**3.2 Gold bullet/gene gun**

Similar to electroporation, another method, called "gene-gun", does not require the presence of complicated and potentially toxic delivery systems [Gardlik et al., 2005]. The gene transfer is mediated by small particles of gold on which the DNA is bounded. These particles are then shot into the cell under great pressure and speed (with the help of compressed helium) and so pass the membrane barrier [Katare and Aeri, 2010]. At first, the gene-gun was developed for gene transfer into plant cells; then, its use has expanded to gene transfer into the mammalian cells. Effective development of the gene-gun was also achieved in the field of DNA vaccination. The latest clinical experiments focus on cancer vaccines against various human tumors [Gardlik et al., 2005]. This method has been successfully used to deliver DNA *in vivo* into liver, skin, pancreas, muscle, spleen and tumors. Expression of reporter genes (e.g. firefly luciferase and β- galactosidase) or therapeutic genes (human growth hormone) have also been reported by this method [Gardlik et al., 2005]. Recently, gene gun-mediated transgene delivery system has been used for skin vaccination against melanoma using tumor-associated antigen (TAA) human gpl00 and reporter gene assays as experimental systems [Aravindaram and Yang, 2009 S]. In addition, the delivery of HPV DNA vaccines using intradermal administration through gene gun was shown to be the most efficient method of vaccine administration in comparison with routine intramuscular injection [Ogris and Wagner, 2002].

Currently, a HPV16 DNA vaccine encoding a signal sequence linked to an attenuated form of HPV16 E7 (E7 detox) and fused to heat shock protein 70 [(Sig/E7detox/HSP70)] has been used in clinical trials. In a previous study, the immunologic and anti-tumor responses have been evaluated by the pNGVL4a-Sig/ E7 (detox)/ HSP70 vaccine administered using three different delivery methods including needle intramuscular, biojector and gene gun. According to obtained results, DNA vaccine administered via gene gun generated the highest number of E7-specic CD8+ T cells as compared to needle intramuscular and biojector administrations in mice model [Trimble et al., 2003].
