**3. Electrogene transfer**

#### **3.1 Mechanisms and application of gene transfer by electric fields**

Naked DNA vaccination emerged as a promising approach for introducing foreign antigens into the host to induce protective immunity. The delivery of DNA vaccine into skeletal muscle is highly preferable as this organ is not only a passive site but can actively take part in immune reactions. However, one important limitation of intramuscular (i.m.) genetic vaccines is their weak performance in large animals as regards the low DNA transfection efficiency of the tissue (Escoffre et al. 2010). For this reason, novel and safe delivery systems have been developed to further improve the vaccine efficiency and immunogenicity. In several reports, electroporation-mediated DNA delivery was described as an effective tool in eliciting immune response in small and large animal models (Babiuk et al. 2002; Otten et al. 2004; Peng et al. 2007), with numerous studies proving that this technique is effective in the stimulation of humoural and cellular immunity (Dupuis et al. 2000; Tollefsen et al. 2002; Widera et al. 2000). Interest in the application of EP to DNA vaccination protocols is greatly growing in these last years for several considerations. It has been demonstrated that EP allows an augmented uptake of DNA in tissue cells especially if used in combination with hyaluronidase (McMahon et al. 2001). A higher DNA uptake *in vivo* is possible thanks to the enhancement of cell membrane permeabilization and electrophoretic movement of DNA molecules into the target cells. Moreover, if EP is applied in muscle cells, these work as a platform for antigen production within the skeletal muscle (Shirota et al. 2007). A combination of both these events facilitates target cell transfection, this resulting in a higher synthesis of the gene of interest and in an intensification of the immune response to the encoded protein.

Many studies have reported the beneficial effect of EP on the activated response by the immune system against the transferred antigen in several animal models (Tsang et al. 2007). Respect to a simple administration of DNA vaccines through i.m. injection, EP is responsible for a significant increase in antibody titre (Buchan et al. 2005), antigen-specific T-cell frequency and induction of several T-cell effector functions (Bachy et al. 2001; Capone et al. 2006). In particular, a study performed on DNA vaccination mediated by EP demonstrated that the concomitant injection of plasmid DNA and EP is crucial for the adjuvant effect exerted by EP, which is responsible for eliciting antigen-specific IgG2a antibody production

process is commonly known as electropermeabilization or electroporation (EP) (Chiarella et al 2010; Favard et al. 2007; Mir et al. 1999). The simultaneous publications in 1998 by Aihara and Miyazaki and Harrison and co-workers, demonstrated EP as being a more efficient method for gene transfer into muscle than the simple i.m. injection of DNA (Aihara and Miyazaki 1998; Harrison et al. 1998). The strategy is not only promising for enhancing the gene delivery of therapeutic proteins and drugs. Infectious disease, cancer gene therapy and chemotherapy are other fields of application, making electrochemogenetherapy relevant in a variety of research branches and promising in the gene therapy field (Mir 2008; Wells 2004). The advantage of DNA electrotransfer is dual. On the one hand, a high number of muscle cells are transfected with the DNA vaccine; on the other hand the damaged muscle cells release danger signals that favour antigen presenting cell recruitment, thus enhancing the immune response (Chiarella et al. 2008b). For this reason we consider the electropermeabilization such an important and very promising tool in the future of DNA

Naked DNA vaccination emerged as a promising approach for introducing foreign antigens into the host to induce protective immunity. The delivery of DNA vaccine into skeletal muscle is highly preferable as this organ is not only a passive site but can actively take part in immune reactions. However, one important limitation of intramuscular (i.m.) genetic vaccines is their weak performance in large animals as regards the low DNA transfection efficiency of the tissue (Escoffre et al. 2010). For this reason, novel and safe delivery systems have been developed to further improve the vaccine efficiency and immunogenicity. In several reports, electroporation-mediated DNA delivery was described as an effective tool in eliciting immune response in small and large animal models (Babiuk et al. 2002; Otten et al. 2004; Peng et al. 2007), with numerous studies proving that this technique is effective in the stimulation of humoural and cellular immunity (Dupuis et al. 2000; Tollefsen et al. 2002; Widera et al. 2000). Interest in the application of EP to DNA vaccination protocols is greatly growing in these last years for several considerations. It has been demonstrated that EP allows an augmented uptake of DNA in tissue cells especially if used in combination with hyaluronidase (McMahon et al. 2001). A higher DNA uptake *in vivo* is possible thanks to the enhancement of cell membrane permeabilization and electrophoretic movement of DNA molecules into the target cells. Moreover, if EP is applied in muscle cells, these work as a platform for antigen production within the skeletal muscle (Shirota et al. 2007). A combination of both these events facilitates target cell transfection, this resulting in a higher synthesis of the gene of interest and in an intensification of the immune response to the

Many studies have reported the beneficial effect of EP on the activated response by the immune system against the transferred antigen in several animal models (Tsang et al. 2007). Respect to a simple administration of DNA vaccines through i.m. injection, EP is responsible for a significant increase in antibody titre (Buchan et al. 2005), antigen-specific T-cell frequency and induction of several T-cell effector functions (Bachy et al. 2001; Capone et al. 2006). In particular, a study performed on DNA vaccination mediated by EP demonstrated that the concomitant injection of plasmid DNA and EP is crucial for the adjuvant effect exerted by EP, which is responsible for eliciting antigen-specific IgG2a antibody production

vaccination therapy that it deserves a dedicated section of this book chapter.

**3.1 Mechanisms and application of gene transfer by electric fields** 

**3. Electrogene transfer** 

encoded protein.

and Th-1 biased immune responses (Gronevik et al. 2005). Another study demonstrated that a single i.m. DNA vaccination in combination with EP enhanced significantly the onset and the duration of the primary antibody response affecting immune memory (Tsang et al. 2007). The use of EP for delivering a DNA vaccine encoding anthrax toxin protective agent has been demonstrated able in a rapid induction of antibodies against the antigen in 2 weeks following a single immunization in several experimental animals (Luxembourg et al. 2008).

Despite these evidences, details on possible mechanisms responsible for the positive effect of EP on the immune response to DNA vaccines were not completely characterised (Escoffre et al. 2009).

Recently EP has been reported as crucial event through which, inducing transient morphological changes and a local moderate damage in the treated muscle, is possible to generate an early production of endogenous cytokines responsible for signalling danger at the local level. The activation of a danger pro-inflammatory pathway and the recruitment of inflammatory cells result in T lymphocyte migration, indicating electropermeabilization *per se* is able to recruit and trigger cells involved in antigen presentation (Chiarella et al. 2008b). Due to these immunostimulating effects, EP is now recognised as a good adjuvant (Chiarella et al. 2007), helpful in DNA vaccination for increasing the potency and safety of this therapeutic approach due to its property to induce a higher DNA uptake and its ability to stimulate both humoural and cellular immunity. At present, numerous findings are clarifying EP mechanism (Golzio et al. 2010), also showing the easy applicability of EP to large animals. In this view, many studies are concentrated to find the most appropriate and tolerable parameters that will make EP suitable for humans (Tjelle et al. 2008; Tjelle et al. 2006). To this purpose, various electroporating devices have been developed for animal and human use (**Fig. 2**).

Because these initial results seem promising, several clinical trials based on DNA vaccination assisted by electropermeabilization are under investigation and the efficacy and tolerability of EP will be deeply studied in the near future both in preclinical and clinical gene therapy protocols.

#### **3.2 Application of electrogene transfer in DNA vaccination protocols**

The use of EP technology for DNA vaccination in the clinical settings takes advantage from the development of new protocols, which are effective in administrating the vaccine with the minimum discomfort and maximum tolerability for the patient.

The type of EP device, intensity of electrical stimulations, number of electrical pulses and administrations, and choice of the target organ, are important parameters taken into consideration for the design of clinical protocols.

Because the need to develop non-invasive or minimally invasive genetic vaccination methods has become an important issue, several studies have been focussed on the needle-free injection of DNA vaccines. A recent patent, issued in 2007, reports the combination of needle-free injection and electroporation, demonstrating that this noninvasive strategy is sufficient to introduce the DNA vaccine in a form suitable for electrotransfer into a region of the host tissue. This needle-free injection may be used in combination with suitable non invasive electrode configurations (Hofmann 2007). Safe EP protocols for human disease therapies are under investigation and many preclinical studies are described in numerous works investigating the potential of EP in both infectious and tumour diseases.

DNA Vaccination by Electrogene Transfer 187

structural region of HCV (from NS3 to NS5B) induces substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than a simple naked DNA injection in mice and in Rhesus macaques. As already discussed, the T-cell responses elicited by the DNAbased electroporation strategy can be useful in prophylactic vaccine approaches against HCV and this work supports this hypothesis (Capone et al. 2006). Because the administration of a plasmid cocktail, encoding antigen and adjuvants in combination with EP, is proposed as an efficient genetic immunisation strategy, the same group designed a protocol in which Hepatitis C virus (HCV) E2 and cytokine encoding plasmids have been co-injected in the mouse quadriceps with or without EP. The vaccination outcome has been evaluated by analysis of antigen-specific cellular- mediated or antibody-mediated immunity. The co-injection of cytokine and HCV E2-encoding plasmids followed by EP, strongly enhanced T- or B-cell responses to various levels, depending on the particular

Respect to cancer, strong cellular immune responses can be induced in both mice and nonhuman primates, following the administration with EP of a novel HPV18 DNA vaccine

Improvement in the efficacy of a cancer vaccine administered by electroporation, could increase its chances for clinical success. A demonstration of the inhibition of tumour growth has been reported by Curcio and collaborators. They demonstrated that a vaccination protocol using a plasmid encoding the extracellular and transmembrane domains of the Neu oncogene delivered by electroporation, prevents longterm tumour formation in cancer-

Since electropermeabilization is considered a promising delivery system for plasmid DNA vaccination, several clinical trials are now experimenting EP as a medical technology in human patients affected by infectious as well as cancer diseases (Bodles-Brakhop et al.

A summary of the clinical trials performed by DNA vaccination and EP are shown in the

**Clinical trial Condition Pathogen Intervention Phase** 

HPV (E6-E7)

**(prophylactic)** HIV ADVAX

**(therapeutic)** HIV-1 PENNVAX-B

falciparum EP-1300 I

HCV CHRONVAC-

VGX-3100

CELLECTRA <sup>I</sup>

C® I/IIa

TriGrid™ <sup>I</sup>

CELLECTRA <sup>I</sup>

combination used (Arcuri et al. 2008).

prone transgenic mice (Curcio et al. 2008).

2009).

following tables.

NCT00685412

NCT00563173

NCT00545987 **AIDS** 

NCT01082692 **AIDS** 

Table 3. Clinical trials in infectious diseases.

encoding an E6/E7 fusion consensus protein (Yan et al. 2008).

NCT01169077 **Malaria** Plasmodium

**Human Papillomavirus infection** 

**Chronic Hepatitis C Virus Infection**

The DNA vaccine is injected into the skeletal muscle of the mouse limb and penetration into the target tissue is achieved by electropermeabilization of the muscle after DNA is injected with a syringe. The electrical impulse is applied by electrodes in contact with the skeletal muscle. Different electroporator devices are shown in the figure. A) BTX ECM 830 Harvard Apparatus; B) IGEA Cliniporator; C) Inovio MedPulser® EPT; D) Ichor TriGridTM.

#### Fig. 2. Administration of DNA vaccine by electropermeabilization of the muscle

HIV vaccine administration has been conducted in several animal models. Electricallymediated delivery technology has been applied to DNA vaccines against HIV virus, and substantially higher immune responses have been achieved in mice and rabbits following vaccination with DNA encoding HIV genes. Vaccines were administered with constant electric current or constant electric voltage, causing up to 20-fold higher immune responses in comparison to the application of DNA vaccines alone (Selby 2000). In another study in mice, *in vivo* EP amplified cellular and humoural immune responses to a HIV type 1 Env DNA vaccine, enabled a 10- fold reduction in vaccine dose, and resulted in increased recruitment of inflammatory cells (Liu et al. 2008). Another study on the development of plasmid DNA vaccine able in eliciting robust cell-mediated immune response to multiple HIV type 1 (HIV-1)-derived antigens has been conducted in Rhesus macaques. Vaccination in combination with *in vivo* EP led to a more rapid onset and enhanced vaccine-specific immune responses (Luckay et al. 2007).

Also the hepatitis C virus disease is object of investigation. It was demonstrated that gene electrotransfer of a novel candidate DNA vaccine encoding an optimised version of the non

A B

C D

The DNA vaccine is injected into the skeletal muscle of the mouse limb and penetration into the target tissue is achieved by electropermeabilization of the muscle after DNA is injected with a syringe. The electrical impulse is applied by electrodes in contact with the skeletal muscle. Different electroporator devices are shown in the figure. A) BTX ECM 830 Harvard Apparatus; B) IGEA Cliniporator; C) Inovio

HIV vaccine administration has been conducted in several animal models. Electricallymediated delivery technology has been applied to DNA vaccines against HIV virus, and substantially higher immune responses have been achieved in mice and rabbits following vaccination with DNA encoding HIV genes. Vaccines were administered with constant electric current or constant electric voltage, causing up to 20-fold higher immune responses in comparison to the application of DNA vaccines alone (Selby 2000). In another study in mice, *in vivo* EP amplified cellular and humoural immune responses to a HIV type 1 Env DNA vaccine, enabled a 10- fold reduction in vaccine dose, and resulted in increased recruitment of inflammatory cells (Liu et al. 2008). Another study on the development of plasmid DNA vaccine able in eliciting robust cell-mediated immune response to multiple HIV type 1 (HIV-1)-derived antigens has been conducted in Rhesus macaques. Vaccination in combination with *in vivo* EP led to a more rapid onset and enhanced vaccine-specific

Also the hepatitis C virus disease is object of investigation. It was demonstrated that gene electrotransfer of a novel candidate DNA vaccine encoding an optimised version of the non

Fig. 2. Administration of DNA vaccine by electropermeabilization of the muscle

MedPulser® EPT; D) Ichor TriGridTM.

DNA vaccine Skeletal muscle

Electrical impulse

immune responses (Luckay et al. 2007).

structural region of HCV (from NS3 to NS5B) induces substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than a simple naked DNA injection in mice and in Rhesus macaques. As already discussed, the T-cell responses elicited by the DNAbased electroporation strategy can be useful in prophylactic vaccine approaches against HCV and this work supports this hypothesis (Capone et al. 2006). Because the administration of a plasmid cocktail, encoding antigen and adjuvants in combination with EP, is proposed as an efficient genetic immunisation strategy, the same group designed a protocol in which Hepatitis C virus (HCV) E2 and cytokine encoding plasmids have been co-injected in the mouse quadriceps with or without EP. The vaccination outcome has been evaluated by analysis of antigen-specific cellular- mediated or antibody-mediated immunity. The co-injection of cytokine and HCV E2-encoding plasmids followed by EP, strongly enhanced T- or B-cell responses to various levels, depending on the particular combination used (Arcuri et al. 2008).

Respect to cancer, strong cellular immune responses can be induced in both mice and nonhuman primates, following the administration with EP of a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein (Yan et al. 2008).

Improvement in the efficacy of a cancer vaccine administered by electroporation, could increase its chances for clinical success. A demonstration of the inhibition of tumour growth has been reported by Curcio and collaborators. They demonstrated that a vaccination protocol using a plasmid encoding the extracellular and transmembrane domains of the Neu oncogene delivered by electroporation, prevents longterm tumour formation in cancerprone transgenic mice (Curcio et al. 2008).

Since electropermeabilization is considered a promising delivery system for plasmid DNA vaccination, several clinical trials are now experimenting EP as a medical technology in human patients affected by infectious as well as cancer diseases (Bodles-Brakhop et al. 2009).

A summary of the clinical trials performed by DNA vaccination and EP are shown in the following tables.


Table 3. Clinical trials in infectious diseases.

DNA Vaccination by Electrogene Transfer 189

without serious adverse effects related to the administration procedure, we strongly support improvements addressed to the efficacy of DNA vaccines administered by electropermeabilization in clinical protocols. This new approach could successfully increase

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Giudice, G. (2005). "Combined conjugate vaccines: enhanced immunogenicity with

Krivulka, G. R., Lifton, M. A., Crabbs, C. L., Heidecker, G., Perry, H. C., Davies, M. E., Xie, H., Nickerson, C. E., Steenbeke, T. D., Lord, C. I., Montefiori, D. C., Strom, T. B., Shiver, J. W., Lewis, M. G., and Letvin, N. L. (2000). "Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys." *Proc Natl Acad Sci U S A*, 97(8),

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chances for clinical success in humans.

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**5. References** 

1048-54.

4192-7.


Table 4. Clinical trials in cancer diseases.

The results seem to be promising and applicable to a large cohort of diseases in the next future. Therefore electroporation would appear to be the more efficient technology for local injection of plasmid DNA vaccine into the tissue (Kato and Nakamua 1965; Wells 2010).
