The mechanism of naked DNA uptake and expression

The administration of naked nucleic acids into animals is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models (Herweijer and Wolff, 2003; Hodges and Scheule, 2003). It is also being used in several human clinical trials for genetic vaccines, Duchenne muscular dystrophy, peripheral limb ischemia, and cardiac ischemia (Davis et al., 1996; Romero et al., 2002; Tsurumi et al., 1997). Naked DNA is an attractive non-viral vector because of its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It shows very little dissemination and transfection at distant sites following delivery and can be readministered multiple times into mammals (including primates) without inducing an antibody response against itself (i.e., no anti-DNA antibodies generated) (Jiao et al., 1992). Also, contrary to common belief, long-term foreign gene expression from naked plasmid DNA (pDNA) is possible even without chromosome integration if the target cell is postmitotic (as in muscle) or slowly mitotic (as in hepatocytes) and if an immune reaction against the foreign protein is not generated (Herweijer et al., 2001; Miao et al., 2000; Wolff et al., 1992; Zhang et al., 2004). With the advent of intravascular and electroporation techniques, its major restriction--poor expression levels--is no longer limiting and levels of foreign gene expression in vivo are approaching what can be achieved with viral vectors. Direct in vivo gene transfer with naked DNA was first demonstrated when efficient transfection of myofibers was observed following injection of mRNA or pDNA into skeletal muscle (Wolff et al., 1990). It was an unanticipated finding in that the use of naked nucleic acids was the control for experiments designed to assess the ability of cationic lipids to mediate expression in vivo. Subsequent studies also found foreign gene expression after direct injection in other tissues such as heart, thyroid, skin, and liver (Acsadi et al., 1991; Hengge et al., 1996; Kitsis and Leinwand, 1992; Li et al., 1997; Sikes and O'Malley 1994; Yang and Huang, 1996). However, the efficiency of gene transfer into skeletal muscle and these other tissues by direct injection is relatively low and variable, especially in larger animals such as nonhuman primates (Jiao et al., 1992). After our laboratory had developed novel transfection complexes of pDNA and amphipathic compounds and proteins, we sought to deliver them to hepatocytes in vivo via an intravascular route into the portal vein. Our control for these experiments was naked pDNA and we were once again surprised that this control group had the highest expression levels (Budker et al., 1996; Zhang et al., 1997). High levels of expression were achieved by the rapid injection of naked pDNA in relatively large volumes via the portal vein, the hepatic vein, and the bile duct in mice and rats. The procedure also proved effective in larger animals such as dogs and nonhuman primates (Eastman et al., 2002; Zhang et al., 1997). The next major advance was the demonstration that high levels of expression could also be achieved in hepatocytes in mice by the rapid injection of naked DNA in large volumes simply into the tail vein (Liu et al., 1999; Zhang et al., 1999). This hydrodynamic tail vein (HTV) procedure is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering RNA (siRNA) (Lewis et al., 2002; McCaffrey et al., 2002). The intravascular delivery of naked pDNA to muscle cells is also attractive particularly since many muscle groups would have to be targeted for intrinsic muscle disorders such as Duchenne muscular dystrophy. High levels of gene expression were first achieved by the rapid injection of naked DNA in large volumes via an artery route with both blood inflow and outflow blocked surgically (Budker et al., 1998; Zhang et al., 2001). Intravenous routes have also been shown to be effective (Hagstrom et al., 2004; Liang et al., 2004; Liu et al., 2001). For limb muscles, the ability to use a peripheral limb vein for injection and a proximal, external tourniquet to block blood flow renders the procedure to be clinically viable. This review concerns itself with the mechanism by which naked DNA is taken up by cells in vivo. A greater understanding of the mechanisms involved in the uptake and expression of naked DNA, and thus connections between postulated mechanisms and expression levels, is emphasized. Inquiries into the mechanism not only aid these practical efforts, but are also interesting on their own account with relevance to viral transduction and cellular processes. The delivery to hepatocytes is first discussed given the greater information available for this process, and then uptake by myofibers is discussed.