**8. References**

88 Non-Viral Gene Therapy

Fig. 6. HAEC (A) and HASMC (B) after electroporation of pEGFP and 48 h incubation. Lefthanded: brightfield; Right handed: fluorescent picture (GFP), middle position: overlay of

caveolae does not automatically result in effective transfection as shown in this study. Although uptake in HAEC was partly achieved by the caveolae route, no transfection occurred. This is important to consider, because obviously it is not enough to focus the research on modifying particulate drug carriers, e.g., by attaching specific ligands, introducing surface charge or pH-sensitivity, or changing the particle size or elasticity in

The use of different methods (flow cytometry, spectral bio-imaging, electroporation) and materials (markers and inhibitors of endocytosis pathways) contributed to the evidence that transfection of primary human aorta endothelium and smooth muscle cells was not effective due to insufficient dissociation of plasmid DNA from the lipid/DNA delivery system. This finding is of great importance for future drug deliver development in the field of gene

The authors thank J. Clement, F. Lewrick, U. Huth and R. Schubert for helpful discussions, B. Erhard.and Z. Khalil for technical support, K. Aktories for supply of C-3 Toxin and M.

DC-30 (DC-Chol, 3ß-[(N´,N´-Dimethylaminoethane)-carbamoyl]-cholesterol-HCl and DOPE, Dioleoylphosphatidylethanolamine 3/7 (w/w); EGFP, enhanced green fluorescent protein; HASMC, human aorta smooth muscle cells; HAEC, human aorta endothelial cells; TM, transfection medium; DAPI, 4',6-Diamino-2-phenylindol-dihydrochlorid; mbCD, methyl-

order to trigger the caveolae mediated endocytosis.

pictures.

**A** 

**B** 

therapy.

**6. Acknowledgments** 

**7. Abbreviations** 

Follo for proof-reading the manuscript.


**5** 

**The Mechanical Agitation Method of Gene** 

Gene therapy is a therapeutic method used to treat diseases by altering genes within a patient's cells. The concept of gene therapy emerged as molecular biology evolved from the mere discipline of studying DNA molecules to the scientific art of virtually manipulating the genes of cells. Explosive worldwide research was conducted after the first introduction of the concept of gene therapy into the scientific community. The original aim was to directly modify patient genes through *in vivo* gene therapeutic approaches. However, DNA molecules introduced into the body by *in vivo* gene therapy are delivered at a very low frequency into terminally differentiated tissue cells, which typically do not have the capability of self-renewal (Tenenbaum et al., 2003). Because of the short-lived nature of *in vivo* gene therapy, a defective gene in patients is only temporarily corrected by *in vivo* gene therapy (Kaloss et al., 1999). The development of gene therapeutic methods in which the

*Ex vivo* gene therapeutic methods have been considered as alternative options to gene therapy to overcome the short-lived nature of the corrected genes of *in vivo* gene therapy. In *ex vivo* gene therapy, the surgically removed adult stem cells, such as mesenchymal stem cells or hematopoietic stem cells, are typically cultured in a laboratory apparatus. The therapeutic DNA molecules are introduced into the isolated cells, and these transfected cells are then introduced into the patients. By using adult stem cells in *ex vivo* gene therapeutic methods, the corrected genes that are introduced are, in most cases, expressed permanently once they are corrected properly because the adult stem cells have the capability of selfrenewal (Dube & Denis, 1995; Muller-Sieburg & Sieburg, 2006; Tseng et al., 2006; Nehlin & Barington 2009). In *ex vivo* gene therapy, genetic manipulation is conducted in a lab outside of the body. However, normal somatic cells, including adult stem cells, do not propagate indefinitely and are vulnerable to epigenetic modification. Therefore, long-term cultures of somatic cells isolated from the body are very difficult to sustain (Beyer & Da sliva, 2006; Tonti & Mannello, 2008). This means that the long-term culture of adult stem cells in *ex vivo* gene therapy should be avoided as much as possible. Therefore, it is absolutely necessary to deliver therapeutic DNA molecules into isolated cells immediately with high efficiency.

corrected patient gene remains permanently has been actively pursued.

**1. Introduction** 

**Transfer for** *Ex-Vivo* **Gene Therapy** 

Hea-Jong Chung1, Hyun-Seo Lee1,

*2Jinis Biopharmaceuticals Co.* 

*Republic of Korea* 

Hyeon-Jin Kim2 and Seong-Tshool Hong1 *1Laboratory of Genetics and Department of Microbiology* 

*Chonbuk National University Medical School* 

