Vladimir G. Budker

High Levels of Foreign Gene Expression in Hepatocytes after Tail Vein Injections of Naked Plasmid DNA

We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml. This technique has great potential for a wide variety of laboratory studies.

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 OMalley 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.

Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process.

Following the initial demonstration that intramuscularly‐injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor‐mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented. Copyright

The efficient expression of intravascularly delivered DNA in rat muscle

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals larger than mice.

Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery

The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a ‘chaser’ injection of pAA 30u2009min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the ‘chaser’ injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

Time course of gene expression after plasmid DNA gene transfer to the liver

High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter‐driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It 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. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. II. Morphological studies

The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA.

Electrostimulated uptake of DNA by liposomes

High molecular mass DNA was efficiently taken up by large unilamellar vesicles exposed to a short pulse of electric field (0.1-1 ms) with an intensity as high as 12.5 kV/cm. The efficiency of uptake increased significantly in presence of Mg2+ ions and was approximately 0.6 and 1.5 micrograms of DNA per mumol of lipid for T7 DNA and plasmid pBR 322, respectively. The results presented indicate that DNA was taken up as a result of the electrostimulated formation of endosome-like vesicles rather than via field-induced membrane pores.

Entrapment and Condensation of DNA in Neutral Reverse Micelles

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of nanowires, have apparently not been previously observed.