including melanomas. Here we compare the effect of delivery of a plasmid encoding mouse interferon α on growth of visible B16 mouse melanomas following electrically mediated delivery to muscle or directly to the tumor. Intratumoral delivery of interferon α plasmid not only slows melanoma growth, but induces complete, long term, regression. This effect was not observed after intramuscular delivery

Evaluation of Delivery Conditions for Cutaneous Plasmid Electrotransfer Using a Multielectrode Array

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field strength and pulse width on transgene expression and duration using a plasmid encoding the luciferase reporter gene delivered by intradermal injection in a guinea pig model followed by EP with the MEA. As expected, the level of luciferase expression increased with the magnitude and duration of the voltage applied. In addition to adjusting transgene expression levels by altering fielding strength, levels could also be controlled by adjusting the plasmid dose. Our results indicate that the design of the MEA is a viable option for cutaneous plasmid DNA delivery by in vivo EP to a large surface area

Upregulation of DNA Sensors in B16.F10 Melanoma Spheroid Cells After Electrotransfer of pDNA

Increased expression of cytosolic DNA sensors, a category of pattern recognition receptor, after control plasmid DNA electrotransfer was observed in our previous studies on B16.F10 murine melanoma cells. This expression was correlated with the upregulation of proinflammatory cytokines and chemokines and was associated with cell death. Here, we expanded our research to include the influence of features of cells in a 3-dimensional environment, which better represents the tumors’ organization in vivo. Our results show that lower number of cells were transfected in spheroids compared to 2-dimensional cultures, that growth was delayed after electroporation alone or after electrotransfer of plasmid DNA, and that DNA sensors DDX60, DAI/ ZBP1, and p204 were upregulated 4 hours and 24 hours after electrotransfer of plasmid DNA. Moreover, the cytokines interferon β and tumor necrosis factor α were also upregulated but only 4 hours after electrotransfer of plasmid DNA. Thus, our results confirm the results obtained in 2-dimensional cell cultures demonstrating that electrotransfer of plasmid DNA to tumor cells in spheroids also upregulated cytosolic DNA sensors and cytokines

Transcriptional Regulation of the Bmp2 Gene: Retinoic Acid Induction in F9 Embryonal Carcinoma Cells and Saccharomyces Cerevisiae

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and β-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1

Staphylococcal Cassette Chromosome mec and Panton-Valentine Leukocidin Characterization of Methicillin-Resistant Staphylococcus Aureus Clones

Staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant Staphylococcus aureus MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCCmec type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCCmec type II complex and did not harbor the PVL genes

Donor Platelet Plasma Components Inactivate Sensitive and Multidrug Resistant Acinetobacter Baumannii Isolates

Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated

Method for the treatment of malignancies

A method of treating cancerous tumors is presented herein. The method includes injecting an effective dose of a plasmid encoded for IL-12, B7-1 or IL-15 into a cancerous tumor and subsequently administering at least one high voltage, short duration pulse to the tumor. The electroporation pulses may be administered at least 700V/cm for a duration of less than 1 millisecond. The intratumor treatments with electroporation may be administered in at least a two-treatment protocol with the time between treatments being about 7 days. The intratumor treatments with electroporation may be administered in a three-treatment protocol with a time of four days between the first and second treatments and a time of three days between the second and third treatments. It was found that the intratumor treatments using electroporation not only resulted in tumor regression but also induced an immune memory response which prevented the formation of new tumors