SPECIAL FOCUS y RNA Vaccines

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Electroporation-enhanced delivery of nucleic acid vaccines Expert Review of Vaccines Downloaded from informahealthcare.com by University of Auckland on 02/25/15 For personal use only.

Expert Rev. Vaccines 14(2), 195–204 (2015)

Kate E Broderick* and Laurent M Humeau Inovio Pharmaceuticals Inc., 660 West Germantown Pike, Suite 110, Plymouth Meeting, PA 19462, USA *Author for correspondence: Tel.: +1 858 410 3161 Fax: +1 858 597 0451 [email protected]

The naked delivery of nucleic acid vaccines is notoriously inefficient, and an enabling delivery technology is required to direct efficiently these constructs intracellularly. A delivery technology capable of enhancing nucleic acid uptake in both cells in tissues and in culture is electroporation (EP). EP is a physical delivery mechanism that increases the permeability of mammalian cell membranes and allows the trafficking of large macromolecules into the cell. EP has now been used extensively in the clinic and been shown to be an effective method to increase both the uptake of the construct and the breadth and magnitude of the resulting immune responses. Excitingly, 2014 saw the announcement of the first EP-enhanced DNA vaccine Phase II trial demonstrating clinical efficacy. This review seeks to introduce the reader to EP as a technology to enhance the delivery of DNA and RNA vaccines and highlight several published clinical trials using this delivery modality. KEYWORDS: DNA vaccine . electroporation . electrotransfer . RNA vaccine

A major obstacle to effective vaccination via gene-based methods is the low efficiency of intracellular delivery. Outside of mice models, the delivery of naked DNA through a standard intramuscular (im.) injection is notoriously inefficient. In past studies, this has led to an inability to achieve strong immune responses in large mammals, such as monkeys and humans immunized with naked DNA [1,2]. Multiple physical and chemical methods for gene delivery have been described by several groups worldwide. Iontophoresis, magnetic nanoparticles, sonication, lipid delivery, microneedle delivery, jet injection and gene gun [3,4] are such examples. A physical method that has shown great success at delivering nucleic acids to mammalian cells is electroporation (EP). In vivo EP is a physical method to temporarily increase cell membrane permeability for DNA upload and this technology has become a vanguard when considering delivery modalities for nucleic acidbased vaccination. Introduction to in vivo EP

The dynamic phenomenon of EP is caused by an externally applied electrical field resulting in a significant increase in the local transmembrane voltage and electrical conductivity causing

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10.1586/14760584.2015.990890

permeability of the plasma and nuclear cell membranes [5–7]. The application of brief electrical pulses results in the creation of transient hydrophilic pathways within the lipid bilayer membranes of mammalian cells. This temporary increase in permeability allows the transport of DNA and other macromolecules across a cell membrane that was previously impermeable to these molecules. Therefore, EP has the ability to increase both the uptake and the extent to which drugs and nucleic acids are delivered to the target tissue of interest [8–12]. Recently, an elegant study using in silico computer modeling was used to generate the first description of a nucleic acid molecule traversing the mammalian cell membrane after the application of a brief electrical pulse [13]. The theoretical model was then validated experimentally in vitro. A similar comparison modeling study was performed by Son et al., 2014 [14]. A defined electrical pulse duration and shape are required to reach the specific transmembrane voltage threshold allowing the manifestation of the EP phenomenon to occur. Therefore, only the cells or areas of a cell membrane reaching this magnitude of electric field threshold will be electroporated. If a

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secondary threshold is reached or surpassed, EP will also result in compromising the viability of the cells. This event is termed irreversible EP and is commonly used clinically as a technique to ablate tumor cells [15]. The use of reversible EP combined with chemotherapeutic agents is also used as a treatment for melanoma and cutaneous tumors [16]. The first published documentation of the use of EP to deliver DNA directly to mammalian cells was in 1982 when Neumann et al., delivered both plasmid and linear DNA expressing the herpes simplex thymidine kinase gene to a suspension of mouse lyoma cells [5]. The procedure has subsequently become a widely used laboratory technique for the transfection of cells in vitro. Although naked gene transfer or gene transfer enhanced by liposomal delivery to muscle tissue had been extensively investigated in the early and mid-1990s [17–19], the application of EP technology to mammalian cells in vivo came 10 years later than its application to cells in culture. Rols et al. demonstrated the efficient permeabilization of murine melanoma by EP, with 80% of the cell population able to take up propidium iodide through EP-enhanced delivery. A plasmid expressing b-galactosidase was transferred and expressed in the cells with an efficiency of 4% [20]. A novel method for high-efficiency and region-controlled in vivo gene transfer was described by Nishi et al., where the combination of in vivo EP and intra-arterial plasmid DNA (pDNA) injection was shown to efficiently transfer and express a plasmid expressing LacZ in mice tumor cells [21]. Heller et al., reported that in vivo EP of rat liver tissue in the presence of reporter genes resulted in the strong expression of these genetic markers in rat hepatocyte cells. Between 30 and 40% of the electroporated rat hepatocytes expressed the b-galactosidase reporter gene 48 h following EP-enhanced delivery [22]. The first published description of EP-enhanced delivery of plasmid to muscle tissues came in 1998. The tibialis anterior muscles of mice were injected with plasmid expressing IL-5 and electroporated. Mice that were injected but did not receive EP had serum levels of 0.2 ng/ml. EP application enhanced the IL-5 serum levels over 20 ng/ml [23]. These results demonstrated that DNA plasmid uptake following im. EP is more efficient than naked im. DNA injection alone. A similar study published later that year confirmed these results, demonstrating a 10-fold increase in transfection efficiency of muscles stimulated by EP [12]. DNA vaccines

DNA vaccines are an example of a next-generation vaccine approach that offers major benefits over their conventional prophylactic and preventive vaccine counterparts for the treatment of infection diseases and cancers [1,2]. Unlike conventional strategies (i.e., protein-based or inactivated virus-based vaccines), many DNA vaccines are gene-based expression plasmids that encode specific antigens, do not require isolated viruses or tumor cells for production and can mimic the immunological effects of infection because they are directly transfected into the host cell, or in the case of cancer, break tolerance against a selfantigen. As a result of this entry into the cell, gene expression 196

occurs via the host’s own cellular machinery, allowing for antigen presentation through both the MHC class I and II pathways. Gene-based vaccines also offer the ability to develop, optimize and manufacture large doses of vaccine [24] in a costeffective, rapid manner and deliver different combinations of antigens to avoid immune responses to the delivery system or vector itself. Because of the inherent stability of DNA vaccines, they do not require cold-chain storage, which is a major logistical issue with some current conventional vaccines and biologics. This has obvious major implications for their distribution and use in developing countries. Most importantly, DNA vaccines with EP are able to generate both a robust antibody and T-cell response [1,2]. This ability means that DNA vaccination offers a therapeutic solution against many complex diseases, such as HIV/AIDS and cancers [1,25,26]. Although the majority of gene-based vaccines are in the form of plasmids, research has also focused on the delivery of linear DNA constructs. Such constructs can be produced though standard PCR-based methods [27] or through enzymatic means [28]. These linear constructs, therefore, eliminate the need for elements such as the bacterial origin of replication and antibiotic resistance genes. Minicircle DNA vectors are another example of a paired down construct [29,30]. To date, no DNA-based vaccine is approved by the US FDA for clinical use in patients for either a prophylactic or therapeutic target [31]. However, three DNA vaccines have been licensed for veterinary use. These vaccines target the prophylaxis of West Nile virus in horses [30,32], the prophylaxis of infectious hematopoietic necrosis virus in salmonid fish [33] and a therapy for malignant melanoma in dogs [34]. Interestingly, the canine melanoma vaccine relies on the expression of a xenogenous tumor-associated antigen (human tyrosinase), which induces the breakdown of tolerance against the endogenous protein. This results in the generation of a humoral response that significantly prolongs the overall survival of melanomabearing dogs. This strategy is currently being evaluated in the clinic as an immunotherapy against multiple cancer antigens. The need to break tolerance or to adapt to strain shift was the motivating factor for the development of the SynCon, vaccine design approach pioneered by Inovio Pharmaceuticals. This technology compiles multiple strains of the targeted infectious disease or cancer and sophisticated algorithms are used to analyze the different genetic sequences for the desired antigen. Alternatively, the Gene Designer freeware software is a technology that allows users to codon optimize their gene or sequence of interest for optimal protein expression in any organism [35]. EP-enhanced DNA vaccine delivery

Both preclinical and clinical data generated during the past two decades has demonstrated the importance of both the route and methodology of DNA vaccine delivery [1,25,36,37]. Whereas naked im. injection of DNA-based vaccines in mice yielded impressive immunogenicity, in part, at least because of the hydrodynamic pressure generated on injection, these results failed to translate in larger animals and in humans in the clinic. Expert Rev. Vaccines 14(2), (2015)

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EP-enhanced delivery of nucleic acid vaccines

This led to a significant research effort into the investigation of enhancing DNA delivery modalities. During the past 20 years, the use of nonviral delivery methods, such as jet injection [38,39], ballistic methods [4,40], microneedle injection [41,42], sonoporation and nano- and lipid-based particle transfer [43], have been extensively investigated both preclinically and clinically. Although all platforms have shown significant promise, in vivo EP has emerged as the forefront technology when considering delivery modalities for DNA-based vaccination. The plasticity of EP device design has allowed the technology to be applied to multiple tissue targets. The most common EP tissue targets are muscle [44] and skin [45] but also include direct tumor delivery [46], liver [47], brain [48,49], mucosa [50], pancreas [47] and kidneys [47]. Although skin and muscle remain popular targets for the delivery of nucleic acid-based vaccines or gene therapy products, intratumoral delivery can additionally encompass the delivery of chemotherapeutic agents. Although EP has been perceived as an uncomfortable procedure in the past, recent clinical studies have highlighted that there appear to be no major side effects associated with multiple EP deliveries and that as a procedure, it is generally well tolerated [25,36]. A comparison between the tolerability of im. and intradermal (ID) EP modalities was recently published [51]. Pain, as assessed by visual analog score, was highest immediately after EP but diminished by about 50% within 5 min. Overall, injection followed by EP with the CELLECTRA, Inovio Pharmaceuticals, EP, Inovio’s clinical device was well tolerated, and no significant safety concerns were identified. The CELLECTRA EP technology has been used in over 550 patients with over 1450 vaccinations having been performed [INOVIO PHARMACEUTICALS, UNPUBLISHED DATA]. Historically, im EP was seen as the methodology that could drive cellular responses, whereas ID EP was associated with robust humoral responses. Recently, multiple preclinical and clinical studies have demonstrated that this is not necessarily the case. Indeed, recent advances in ID EP devices have shown equivalency between targeting the skin versus targeting the muscle. A study by El Kamary et al. 2012 investigated the safety and tolerability of the Easy Vax dermal EP system manufactured by Cyto Pulse Sciences Inc (now Cellectis Therapeutics), alone (no DNA delivered) in healthy adults in a single site trial. Three treatments with the device were administered to 10 subjects in two body site areas. Two subjects complained of shooting pain, burning and/or tingling when the treatments were administered to the forearm region but not the lateral deltoid region. Tolerability pain scores never exceeded 3 of 10 in the 11-point pain rating scale and 12 of 100 in the visual analog score. Electrical properties of the skin, measured automatically by the EP device, demonstrated no correlation between pain intensity and skin conductance. In conclusion, the investigating team found that the Easy Vax EP device was safe and well tolerated when administered over the lateral deltoid skin regions in healthy volunteers [45]. A recent search of clinicaltrials.gov website [52] reveals that of the 898 trials listing DNA vaccines as a keyword, 43 also use EP as an enabling delivery technology. This number has risen informahealthcare.com

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steadily during the past 5 years and, because of some promising ongoing clinical programs, is likely to continue its upward trajectory. Although the bulk of literature describing the combination of DNA vaccines with EP is in the preclinical realm, in the interests of space, this review has chosen to highlight only published articles pertaining to clinical trials performed using EP-enhanced delivery of DNA-based vaccines. For ease of separation, two sections have been compiled: one summarizing published trials on the use of DNA vaccines enhanced by EP for oncological targets and the second, describing the clinical use of DNA vaccine and EP for infectious diseases. DNA vaccines & EP in the clinic: oncology targets

The clinical electroporation devices discussed in the review are summarized in TABLE 1. Here the relevant references are cited and the electrode type and EP parameters are described in more detail. Therapeutic vaccination could become an important modality to fight cancer because there is now an FDA-approved therapeutic cancer vaccine – Provenge from Dendreon. Multiple approaches to vaccinate cancer patients against their own diseases have been attempted, including the administration of DNA constructs coding for one or more tumor-associated antigens. The function of these DNA-based cancer vaccines is to elicit a tumor-specific immune response. There is a plethora of preclinical data demonstrating tumor stabilization and tumor reduction in a number of animal cancer models following DNA vaccination. Historically, however, clinically, this strategy has failed to generate therapeutically relevant clinical responses. A recent review by Pol et al. 2014 [53] discussed the latest advances on the use of DNA-based vaccines in cancer therapy, covering the literature that has been generated around this topic during the last year as well as clinical studies that have been launched during this time frame to assess the actual therapeutic potential of this intervention. Of all the clinical trials testing naked DNA vaccines as therapeutic cancer targets, approximately half used EP as an enabling technology delivering the vaccine constructs to either the skin or muscle. The EP-related trials ranged in disease target from cervical intraepithelial neoplasia [54], colorectal cancer [55], melanoma [56] and prostate cancer [57]. A prostate cancer Phase I study involved 15 patients with biochemical relapse of prostate cancer without macroscopic disease [58]. A DNA vaccine coding for rhesus prostate-specific antigen (rPSA) was delivered by ID injection and skin EP and the safety, clinical efficacy and immunogenicity of the vaccine was assessed. No systemic toxicity to the vaccine or delivery process was observed and discomfort from EP did not require analgesia or topical anesthetic. No clinically significant changes in PSA kinetics were observed because all patients’ required antiandrogen therapy following the completion of the vaccination regime because of rising PSA. Immunogenicity, measured by T-cell reactivity to the modified PSA peptide and to a mix of overlapping PSA peptides representing the full-length protein, was observed in some patients. All but one patient had prestudy PSA-specific T-cell reactivity [58]. 197

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Table 1. Summary of clinical electroporation devices discussed in the review. Study (year)

EP device name

Manufacturer

Target tissue

Electrode type

EP parameters

Ref.

El Kamary et al. (2012), Eriksson et al. (2013)

Easy Vax

Cytopulse

Skin

80-microneedle array arranged in eight rows of 10 needles

Six pulses of 100 ms of 100 V

[45,58]

Diehl et al. (2013), Bagarrazzi et al. (2012), Kalmas et al. (2013)

CELLECTRA

Inovio Pharmaceuticals

Muscle

Five penetrating needle electrodes with a depth penetration of 19 mm

Three 52 ms pulses of 0.5 Amps

Diehl et al. (2013)

CELLECTRA

Inovio Pharmaceuticals

Skin

Three penetrating needle electrodes with a depth penetration of 3 mm

Four 52 ms pulses of 0.2 Amps

[51]

Vasan et al. (2011), Hooper et al. (2014)

TriGrid Intramuscular Delivery System

Ichor Medical Systems

Muscle

Four penetrating electrodes in a diamond formation

Data not available

[80,85]

Diaz et al. (2013), Weiland et al. (2013)

Medpulser

Inovio Pharmaceuticals

Muscle

Four penetrating electrodes in a rectangular pattern

Two 60 ms pulses of 106 V

[60,87]

Chudley et al. (2012)

ELGEN 2000

Inovio Pharmaceuticals

Muscle

Two penetrating needle electrodes with a 4-mm spacing

Two 60 ms pulses of 0.4 Amps

[59]

Spanggaard et al. (2013)

Clinporator

IGEA

Tumor

Linear needle electrode (4 mm between needle arrays)

One high-voltage pulse of 1250 V/cm, 100 ms and one low-voltage pulse 140 V/cm, 400 ms

[62]

[51,61,82]

EP: Electroporation.

Another Phase I/II trial using EP for DNA delivery to patients with prostate cancer was reported in 2012 [59]. The authors reported the immunogenicity and clinical effects of a dose escalation trial of a DNA fusion vaccine delivered by im. EP in 32 patients. The vaccine encoded a domain from fragment C of tetanus toxin linked to a human leukocyte antigen-A2-binding epitope from the human prostate-specific membrane antigen. The team evaluated the effect of im. vaccination without or with EP on vaccine potency. DNA vaccination procedure was considered safe and well tolerated. At week 24, delivery of the vaccine with and without EP led to increased CD4+ and CD8+ vaccinespecific T cells with a trend toward greater effect with EP. PSA doubling time increased significantly from 11.97 months pretreatment to 16.82 months over the 72-week follow-up (p = 0.0417), with no clear differential effect of EP [59]. In 2013, Diaz et al. reported the results of two multicenter Phase I studies involving 33 adult patients with solid tumor cancer and stage II–IV disease. Patients were vaccinated with V930 alone, a DNA vaccine containing equal amounts of plasmids expressing the extracellular and trans-membrane domains of human human epidermal growth factor 2 and a plasmid expressing carcinoembryonic antigen fused to the B subunit of Escherichia coli heat labile toxin (Study 1) enhanced by EP, or a heterologous prime-boost vaccination approach with 198

V930 followed by V932, a bicistronic adenovirus subtype-6 viral vector vaccine coding for the same antigens (Study 2). The V930 vaccination with EP alone or in combination with the adenovirus-based vector vaccine was well tolerated without any serious adverse events. The most common vaccine-related side effects were injection site reactions and arthralgias. No measurable cell-mediated immune response to carcinoembryonic antigen or human epidermal growth factor 2 was detected in patients by enzyme-linked immunosorbent spot. However, a significant increase of both cellular immunity and antibody titer against the bacterial heat labile toxin was observed following vaccination [60]. A study investigating the impact of a DNA vaccine targeted against HPV serotypes 16 and 18 delivered by EP in women with cervical dysplasia was published in 2012 [61]. The authors reported encouraging Phase I safety, tolerability and immunogenicity results for the therapeutic HPV16/18 candidate DNA vaccine, VGX-3100, delivered by EP. Eighteen women who were previously treated for cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) received an im. three-dose regimen of pDNA encoding HPV16 and HPV18 E6/E7 SynCon antigens followed by EP in a dose escalation study (0.3, 1 and 3 mg per plasmid). The immunizations were well tolerated with reports of mild injection site reactions and no study-related serious or grade 3 and 4 adverse events. No dose-limiting toxicity was noted, and pain was assessed Expert Rev. Vaccines 14(2), (2015)

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EP-enhanced delivery of nucleic acid vaccines

by visual analog scale, with average scores decreasing from 6.2/ 10 to 1.4 within 10 min. Average peak IFN-g enzyme-linked immunospot magnitudes were reported as highest in the 3 mg cohort in comparison to the 0.3 and 1 mg cohorts, suggesting a trend toward a dose effect. Flow cytometric analysis revealed the induction of HPV-specific CD8+ T cells that efficiently loaded granzyme B and perforin and exhibited full cytolytic functionality in all cohorts. Excitingly, at the time of press, the successful results of the VGX-3100 (3 mg per plasmid) Phase II study have just been announced publically. The team from Inovio Pharmaceuticals has released and discussed the results from the randomized, double-blind, placebo-controlled Phase II trial of VGX-3100 in women with biopsy-proven CIN2/3 associated with HPV types 16 or 18. Treatment with VGX-3100 resulted in histopathological regression of CIN2/3 to CIN1 or no disease, meeting the study’s primary endpoint. In addition, the trial demonstrated clearance of HPV in conjunction with regression of cervical lesions, meeting the study’s secondary endpoint. Robust T-cell activity was detected in subjects who received VGX-3100 compared with those who received placebo. A study by Spanggaard et al. investigated the safety and tolerability of intratumoral EP enhanced delivery of plasmid encoding antiangiogenic metargidin peptide (AMEP) into cutaneous metastatic melanoma [62]. AMEP is a novel anticancer peptide, which can have both antiproliferative and antiangiogenic effect through binding to aVb3 and a5b1 integrins. Five patients with disseminated melanoma were treated at two dose levels (1 and 2 mg DNA). In each patient, two cutaneous lesions were identified (one treated and one control). At day 1 and day 8, plasmid encoding AMEP was injected intratumorally followed by EP. Patients were monitored weekly until day 29 and at day 64. Local efficacy was assessed at day 29 by direct measurement, and post-treatment biopsies for AMEP mRNA levels were evaluated by reverse transcriptase quantitative polymerase chain reaction. Minimal systemic toxicity was observed, including transient fever and transitory increase in C-reactive protein. No related serious adverse events occurred. AMEP mRNA was found in three of five treated lesions and none of the control lesions. At day 29, all five treated lesions were stable in diameter, whereas four of five control lesions increased more than 20%. No response occurred in distant lesions. This first-in-man study using EP-enhanced delivery of plasmid encoding AMEP into cutaneous melanoma demonstrated that the procedure and drug were safe and that local transfection was obtained. Two review articles by Aurisicchio et al. 2013 and Fioretti et al. 2013 summarize the clinical data generated through the use of DNA vaccines against cancer [63] and cancer and neurodegerative disease [64] enhanced by EP and further discusses the impact of optimized vector design strategies and enhanced delivery platforms and their impact on the generated immune response. Importantly, they both touch on future implications for the field and discuss the importance of an enabling delivery technology for the success of DNA vaccines in the clinic. informahealthcare.com

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DNA vaccines & EP in the clinic: infectious diseases

The plasticity of the immune responses generated by DNA vaccine immunization has the ability to both prevent and treat infectious diseases adding a new dimension to vaccination against currently unmet disease targets. There is a vast battery of preclinical data published on the use of DNA vaccines for a spectrum of infectious disease targets, including HIV/AIDS [65–69], malaria [70], HCV [71–73], HBV [74,75], Clostridium difficile [76], influenza [77] and Ebola [78,79]. Although the majority of the published data for infectious disease DNA vaccines combined with EP is in preclinical animal models, this review will focus on some of the most pertinent published clinical studies. A study by Vasan et al. determined the safety, tolerability and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly enhanced by in vivo EP in a Phase I, double-blind, randomized, placebo-controlled trial in healthy volunteers [80,81]. Eight volunteers each received 0.2, 1 or 4 mg ADVAX or saline placebo via EP using the Ichor Medical Systems TriGrid im. delivery system or 4 mg ADVAX via standard im. injection (no EP) at weeks 0 and 8. A third vaccination was administered to 11 volunteers at week 36. EP was found to be safe, well-tolerated and considered acceptable for a prophylactic vaccine. Importantly, EP delivery of ADVAX increased the magnitude of HIV-1-specific cellmediated immunity by up to 70-fold over im. injection only, as measured by IFN-g enzyme-linked immunosorbent spot. In addition, the number of antigens to which the response was detected was improved with EP and increased plasmid doses. Intracellular cytokine staining analysis of IFN-g enzyme-linked immunosorbent spot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. The authors believe that this study was the first published 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 [80]. In 2012, Kalams et al. described two multicenter HIV Vaccine Trials Network (HVTN) clinical studies (070 and 080) [82]. The trials used the PENNVAX-B (PV) HIV-1 DNA vaccine, which consisted of a mixture of three plasmids encoding HIV-1 Clade B Env, Gag, and Pol and a DNA plasmid encoding IL-12, which expressed the human IL-12 proteins, p35 and p40. The subjects enrolled in the trial were healthy HIV-1-uninfected adults in the age range of 18– 50 years. Four im. vaccinations were administered in HVTN 070 and three im. vaccinations enhanced by EP were administered in HVTN 080. Immunogenicity read-outs for this study were cellular immune responses as measured by intracellular cytokine staining after stimulation with HIV-1 peptide pools. Overall, the vaccination was safe and well tolerated. The administration of PV plus IL-12 with EP had a significant dose-sparing effect and provided immunogenicity superior to that observed in the trial without EP, despite there being fewer vaccinations. A total of 71.4% of individuals vaccinated with PV plus IL-12 plasmid with EP developed either a CD4+ or 199

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CD8+ T-cell response after the second vaccination, and 88.9% developed a CD4+ or CD8+ T-cell response after the third vaccination. In conclusion, the study noted that the use of EP following PV administration provided superior immunogenicity than delivery without EP. The HVTN 070 and 080 studies illustrated the power of a combined DNA approach to generate impressive immune responses in humans [82]. A good summary of both prophylactic and therapeutic DNA vaccines pertaining to HIV can be found in the review entitled ‘Therapeutic and prophylactic DNA vaccines for HIV-1,’ where additional attention is paid to studies using EP as an enabling technology [83]. Although DNA vaccines against hemorrhagic fever with renal syndrome (HFRS) had previously been investigated in the clinic using the particle-mediated epidermal delivery device [79], a study in 2014 detailed the delivery of a HFRS DNA vaccine enhanced by EP [84,85]. The USAMRIID-based team developed a candidate DNA vaccine for HFRS expressing the Gn and Gc genes of the Hantaan (HTNV) and Puumala (PUUV) viruses and evaluated them in an open-label, single-center Phase I study. Three groups of nine participants each were vaccinated on days 0, 28 and 56 with the DNA vaccines for HTNV, PUUV or a mixture of both vaccines using the Ichor Medical Systems TriGrid im. delivery system. All vaccinations consisted of a total dose of 2.0 mg DNA in an injected volume of 1 ml saline. For the combined vaccine, the mixture contained equal amounts (1.0 mg) of each DNA vaccine. No study-related serious adverse events were reported. The immunology read-out for the trial was the generation of neutralizing antibody responses, and these were measured by a plaque reduction neutralization test. Neutralizing antibody responses were detected in five of nine and seven of nine individuals who completed all three vaccinations with the HTNV or PUUV DNA vaccines, respectively. In the combined vaccine group, seven of the nine volunteers receiving all three vaccinations developed neutralizing antibodies to PUUV. The three strongest responders to the PUUV vaccine also had strong neutralizing antibody responses to HTNV. These results demonstrated that the HTNV and PUUV DNA vaccines delivered by EP separately or as a mixture are safe. In addition, both vaccines were immunogenic, although when mixed together, more participants responded to the PUUV than to the HTNV DNA vaccine. These studies are also summarized in a review titled ‘DNA vaccines for HFRS: Laboratory and clinical studies’ [86]. Clearance of infections caused by the HCV virus correlates with HCV-specific T-cell function. A Phase I/IIa study was performed at the Karolinska Institutet evaluated therapeutic DNA vaccination schedule enhanced by EP in 12 patients with chronic HCV infection. Eight patients also underwent a subsequent standard-of-care (SOC) therapy with pegylated IFN-a2a and Ribavirin. The clinical trial was performed in treatment of naive HCV genotype 1 patients who received 4 monthly vaccinations in the deltoid muscles with 167, 500 or 1500 mg codon-optimized HCV nonstructural 3/4A-expressing DNA 200

vaccine delivered by in vivo EP. Overall the treatment was safe and well tolerated and the vaccinations significantly improved IFN-g-producing responses to HCV nonstructural 3 during the first 6 weeks of therapy. Five patients experienced 2–10 weeks 0.6–2.4 log10 reduction in serum HCV RNA. Six of eight patients starting SOC therapy within 1–30 months after the last vaccine dose were cured. The authors believe this to be a first-in-man therapeutic HCV DNA vaccine study, where the vaccine was delivered by in vivo EP and resulted in transient effects in patients with chronic HCV genotype 1 infection. The interesting result noted after SOC therapy suggests that therapeutic vaccination can be explored in combination with SOC treatment [87]. EP-enhanced RNA vaccine delivery

In 1990, Wolff et al. demonstrated that direct injection of mRNA and pDNA into the skeletal muscle of a mouse resulted in expression of the encoded protein [16]. However, because of the inherent stability issues associated with mRNA and difficulties involved in its large-scale manufacture, much of the subsequent effort on the development of nucleic acid vaccines focused on pDNA. Recently, many of these perceived hurdles have been overcome and a resurgence of interest in RNA-based vaccines has appeared. RNA vaccines are based on mRNA or RNA replicons and like DNA vaccines could offer specific advantages over conventional and viral vector-based vaccines. In addition, unlike DNA vaccines, RNA vaccines do not require full passage into the nucleus, so delivery to the cytoplasm is sufficient for their mode of action and are also unable to integrate into the host genome. However, from a stability perspective, RNA is generally considered to be more susceptible to degradation than DNAbased entities. Several clinical trials delivering mRNA vaccines to melanoma patients without EP [88,89] and patients with stage IV renal cell cancer [90] have resulted in the induction of antitumor immunity, demonstrating proof of concept clinical efficacy. The German company CureVac is currently pursuing multiple mRNA based vaccines as an immunotherapy for oncological targets and as prophylactic vaccines for a number of infectious disease targets. RNA vaccines can also be engineered in the form of selfreplicating RNA replicons. These RNA vaccines are derived from RNA viruses lacking viral structural proteins making them capable of self-replication on delivery to the cytoplasm [91,92]. The RNA amplification process also leads to the production of double-stranded RNA intermediates, which are potent stimulators of innate immunity and as such may have inherent adjuvanting capabilities. Much of the recent effort to deliver these RNA vaccines has focused on nonviral mechanisms, such as lipid nanoparticle formulations, and has shown promising results [91,93–97]. A substantial body of work has focused on pairing the delivery of siRNA molecules with EP and this has shown significant promise [97–103]. However, less attention appears to have been Expert Rev. Vaccines 14(2), (2015)

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EP-enhanced delivery of nucleic acid vaccines

paid to the combination of RNA-based vaccines and EP. A method-based article [104] describes the in vivo delivery of conventional mRNA or alphaviral replicon RNA via ID EP. The authors discuss that ID EP many be an ideal route for the administration of RNA-based vaccines because of the high number of antigen-presenting cells residing in the skin. An article published in 2013 by Cu et al. explored the use of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope proteins [105]. The work detailed in this article demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable with those induced by EP delivery of pDNA. Specifically, the use of EP for the in vivo delivery of large, self-amplifying mRNA was investigated, as measured by both green fluorescent protein reporter gene expression and immunogenicity of genes encoding HIV envelope protein. The work detailed in this article demonstrated that like EP delivery of pDNA, EP delivery of self-amplifying mRNA elicited improved stronger and broader immune responses relative to non-EP delivered RNA. As with DNA vaccines, formulation and enabling delivery technologies will be an important area of research for RNA vaccines. As this vaccine technology enters the clinic over the next few years, harnessing the optimal delivery strategy is likely to be key to its success. Expert commentary

This is an extremely exciting time in the field of nucleic acid vaccines with the clinical success of the Phase II VGX-3100 therapeutic cervical dysplasia DNA vaccine a pivotal milestone in the field. VGX-3100 combined with EP was able to induce regression of precancerous cervical disease and clear HPV infection through the induction of robust T-cell responses. Meeting both the primary and secondary endpoints of this Phase II trial were paradigm shifting moments for the field because this was the first time that a DNA vaccine combined with EP was able to show clinical efficacy. Although this pioneering trial is a major milestone in the clinical development of DNA vaccines, the authors believe that many more successes will follow in its wake. Indeed, a recent publication in Nature by Kim et al. 2014 [106], which demonstrates clearance of persistent HPV infection through the use of a therapeutic DNA vaccine, supports breakthough studies in the DNA vaccine field and is a confirmation of the Phase II VGX-3100 therapeutic trial by other researchers in the field. Although DNA-based vaccines are commercially available for veterinary purposes, currently there are no FDA-approved nucleic acid vaccines for human use. Clearly, achieving this milestone is paramount to the clinical/public acceptance of this

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technology. However, because of the length of late-stage clinical trials and the regulatory and commercialization processes, this is unlikely to be achieved in the near-future (2–4-year time frame). An interesting area for consideration is that this commercialized product is likely to consist of the vaccine component combined with the enabling delivery technology because it is likely that the delivery mechanism is fundamentally tied to the clinical success. The need for an additional piece of delivery equipment over and above a needle and syringe could be viewed as a limitation to the use of this technology. However, the quality of the immune response generated and the efficacy of the vaccine platform should be the scientific and clinical measure. Accordingly, significant work is underway by a number of teams in the field to make the EP component as small a footprint as possible and make it applicable to widespread use in the field even under developing conditions. The authors are enthused by the development of new and novel nucleic acid vaccine candidates against a spectrum of diseases, as well as the significant strides being made in the EP device development arena. Combining optimized vaccine constructs with optimized EP delivery platforms will ensure that this field delivers on the promise that it has shown thus far. Five-year view

The next 5 years will be crucial to establish nucleic acid vaccines as effective therapeutic strategies and will be the true test of the technology as a whole. The translation from a promising technology on a scientific bench or in an animal model to success in the clinic against multiple disease targets is key. The use of enabling delivery technologies will be fundamental to this clinical transformation. EP is clearly leading the field as the enabling delivery for DNA vaccines and also shows promise for RNA-based vaccines. The increasing numbers of successful clinical studies with EP delivery suggest that the perception of tolerability of the procedure in a clinical setting has not proven to be a deterrent. Although the clinical proof of concept work has been performed using muscle EP, the significant effort geared toward alternative, less invasive strategies such as ID EP is likely to hold the key for the widespread use of this technology for prophylactic purposes. Acknowledgements

We would like to thank A Gomez and A Slager for manuscript assistance. Financial & competing interests disclosure

Both of the authors are current employees of Inovio and as such have financial interest (in the form of salary compensation, stock options and/or stock ownership) in the work described in this review article.

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Key issues .

Multiple trials demonstrating the safety and tolerability of electroporation (EP) as an enhancing delivery technology for DNA vaccines exist in the literature.

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DNA vaccines combined with EP have been extensively shown to generate robust immune responses both preclinically and clinically over naked delivery alone.

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Multiple DNA-based cancer vaccines combined with EP have been and are being evaluated in the clinic.

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Multiple DNA-based vaccines against a range of infectious diseases combined with EP have been and are being evaluated in the clinic.

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The combination of a therapeutic DNA vaccine and EP was shown to elicit clinical efficacy in a Phase II trial.

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Self-replicating RNA vaccines are a promising technology, but clinical data are lacking. The entry of this technology into the clinic is

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likely to be soon, but a delivery technology will be required.

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Electroporation-enhanced delivery of nucleic acid vaccines.

The naked delivery of nucleic acid vaccines is notoriously inefficient, and an enabling delivery technology is required to direct efficiently these co...
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