**5. Acknowledgements**

The authors are thankful to Conselho Nacional de Desenvolvimento Cientifico- CNPq and Fundacao de Amparo a Pesquisa do Rio Grande do Sul- FAPERGS.

We also would like to thank our colleagues Fabiana Quoos Mayer, Carlos Oscar Kieling, Valeska Lizzi Lagranha and Raquel Balestrin for contributing with data to this paper.

#### **6. References**

162 Non-Viral Gene Therapy

Based on those findings, a new technology has emerged, known as the minicircle (MC) gene therapy. This system uses a phiC31 integrase recombination event to remove the bacterial backbone elements of the plasmid resulting in a DNA circle (the MC), encoding the mammalian expression cassette of choice and a small attR footprint (Chen et al, 2003). This has proven to be resistant to gene silencing *in vivo*, is maintained as an extrachromosomal episome, and therefore represents an interesting platform for gene replacement strategies

This technology was recently used for treatment of MPS I mice in a proof-of-concept study (Osborn et al, 2011). In this study, the researchers performed a hydrodynamic injection of a minicircle plasmid containing the IDUA gene combined with immunomodulation, achieving stable expression of the transgene, increased IDUA tissue levels and reduction in GAG storage. As a recent technology, this is the only study performed on LSDs so far, but

Fig. 8. Production of a minicircle plasmid. This simplified version of the process shows the parental plasmid containing both the gene of interest and the bacterial components,

from the bacterial backbone, which then is degraded.

clinically relevant results were obtained in some cases.

**4. Conclusions** 

including origin of replication (ORI), genes that confer resistance to antibiotic (Ab resist) and sites that allow attachment of the integrase (att B and P). After activation of the integrase, a cis-recombination event occurs, separating the gene of interest and its regulatory elements

Still nowadays most lysosomal storage disorders do not have an effective treatment. Moreover, treatments currently available are not able to correct all the manifestations of these multisystemic diseases. Despite the small number of protocols (if compared to other areas, like oncology), gene therapy approaches have shown their potential to be helpful in many of these diseases. Not only proof of concept experiments have been performed, but

results are encouraging and should be tested on other diseases soon.

for lysosomal storage disorders (figure 8).


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**8** 

*Italy* 

**DNA Vaccination by Electrogene Transfer** 

*2Laboratory of Molecular Medicine and Biotechnology CIR, University Campus Bio-Medico of Rome, Rome* 

*3Laboratory of Oncology, IRCCS 'Casa Sollievo della Sofferenza'* 

Pieranna Chiarella1,2, Vito Michele Fazio1,3 and Emanuela Signori1,2 *1Laboratory of Molecular Pathology and Experimental Oncology, CNR-IFT, Rome* 

Vaccination is historically one of the most important methods for the prevention of infectious diseases in humans and animals. When Edward Jenner inoculated James Phipps with a bovine poxvirus to induce protection against the closely related human pathogen smallpox virus in 1796 and then, almost a century later, Pasteur developed a live attenuated vaccine against rabies, the basic principles for vaccine development were established (Fraser and Rappuoli 2005). Traditionally, a vaccine is known as a preparation of attenuated or killed microorganisms or of subunit vaccines (purified components of a pathogen including the protein-conjugated capsular polysaccharides, toxoids, cell-free extracts, recombinant proteins and stand-alone capsular polysaccharides) administered for inducing active

Two types of immunization exist with intrinsic differences between them: prophylactic vaccination initiates a response against an antigen to which the immune response is naïve, leading to a long-term memory cell maintenance and protective efficacy; therapeutic vaccination stimulates the immune system to a chronically displayed antigen, leading to a

Several infectious diseases can be prevented by vaccines produced with conventional approaches. These methods are based on the cultivation in laboratory conditions of the microorganism from which single components are isolated individually by using biochemical, microbiological and serological techniques. Each antigen is produced in pure form either directly from the bacterium or using the DNA recombinant technology, and finally tested for its ability to induce an immune response (Serruto and Rappuoli 2006). Conventional approaches provided the basis of vaccinology and led to great achievements such as the eradication of smallpox and the virtual disappearance of diseases like diphtheria, tetanus, poliomyelitis, pertussis, measles, mumps, rubella and invasive *Haemophilus influenzae* B, increasing the life quality and expectancy (Andre 2003). Nevertheless, they present major disadvantages such as to be time-consuming and, more important, to be impractical in some circumstances due to the difficulty in cultivating some microorganisms *in vitro* and to the fact that even attenuation may result in detrimental or unwanted immune responses (Purcell et al. 2007). Moreover, in many cases the antigens

**1. Introduction** 

immunity to a specific disease.

clearance of an established infection.

**Vaccination: Traditional and new generation vaccines** 

