A combination of intradermal jet-injection and electroporation overcomes in vivodose restriction of DNA vaccines

Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.     

Numerical modeling of in vivo plate electroporation thermal dose assessment

Electroporation is an approach used to enhance the transport of large molecules to the cell cytosol in which a targeted tissue region is exposed to a series of electric pulses. The cell membrane, which normally acts as a barrier to large molecule transport into the cell interior, is temporarily destabilized due to the development of pores in the cell membrane. Consequently, agents that are ordinarily unable enter the cell are able to pass through the cell membrane. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with joule heating. This paper explores the thermal effects of various geometric, biological, and electroporation pulse parameters including the blood vessel presence and size, plate electrode configuration, and pulse duration and frequency. A three-dimensional transient finite volume model of in vivo parallel plate electroporation of liver tissue is used to develop a better understanding of the underlying relationships between the physical parameters involved with tissue electroporation and resulting thermal damage potential.     

Enhancement of DNA vaccine potency by electroporation in vivo

The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or beta-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals.     

Increased DNA vaccine delivery and immunogenicity by electroporation in vivo

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.     

In vivo single-cell electroporation for transfer of DNA and macromolecules

Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.     

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.     

Needleless in vivo gene transfer into muscles by jet injection in combination with electroporation

Previously, we have established an in vivo electroporation method for gene transfer into muscle by injection of DNA with a needle followed by electric pulse delivery using needle-type electrodes and proved that this method is effective for the systemic delivery of cytokines. To perform the needleless gene delivery, we combined jet injection of DNA with electroporation using plate-type electrodes. For delivery of beta-galactosidase- and enhanced green fluorescent protein (EGFP)-expressing plasmids into muscles, there was no significant difference between the previous needle-mediated method and the newly developed jet-injection method. When pCAGGS-IL-5 was introduced into tibialis anterior, quadricipital and back sural muscles by this new method, the serum IL-5 levels reached 3.4 +/- 0.9, 5.7 +/- 1.7 and 8.4 +/- 2.7 ng/ml at day 5, respectively. Although the peak values of IL-5 achieved by the jet-injection method in these muscles were lower than that of the highest value achieved by needle-mediated gene delivery into anterior tibial muscle, this new method could deliver plasmid into relatively large muscles with better efficiency than the needle-mediated method. Thus the jet-injection method provides a useful means of gene delivery into large muscles, which is essential for future use in 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.     

DGAT1 overexpression in muscle by in vivo DNA electroporation increases intramyocellular lipid content

In adipose tissue, the microsomal enzyme 1,2-acyl CoA:diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage. Because DGAT1 is expressed in skeletal muscle as well, we aimed to directly test the effect of DGAT1 on muscular triglyceride storage by overexpressing DGAT1 using in vivo DNA electroporation. A pcDNA3.1-DGAT1 construct in saline was injected in the left tibialis anterior muscle of rats, followed by the application of eight transcutaneous pulses, using the contralateral leg as sham-electroporated control. Electroporation of the DGAT1 construct led to significant overexpression of the DGAT1 protein. The functionality of DGAT1 overexpression is underscored by the pronounced diet-responsive increase in intramyocellular lipid (IMCL) storage. In chow-fed rats, DGAT1-positive myocytes showed significantly higher IMCL content compared with the control leg, which was almost devoid of IMCL (1.99 +/- 1.13% vs. 0.017 +/- 0.014% of total area fraction; P <0.05). High-fat feeding increased IMCL levels in both DGAT1-positive and control myocytes, resulting in very high IMCL levels in DGAT1-overexpressing myocytes (4.96 +/- 1.47% vs. 0.80 +/- 0.14%; P <0.05). Our findings indicate that DGAT1 contributes to the storage of IMCL and that in vivo DNA electroporation is a promising tool to examine the functional consequences of altered gene expression in mature skeletal muscle.     

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.     

Co-transfer of restriction endonucleases and plasmid DNA into mammalian cells by electroporation: effects on stable transformation

The antimutagenic principle of the green fruits of Momordica charantia was shown by the micronucleus test to be an intractable mixture of novel acylglucosylsterols. The antimutagens were extracted from the green fruits with ethanol and isolated from the bioactive petroleum ether and carbon tetrachloride extracts by repeated and sequential flash column chromatography. The major component of the mixture is

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and the minor component is the stearyl derivative (Guevara, 1989). At a dosage range in mice of 50–12.5 μg extract/g, the mixture reduced by about 80% the number of micronucleated polychromatic erythrocytes induced by the well-known mutagen mitomycin C. Structure-activity correlation studies suggested that the antimutagenic activity may reside in the peculiar lipid-like structure of the acylglucosylsterols. Ingestion of these compounds may result in their absorption in the plasma membrane lipid bilayer which could adversely affect the membrane permeability towards mitomycin C and disrupt the cellular activity of the latter.     

A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo

ObjectiveMultidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemo-resistant cancers patients.MethodsWe investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer.ResultsNF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer.ConclusionThis novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1:200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1:102400 after the second injection and decreased to 1:12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1:25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-λ responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Tuberculosis is an ancient scourge of mankind. According to statistics, there are more than 10 million new cases of tuberculosis each year and the annual death toll for tuberculosis exceeds three millions. The current available BCG is of questionable efficacy, and its protection ranges from 0 to 85%. Therefore, developing a safe and effective vaccine against this scourge is very important. Previous studies have shown that the secreted proteins of Mycobacterium tuberculosis (M. TB) can induc…     

In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA.

The skin cells of newborn mice were stably transformed in vivo with the aid of electroporation. The plasmid DNA was introduced subcutaneously followed by high-voltage pulses applied to the skin pleat. NEO-resistant colonies were found in primary cell cultures obtained from the treated skin. The experiments show that in vivo electroporation can be used for the introduction of plasmid DNA into skin cells of mouse.     

In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers

Background

DNA-based vaccines have been safe but weakly immunogenic in humans to date.

Methods and Findings

We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines.

Conclusions

This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00545987","term_id":"NCT00545987"}}NCT00545987     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.     

Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.