**4. Conclusions**

The last decade has witnessed a revolution in the approach to vaccine design and development. Despite considerable success in the prevention field, vaccinations against intracellular organisms, which require a cell-mediated immunity, are not yet available, while infectious diseases such as tuberculosis and malaria remain a serious problem in the Third World. Following the studies of Wolff and colleagues, in recent years immunisation with naked plasmid DNA encoding antigens has revealed a number of advantages, making DNA vaccination a promising therapeutic approach against infectious diseases and cancer. Of course improvement of vaccine efficacy has become a goal in the development of DNA vaccination protocols. Electropermeabilization has been shown to increase both the number of transfected cells and also the number of plasmids that permeate into each cell, therefore, electropermeabilization is now regarded as a promising delivery system for plasmid DNA vaccination. Intramuscular DNA vaccination combined with electropermeabilization has been described as effective in activating both humoural and cellular immune response in the host as well as in enhancing expression of the encoded antigen. Several reports showed that EP has adjuvant-like properties when combined with plasmid DNA injection. This approach is currently used not only in preclinical protocols in animals but also in humans, and studies for evaluating pain and stress induced by the treatment are currently under investigation indicating this approach as applicable and promising. Because this procedure is used safely 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 chances for clinical success in humans.

#### **5. References**

188 Non-Viral Gene Therapy

**Melanoma** Tyrosinase TriGrid I

tetwtCEA DNA wt CEA with tetanus toxoid Th epitope Derma Vax (electroporation device)

(SCIB-1) EP device I/II

(Tjelle 2006)

V934/V935

DERMA VAX™ I/II

DNA <sup>I</sup>

I/II

I/II (Closed 1.4.2008)

**Clinical trial Condition Antigen Intervention Phase** 

**Cancer** CEA

NCT00859729 **Prostate cancer** PSA pVAXrcPSAv53l

V934/V935 hTERT

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).

The last decade has witnessed a revolution in the approach to vaccine design and development. Despite considerable success in the prevention field, vaccinations against intracellular organisms, which require a cell-mediated immunity, are not yet available, while infectious diseases such as tuberculosis and malaria remain a serious problem in the Third World. Following the studies of Wolff and colleagues, in recent years immunisation with naked plasmid DNA encoding antigens has revealed a number of advantages, making DNA vaccination a promising therapeutic approach against infectious diseases and cancer. Of course improvement of vaccine efficacy has become a goal in the development of DNA vaccination protocols. Electropermeabilization has been shown to increase both the number of transfected cells and also the number of plasmids that permeate into each cell, therefore, electropermeabilization is now regarded as a promising delivery system for plasmid DNA vaccination. Intramuscular DNA vaccination combined with electropermeabilization has been described as effective in activating both humoural and cellular immune response in the host as well as in enhancing expression of the encoded antigen. Several reports showed that EP has adjuvant-like properties when combined with plasmid DNA injection. This approach is currently used not only in preclinical protocols in animals but also in humans, and studies for evaluating pain and stress induced by the treatment are currently under investigation indicating this approach as applicable and promising. Because this procedure is used safely

GTAC No 89 **Prostate cancer** PSMA EP device

NCT01064375 **Colorectal** 

NCT00471133 **Intraocular** 

Table 4. Clinical trials in cancer diseases.

NCT00753415

**4. Conclusions** 

NCT01138410 **Melanoma** Antibody

**Colon cancer Breast Cancer Melanoma** 


DNA Vaccination by Electrogene Transfer 191

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**1. Introduction**

genetic materials such as siRNA (Zhigang).

**9** 

*USA* 

**Gene Delivery with Ultrasound** 

Gene therapy and treatment with siRNA hold potential to treat a wide variety of different diseases. Genetic material is not usually stable and is generally hydrolyzed following intravascular administration. This makes the delivery of genes somewhat problematic since intact genetic material must usually be delivered intracellularly for therapeutic effect. For gene therapy, in most cases the gene construct must reach the cellular intranuclear subcompartment in order to elicit the desired biological effect. Viruses have evolved to deliver DNA and RNA to cells but viral vector-based gene therapy has been associated with effects inherent in biological systems (Marshall, Muruve, Thomas). Non-viral based systems afford the potential to deliver genetic materials without the adverse biological effects of viral-based systems. In most cases, however, non-viral based gene delivery systems have been less effective than viral based systems, yielding lower levels of gene expression (Litzinger). Microbubbles, e.g. acoustically active carriers, in concert with ultrasound (Unger, Zhou) may afford potential for highly effective site directed gene therapy and delivery of other

**2. Summary of gene delivery with microbubbles and ultrasound** 

contrast agents with approved claims for echocardiography.

The basic outline of a microbubble is shown in Figure 1. Microbubbles are composed of gas with a stabilizing shell material oftentimes consisting of lipids, albumin, or biocompatible polymers. For biomedical application they range in size from several microns in diameter to several hundred nanometers in diameter. The original biomedical application was as ultrasound contrast agents for echocardiography. Two agents, Definity®, phospholipidcoated perfluoropropane microbubbles (Lantheus, Billerica, MA) and Optison®, albumincoated perfluoropropane microbubbles, are approved by the FDA in the US and are sold as

Because of the large impedance mismatch between liquid and gas, when sound waves strike a microbubble, the waves are efficiently scattered back (microbubbles are excellent acoustic reflectors) and this is the basis for the use of microbubbles as ultrasound contrast agents. They are excellent reflectors of sound energy and hence are outstanding contrast agents for biomedical ultrasound imaging. Furthermore, the design of special ultrasound pulse sequences as 2nd harmonic imaging and phase inversion harmonic imaging has helped to increase ultrasound imaging by eliminating significant amounts of noise from tissue reflection.

**and Microbubbles** 

*University of Arizona* 

Evan Unger and Terry Matsunaga

Yoon, H. A., Aleyas, A. G., George, J. A., Park, S. O., Han, Y. W., Lee, J. H., Cho, J. G., and Eo, S. K. (2006). "Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses." *Microbiol Immunol*, 50(2), 83-92.
