Plasmid DNA naked

Presently, 1,260 gene-therapy trials are underway worldwide (Tables 1 and 2). For details see http // www.wiley.co.uk/genetherapy/clinical/. Almost three fourths of all trials are based on viral vectors. The vast majority of nonviral gene-therapy trials use naked/ plasmid DNA (18% of all gene-therapy trials). 

The initial approach adopted entailed administration of naked plasmid DNA housing the gene of interest. This avenue of research was first opened in 1990, when it was shown that naked plasmid DNA was expressed in mice muscle cells subsequent to its i.m. injection. The plasmid DNA concerned housed the P-galactosidase gene as a reporter. Subsequent expression of P-galactosi-dase activity could persist for anything from a few months to the remainder of the animal s life. The transfection rate recorded was low (1-2 per cent of muscle fibres assimilated the DNA), and the DNA was not integrated into the host cell s chromosomes. 

The simplest nonviral gene transfer system in use for gene therapy is the injection of naked plasmid DNA (pDNA) into local tissues or the systemic circulation (88, 100). Naked DNA systems are composed of a bacterial plasmid that contains the cDNA of a reporter or therapeutic gene under the transcriptional control of various regulatory elements (101, 102). In recent years, work in several laboratories has shown that naked plasmid DNA (pDNA) can be delivered efficiently to cells in vivo either via electroporation, or by intravascular delivery, and has great prospects for basic research and gene therapy (101). Efficient transfection levels have also been obtained on direct application of naked DNA to the liver (103, 104), solid tumours (105), the epidermis (106), and hair follicles (106). 

Zhang, G., Vargo, D., Budker, V., Armstrong, N., Knechtle, S., Wolff, J.A. (1997). Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers. Hum. Gene Ther., 8, 1763-1772. 

Kawase, A., Nomura, T., Yasuda, K., Kobayashi, N., Hashida, M., Takakura, Y. (2003). Disposition and gene expression characteristics in solid tumors and skeletal muscle after direct injection of naked plasmid DNA in mice. J. Pharm. Sci., 92(6), 1295-1304. 

Nomura, T., Yasuda, K., Yamada, T., et al. (1999). Gene expression and antitumor effects following direct interferon (IFN)-gamma gene transfer with naked plasmid DNA and DC-chol liposome complexes in mice. Gene Ther., 6(1), 121-129. 

Naked plasmid DNA was not only trapped in the cytoplasm around the site of microinjection, as visualized by fluorescence in situ hybridization (FISH) or using FITC-labeled DNA, but was also eliminated rapidly at physiological temperature (Lechardeur et al., 1999). The disposal of the DNA was completely prevented when the cells were kept at 4°C (Lechardeur et al., 1999). A similar conclusion was reached by monitoring the amount of microinjected expression cassette by the polymerase chain reaction (PCR) (Pollard et al., 2001), suggesting that the metabolic instability of naked DNA contributes to the low efficiency of gene transfer (Lechardeur et al., 1999 Mirzayans et al, 1992 Neves etal., 1999 Pollard et al., 2001). 

Nomura, T., Nakajima, S., Kawabata, K., Yamashita, F., Takakura, Y. and Hashida, M. (1997) Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer. Cancer Res., 57, 2681-2686. 

Budker, V., Budker, T., Zhang, G., Subbotin, V., Loomis, A. and Wolff, J.A. (2000) Hypothesis naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process. J. Gene Med., 2, 76-88. 

The in-vivo disposition and tissue distribution of naked plasmid DNA has been studied by several investigators after administration via various routes, including intravenous (IV), intramuscular (IM), intradermal, and intranasal. It is generally difficult to determine pharmacokinetic parameters for naked DNA as it is rapidly and extensively degraded in plasma. Our studies indicated that supercoiled DNA is degraded with a half-life of 1.2 min upon incubation in isolated rat plasma at 37 °C [3], The open-circular form has a half-life of 21 min, while the linear form degrades with a half-life of 11 min. Other studies show similar results, with most of the supercoiled form of plasmid converting to open-circular and linear forms by 15 min [4], The... 

Liu et al. [7] found significant differences between the disposition of complexed and uncomplexed [1251]-labeled plasmid DNA in mice. An equal amount of radioactivity was found in blood and liver at 15 min after injection of naked plasmid DNA. In contrast, at the same time-point, a major portion of injected DNA was in the lung when DNA-lipid complexes were injected. 

Several attempts in the past have been made to achieve local gene delivery via coated balloon catheters or infusion catheters using both naked plasmid DNA and adenoviral vectors for the transfer of VEGF-1 or -2 (69,70). Walter et al. (69) demonstrated that local VEGF-2 plasmid gene delivery of a therapeutic gene via coated stents appears feasible, safe, and effective. 

Table 21.3 shows the clinical studies that have been conducted worldwide, with their different applications, showing that therapies intended for monogenic disease treatment are the second most assessed group, after therapies for tumor treatment. The most used vectors in gene therapy clinical studies are viral vectors (68%), and among those, retroviruses and adenoviruses are the viruses of choice. Synthetic vectors were used in 25% of the studies performed, and about 16% correspond to the use of naked plasmid DNA (Table 21.4). 

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