Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
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ABSTRACT |
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electroporation; liposomes; plasmids; transfection; gene expression
Perhaps the most powerful way to do this is to utilize gene transfer strategies to deliver genes and other DNAs or RNAs to cells within tissues to study their roles in the context of the animal itself. Such approaches can be used to upregulate gene expression, to study transcriptional and posttranscriptional regulation of various genes and gene products, and to downregulate expression of desired targets. However, to succeed in all of these studies, efficient ways to deliver nucleic acids to muscle cells in vivo must be used. This review focuses on the techniques used to deliver genes to skeletal, smooth, and cardiac muscle in vivo and discusses various approaches to upregulating and downregulating expression in living systems with gene transfer.
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TRANSFECTION STRATEGIES |
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SKELETAL MUSCLE |
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An approach to increasing the efficiency of this method was developed by applying external electric fields to the injected muscle. Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 20 years (6, 99). More recently, it has been applied to intact tissues in living animals, including skeletal, smooth, and cardiac muscle. Electroporation uses electrical fields to create transient pores in the cell membrane that exist for the lifetime of the electric field and allow the entry of normally impermeant macromolecules into the cytoplasm (111). The electric field also results in the electrophoretic movement of the added DNA, aiding in gene delivery (104). Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in relatively little damage or trauma, although some low level of necrosis and changes in gene expression may be detected (8, 39, 81, 88). When a square-wave electric field at the appropriate strength was applied to mouse quadriceps muscles after direct injection of the plasmid, the levels of gene expression jumped between 100- and 1,000-fold (2, 81, 86). The distribution of cells taking up and expressing the DNA increases with electroporation, as does the absolute amount of gene product per cell (this is likely due to increased delivery of plasmids into each cell) (Fig. 1). Indeed, many reports demonstrate that between 50% and 80% of fibers within a given muscle group can be transfected with this approach (29, 81, 86). As with direct injection, gene expression is long lasting. Furthermore, it has been used successfully in a number of species, up to nonhuman primates (93).
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Although electroporation greatly increases gene transfer and expression in skeletal muscle, a number of studies have evaluated the use of adjunct reagents to increase gene transfer even further, in both electroporated and nonelectroporated muscles. Early studies from Wolff's laboratory (20), which developed the naked DNA injection technique, demonstrated that injection of the muscles with bupivacaine at doses that induced limited muscle degeneration, before DNA injection, resulted in increased gene transfer and expression. More recently, several groups have shown that injection of hyaluronidase into muscle, before DNA delivery and electroporation, also increased gene transfer and expression (36, 42, 84, 85, 88). In this case, the enzyme partially degrades the extracellular matrix surrounding the myotubes, and this is thought to aid in uptake of the DNA into the cells, resulting in up to fourfold-higher gene transfer versus electroporation alone.
Apart from direct DNA injection with or without electroporation, a number of chemical methods and techniques have been tested for DNA delivery to skeletal muscle in vivo, with varying degrees of success. Cationic liposomes, such as Lipofectin, N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP)-l--phosphatidylethanolamine, and dioleoyl (DOPE), have revolutionized transfections of cultured cells in the laboratory because of their ease of use and typically high transfection efficiency. However, although many of these reagents can be used to productively transfect myoblasts and myoblast cell lines in culture, they show almost no transfection activity in differentiated myotubes in culture or in skeletal muscle in vivo (48, 72). Similarly, cationic polymers including poly(ethyleneimine) (PEI), which are highly efficient for in vitro delivery and delivery to other tissues such the lung, have also not performed well in differentiated myotubes, in vitro or in vivo (14, 68).
Several other physical methods are worth mentioning. First, particle bombardment has been used in several studies to transfer genes to various skeletal muscles with success (62, 133). This technique coats gold particles with plasmids, and the particles are delivered into the tissue with a "gene gun" that uses pressure to shoot the particles into the tissue. In one study in which this method was compared to direct DNA injection, the gene gun performed best in the muscle of young rats and yielded between 10- and 100-fold more expression than direct DNA injection (62). The only drawback to this technique is that it traditionally requires the muscle to be exposed (although a new device can penetrate multiple layers of the skin and transfect subepithelial tissues; Ref. 27) and induces some degree of trauma. Microbubble ultrasound has also been used to deliver DNA to skeletal muscle. DNA is delivered directly or with a microbubble agent such as Optison, and ultrasound waves are concentrated on the area of the tissue where gene transfer is desired (73). Wide distribution is achieved, as is high-level gene expression, with the benefit of being able to direct the waves to specific regions of interest. Finally, hydrostatic pressure, or hydroporation, has been used successfully to transfer genes to skeletal muscle in a number of organisms. The original versions of this technique delivered a large volume of DNA via the tail vein over a short period of time and resulted in tremendous expression in the liver (70). Wolff and colleagues (11, 134) and others (19, 64) have applied this method to isolated limbs to target skeletal muscle by injecting large volumes of DNA into the circulation over a short period of time and have achieved relatively uniform, high-level expression in most muscle groups fed by the vasculature in the limb. This has now been applied to animals from mice to nonhuman primates with success. However, the only drawback to this approach is that some tissue damage does occur, but the ability to target the majority of muscles with a given limb is attractive.
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SMOOTH MUSCLE |
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Another limitation for smooth muscle transfection in vivo is that direct DNA injection cannot be used effectively because in most instances the smooth muscle layers are too thin to inject reliably. This is especially true in the vasculature. With the exception of a few large vessels, the vessel wall is too thin to be injected with DNA. Moreover, even if DNA could be delivered to the walls by injection, the architecture of the vessel wall would prevent the even distribution of the DNA throughout it. Similar limitations apply in airways, bladder, digestive tract, and uterus. However, despite these limitations, several approaches have been developed to transfect SMCs in vivo.
One way around the inaccessibility of SMCs within a tissue is to transfect cultured SMCs and then engraft them back into the host in hopes that they will home to the appropriate smooth muscle layer. The advantage of such an approach is that it is much easier to transfect cells in culture than it is in vivo. Several attempts at this have been made with little success, because although the cells can be transfected and express after delivery in vivo, they do not necessarily engraft into the smooth muscle layer to any great extent. In one study, transfected SMCs were injected intravenously, intraperitoneally, or intramuscularly (34). Although cells injected by all routes expressed for 47 days, there were no data to show where these cells actually localized because the gene expressed encoded for a secreted protein. In another case, a stent was embedded with transfected SMCs and placed (with accompanying endothelial cell injury) into the coronary arteries of pigs (94). These cells continued to express their gene product for up to 1 mo after stent placement, but the expressing cells were detected only within the mesh of the stent; no engraftment of transfected cells into the vascular smooth muscle layer occurred.
As seen in skeletal muscle, pressure can be used to deliver genes and other nucleic acids to the vascular wall. Surprisingly, hydrostatic delivery of DNA via the circulation does not result in significant delivery and expression of genes within the vessel wall (11, 69, 134). Thus, to target the vessel wall, a different method that transfects cells in explanted vessels that are then transplanted into the host has been developed (121). Vessel segments are removed and placed in a container along with the plasmid or other nucleic acid, and the container is briefly pressurized (typically vessels are placed in a dialysis bag that is tied off at one end and attached to a syringe at the other). After gene transfer, the vessel is then either cultured ex vivo for study or transplanted into a recipient. The nice feature of this approach is that gene transfer to all cell types within the vessel is achieved without injuring the endothelial layer. This approach has been used to transfer plasmids for vascular SMC gene expression, antisense RNA to inhibit gene expression, and decoy oligonucleotides to downregulate transcription factor activity (32, 77, 78, 121).
Ultrasound has also been used in the vasculature to transfer genes to SMCs. As seen in other tissues, the use of a microbubble contrast agent such as Optison greatly increased gene transfer with ultrasound over DNA alone or ultrasound using DNA without microbubbles (52, 115). Microbubble ultrasound was able to give high-level gene expression of either of two reporter genes in the vessel wall. Furthermore, the levels of expression were roughly the same for balloon angioplasty-injured and uninjured vessels. Unfortunately, in both studies, cellular localization of gene expression after transfer in uninjured vessels was not determined. Because the levels of expression were roughly equivalent in denuded and intact vessels in these studies, it is likely that the SMCs are targeted to some degree without injury. However, as with chemical transfection methods, in the injured vessels, significant gene delivery and expression was found in the smooth muscle layer (115).
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ELECTROPORATION TO TARGET SMOOTH MUSCLE |
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Vascular smooth muscle. The first use of electroporation for these tissues was described by Martin et al. (79), who transferred genes to the mesenteric vasculature of rats with adventitial DNA delivery. Vessels of the rat mesenteric vascular tree were exteriorized in anesthetized animals, placed in an electrode resembling a spoon with two wires flanking a notch for the vessel (Figs. 2 and 3), covered with a solution of plasmid, and electroporated with a series of eight square-wave electric pulses of 10-ms duration at an optimal field strength of 200 V/cm. After electroporation, the vessel was removed from the electrode, the vessels and the intestine were returned to the abdomen, and the animals recovered without incident. Gene expression was detected as early as 6 h after electroporation. With the CMViep used to drive the reporter gene, expression peaked between days 1 and 3 and then dropped to baseline by day 7, after which gene expression cannot be detected (79, 131). Such short duration of gene expression with the CMV promoter has been seen in other tissues and is likely the result of promoter inactivation (see below). The use of other promoters, such as the ubiquitin C promoter, can drive much longer gene expression (on the order of weeks, not days; see below). Gene transfer using this approach is dose dependent, time dependent, and highly dependent on field strength (79). No gene transfer and expression above background was seen without an applied electric field or at 50 V/cm. At 100 V/cm reasonable, but variable, gene expression was detected, and at 200 V/cm consistent, high-level gene transfer and expression was seen. When the field strength was increased to 400 V/cm, tissue damage was detected and the levels of gene expression declined.
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Although this technique may seem traumatic, electroporation at the optimal field strength parameters does not induce any histological changes, inflammatory response, or trauma (79). Furthermore, when vasoreactive responses of the vessels were measured at the peak of gene expression (2 days) or long after gene expression had subsided (40 days) after electroporation, the vessels responded in a manner indistinguishable from control vessels in terms of constriction to phenylephrine and relaxation to adenosine and isoproterenol (79). Finally, our lab (131) has also performed DNA microarray analyses on pre- and postelectroporation vessel segments and found that the act of electroporation does not affect the global patterns of gene expression to any degree.
Several other electroporation approaches for the vascular wall have also been developed by other groups using DNA delivered via the lumen. The advantage to this method is that with the appropriate device (e.g., single or double balloon catheter), vectors can be delivered to defined regions of the vasculature with relatively simple methods that are clinically routine. The disadvantages are that for vectors to be delivered to the vessel lumen with double balloon catheters, blood flow must be restricted, which may cause ischemia of the downstream vessels and tissues. Several studies from one group have used a porous balloon catheter that also contains two electrodes to deliver heparin or DNA through the porous balloon and electroporate at the same time (Fig. 2; Refs. 25, 26). The electrode system uses the guide wire as one electrode and an internal wrapped wire contained entirely within the balloon as the second electrode. When a voltage is applied, an electric field develops between the two electrodes, causing electroporation of the vessel wall and delivery of heparin or DNA to cells within the vessel wall. As with adventitial delivery and electroporation, transfer is detected within the endothelial cells and both the intimal and medial smooth muscle layers. Another group delivered DNA with a double balloon catheter and applied the electric field from the adventitial surface of the rabbit carotid with two "T"-shaped electrodes (2.5 cm long x 0.5 cm wide) placed on either side of the vessel segment (82). In both of these cases, gene transfer was dependent on the electric field. No expression was seen at 0 V/cm, but gene transfer and expression increased as the field strength was raised to 100 and 200 V/cm and then decreased at 300 and 400 V/cm. Again, as in the studies by Dev and colleagues (25, 26), gene transfer was detected in the endothelial and smooth muscle layers. More recently, a similar study was performed in the abdominal aorta of rats in which DNA was administered to the lumen and the vessel was electroporated from the adventitial surface, resulting in gene transfer and expression to all cell types in the aorta wall (51). With all of these methods, the levels of gene product expressed have been sufficient to elicit physiological or therapeutic responses (25, 26, 51, 110).
Airway smooth muscle. Airway smooth muscle also suffers from the fact that it is buried beneath other cells and is hidden from standard gene transfer reagents. Our lab has recently shown (21, 23, 75) that electroporation can also be used to target these cells without inducing any injury to the airways to make the cells accessible. Purified plasmid suspended in a physiologically compatible buffer containing 140 mM NaCl is administered to the airways of anesthetized mice or rats and the animals are electroporated with flat electrodes placed on either side of the chest. As for the vasculature, a series of eight square-wave pulses are delivered to the animals at an optimal field strength of 200 V/cm and pulse lengths of 10 ms, and the animals recover without incident.
Gene transfer is dependent on field strength, with very little gene transfer and expression seen at values <100 V/cm and an optimum at 200 V/cm. However, it should be stated that other field strength and pulse length combinations may work very well, as has been seen with skeletal muscle (i.e., higher field with shorter pulses). Indeed, in one study, the pulse length was varied from 10 µs to 10 ms with a constant field strength of 200 V/cm in the rat (75). Although gene transfer was seen with the 10-µs pulses, expression increased by >100-fold when the pulse length was raised to 10 ms. However, by increasing the dose of DNA, equivalent levels of expression can be achieved with the short pulse length.
Gene transfer using transthoracic electroporation was DNA dose dependent, with 100 µg of DNA giving 10 ng of gene product per gram of lung wet weight in mice (23). In rats, the levels of expression are lower, with 100 µg giving 150 pg of gene product per gram of wet weight at the longer pulse length (75). The level of gene expression is more than enough to elicit physiological effects, as demonstrated by increases in alveolar fluid clearance following electroporation-mediated transfer of plasmids encoding a subunit of the Na+-K+-ATPase (75). Indeed, the physiological response was equivalent to that seen with recombinant adenoviruses expressing the same gene product (35, 75). Most importantly, gene expression was detected throughout all cell types in the lung (21, 23, 75). Cell types that received and expressed DNA included alveolar type I and type II epithelial cells, endothelial cells, airway epithelial cells, and vascular and airway SMCs. By immunohistochemistry for the expressed gene product, there appeared to be little to no difference between the levels of gene expression in the various cell types. As for uniformity of gene transfer, there appears to be transfer and expression to all regions of the lung that is relatively homogeneous over a two- to threefold range (21, 75).
Using smooth muscle specific promoters, we have also been able to limit gene expression after electroporation to airway SMCs (T. Kuzniar and D. A. Dean, unpublished observations). This has been done in the rat and the mouse, both in vivo with transthoracic electroporation after tracheal delivery of DNA and in lung explants. Expression from plasmids utilizing smooth muscle-specific promoters was detected only in the smooth muscle layer, whereas expression from plasmids containing the CMViep or SV40 early promoter was seen in all cell types. When quantified, expression in the smooth muscle accounted for 5% of total lung expression, which is slightly more than the relative ratio of SMCs to total cells. Thus not only can the airway SMCs be transfected along with all other cell types in the lung, they can also be specifically targeted with the appropriate promoters and electroporation.
Bladder smooth muscle. Electroporation-mediated transfer of genes to bladder smooth muscle in rats has also been reported (53). Genes for luciferase, green fluorescent protein (GFP), and neuronal nitric oxide synthase (nNOS) were transferred to the bladder by injecting 50 µl of the DNA into the subserosal region of the exposed bladder wall after removal of urine from the bladder. Flat, tweezerlike electrodes were placed on either side of the bladder, and square-wave pulses were delivered to the tissue. The abdomen was then closed, and gene expression was evaluated at later times. Gene transfer and expression were dependent on the field strength, with optimal expression seen at 225 V/cm with eight pulses of 50-ms duration each. As seen in the lung (75), expression was also pulse length dependent, although the dependence was not as pronounced as in the lung. Also, the optimal pause between pulses was found to be 1 s; shorter pauses gave lower levels of expression. Furthermore, although there was no trauma or tissue damage under the optimal conditions, at higher field strengths, tissue damage was detected.
Transfer of GFP resulted in significant levels of gene expression in the smooth muscle layer of the bladder. Not only was the reporter gene expressed, but transfer of nNOS resulted in enhanced staining for the transferred nNOS in the smooth muscle of the tissue sections. Furthermore, NO production was increased by >50% in bladder strips isolated from electroporated animals that had received the nNOS plasmid but not in control strips that received plasmid without electroporation or in strips that were electroporated without added DNA. Together, these results suggest that electroporation can be used to target SMCs within a number of different tissues.
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CARDIAC MUSCLE |
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For delivery via the circulation, in most cases the plasmid is complexed with either liposomes or polymers for two reasons. First, the complexed cationic lipids or polymers condense the DNA to a smaller size and mediate interactions between the DNA and the cell membrane. Without such reagents, very little DNA would interact with the plasma membranes of cells, and almost no DNA uptake would be observed. These complexes also serve to provide stability to the DNA and protection from degradation. In the absence of liposomes, the half-life of free DNA in the serum is on the order of several minutes at best (54). Thus, without the protective function of these chemical transfection reagents, the DNA would be degraded long before it ever got to the target cells. DNA has been complexed with a number of different lipids for gene transfer to the heart via the circulation. A number of studies have used a mixture of phosphatidylserine, cholesterol, and phosphatidylcholine mixed with inactivated hemagglutinating virus of Japan (HVJ) particles (33, 105, 112). These HVJ liposomes are fusogenic and promote fusion of the liposome with the endosomal membrane and uptake of the DNA into the cell. In several studies, mixtures of plasmid and HVJ liposomes were infused over the course of 10 min into either the aorta or coronary arteries of excised hearts maintained at 4°C, and then the hearts were transplanted into recipient mice or rats (105, 112). In one study, this method resulted in up to 50% of cardiomyocytes receiving and expressing the transgene for up to 14 days (the -actin promoter was used to drive gene expression in these studies) (105). However, infusion through the coronary artery or aorta in vivo (i.e., an in situ heart) appeared to result in much less efficient uptake of DNA (<1.6% of cardiomyocytes)(33, 109). Unlike the study of Sawa et al. (105) using HVJ liposomes in excised, transplanted hearts, the use of other lipids [N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (DLRIE)-DOPE] or dendrimers to complex with the DNA resulted in very low levels of gene transfer to either endothelial cells or cardiomyocytes (<1% of cells) when a similar infusion/transplant method was used (24, 108, 125).
Direct injection of DNA, either as naked DNA or complexed with lipids or polymers, into the wall of the heart has been shown to give much higher levels of gene expression in cardiomyocytes, but usually with a more limited distribution of gene expression. Most studies have used naked DNA and have injected the DNA into multiple sites within the wall of the heart. In some cases, injections were performed after thoracotomy (58, 122) or by a subdiaphragmatic approach after a midline abdominal incision (1), but in most cases, less invasive procedures were used that employed echocardiography to guide percutaneous injections into the ventricular wall (95, 103, 116, 117). In the majority of studies, gene expression was limited to a relatively small number of cells around the injection sites (95, 116, 117). However, several studies have shown that the heart can express very high levels of gene product. In one study following gene transfer in rat hearts after direct injection of DNA, up to 800 ng of luciferase or 2 ng of VEGF per heart was expressed (103). This study used the CMV promoter to drive expression, and, as found in other studies, expression peaked within the first 3 days and then dropped off dramatically. Furthermore, this robust gene expression was detected with a dose of only 30 µg of DNA (103). Similarly, Kitsis and colleagues (58) found that the heart was even 10100 times more efficient at gene expression than skeletal muscle when the same dose of DNA was delivered by direct injection. The limitation to this approach is not that inadequate levels of gene product are produced, but rather that the distribution of expression is not uniform throughout the myocardium. One method that may be useful for increasing the distribution of gene expression after direct injection is to inject DNA complexed with PE6400 block copolymer or HVJ liposomes (4, 98). With the block copolymer, much better distribution of gene expression compared with naked DNA was seen (98); although the absolute levels of expression (40 ng luciferase/heart in rats) were not as high as in some other reports, this is still extremely good expression.
Several groups have also begun to use microbubble-enhanced ultrasound to mediate gene delivery to the heart in vivo (18, 60). In this approach, DNA mixed with Optison resulted in gene transfer to the heart after infusion into the carotid artery or jugular vein. However, in both studies, no absolute levels of gene expression are reported (only relative light units for luciferase transfer) and no histological analysis was performed to localize the transferred genes. Thus it is unclear whether this method yields high-level gene transfer or whether it targets the cardiomyocytes or only the endothelial cells.
Finally, two studies have been reported using electroporation to transfer DNA to the heart (46, 124). In one study, hearts were removed from stage 18 embryonic chickens, placed in a bath of DNA, and electroporated at 200 V/cm with 10-ms square-wave pulses delivered with a Grass stimulator. The hearts were then cultured ex vivo on a collagen gel in the presence of cell culture medium and analyzed for gene expression 13 days later. When no electric field was applied, no gene expression was detected in any of the hearts. However, when either six or eight pulses were delivered to the hearts, significant levels of GFP or luciferase expression could be detected. Indeed, up to 30% of the cells in the heart showed GFP expression, almost all of which was in cardiomyocytes. Another group performed very similar studies on excised and transplanted mouse hearts (124). They found good expression by using naked DNA coupled with electroporation but found that complexation of the DNA with dendrimer complexes increased the levels of gene transfer between 10- and 45-fold, with very similar electroporation parameters.
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REGULATING GENE TRANSFER AND EXPRESSION |
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As an alternative to these strong viral promoters, ubiquitously active cellular promoters are often used to drive gene expression. The -actin promoter has been used extensively in a variety of tissues, such as the heart, and gives levels of expression that are at the same level as those seen with the CMV promoter (105). The elongation factor 1
(EF-1
) promoter has also been used effectively to drive gene expression in heart and other tissues (103). Other ubiquitously active endogenous promoters include the GAPDH, heat shock protein (HSP)70, and ubiquitin C promoters. The major advantage to these promoters is that they are usually not subject to rapid silencing, as are the viral promoters (see below), and can express genes for a much longer time.
One of the problems with many of these "strong" viral promoters is that they often express for only a limited duration of time in many tissues. For example, the CMViep gives a rapid burst of gene expression that usually lasts for less than a week in vivo, despite the fact that the plasmids can still be detected in the tissues. This has been seen in smooth muscle in the vasculature and airways as well as in cardiomyocytes and other tissues (23, 98, 131). Similar findings have been reported for the SV40 early and EF-1 promoters in heart (103). However, it should be stressed that this is not the case in skeletal muscle, where gene expression from the CMV promoter (and many other promoters) persists for quite a long time. Indeed, it has even been shown that gene expression can be detected up to 19 mo after intramuscular injection in mice (126). There are a number of potential explanations for the limited duration of expression in these and other tissues, including promoter inactivation (49), DNA methylation (50), altered plasmid chromatin structure, inflammation caused by CpG motifs in the DNA (130, 136), and immune responses to the transgene product (49, 61).
Several approaches have been taken to increase the duration of gene expression in vivo (41, 128, 129). The most straightforward approach has been to evaluate alternative promoters for long-term gene expression. One of the most durable promoters identified to date has been that of the ubiquitin C gene (41, 129). This promoter can drive gene expression at levels roughly equal to those seen with the CMV promoter, but expression persists for up to 6 mo in a number of tissues, including vascular and airway smooth muscle (Refs. 41, 129; unpublished observation). Thus, by using the appropriate promoter, either short- or long-term gene expression can be achieved in vivo.
Apart from high-level and either short- or long-term gene expression, many times it is crucial to have gene expression restricted to a certain cell type within a tissue. One way to restrict gene expression to a desired cell type is to use a promoter that is active only in that cell type. Such cell-specific promoters abound. For example, in smooth muscle, the promoters for smooth muscle -actin, SM22
, and smooth muscle myosin heavy chain have all been used to drive high-level expression that is cell restricted. Other promoters may be generally restricted to all types of muscle in general, such as that from the gene for desmin (65). Synthetic promoters have also been developed that work in specific cell types, such as the synthetic C512 promoter that was constructed by randomly combining skeletal muscle-specific transcription factor consensus binding sites to form a promoter that acts uniquely in skeletal muscle (67). Similar promoters have been constructed for smooth muscle-specific expression (56, 100).
A second method for restricting gene expression to specific cell types relies on the fact that the nuclear import of plasmids in nondividing cells is sequence specific (137). Our lab (131) and others (64) have demonstrated that plasmids containing certain sequences can enter the nucleus in a nondividing cell in the absence of mitosis, whereas plasmids lacking such sequences remain in the cytoplasm, fail to express their genes, and are degraded. Vacik and colleagues (119) identified such a DNA sequence that mediates DNA nuclear import uniquely in nondividing smooth muscle cells. When carried on a plasmid that is transfected into the cell, the smooth muscle -actin (SMGA) promoter binds to several SMC-specific transcription factors in the cytoplasm (which is where all transcription factors and other proteins are synthesized) to form a DNA-protein complex. Because these proteins are destined for the nucleus, they contain specific nuclear localization signals for their nuclear import. As a result of the proteins binding to the SMGA promoter on the plasmid, the plasmid becomes covered with nuclear localization signals for nuclear entry. Because the transcription factors are expressed only in SMCs, the import complexes only form in these cells. Universally active DNA nuclear import sequences have been shown to act in vivo to increase gene transfer in all cell types, including both skeletal muscle and smooth muscle (9, 66, 131), whereas the SMGA sequences act specifically to restrict DNA nuclear import (and subsequent expression) to SMCs in the vasculature and airways (J. L. Young, W. E. Zimmer, and D. A. Dean, unpublished observations; T. Kuzniar and D. A. Dean, unpublished observations).
Another aspect of regulation is the ability to turn gene expression on and off at will. A number of systems have been developed to do just this, but the three most developed are the "Tet on/off," Geneswitch, and ecdysone-regulated systems. In all cases, a combination of ligand-binding, synthetic inducer/repressor proteins and promoter control regions are used to regulate transgene expression in a controllable fashion. The most-used system is based on variations of the tetracycline resistance operon from bacteria and is called the "Tet on/off" system (43). The gene of interest whose level is to be controlled is placed behind a basal promoter that also contains binding sites for the tetracycline repressor or inducer (the Tet response element, or TRE). One of two fusion proteins, both of which bind doxycycline as an inducer, is then introduced into the cell, typically encoded for either in the genome (i.e., in a transgenic mouse) or on a second plasmid. The first fusion protein, the tetracycline transactivator, binds to the TRE and activates transcription of the gene of interest in the absence of doxycycline. Thus addition of doxycycline renders the Tet activator unable to bind to the TRE and expression is turned off (Tet-off). The second fusion protein that can be used is the reverse tetracycline transactivator, which only binds to the TRE in the presence of doxycycline. In this case, in the absence of drug, expression is off, but addition of drug causes gene expression to be induced (Tet-on). One advantage of this system is that expression can be controlled in a graded manner: the more doxycycline that is administered, the greater the level of induction or suppression. However, doxycycline is also a potent antibiotic and can result in side effects in animals.
The second system is very similar but is based on a protein/DNA sequence from Drosophila. The ecdysone-inducible system again consists of a fusion protein, in this case the transactivation domain of the glucocorticoid receptor fused to the ecdysone-binding nuclear receptor protein, and an ecdysone response element that is placed upstream of a minimal promoter driving expression of the desired gene product. On addition of ecdysone, an insect hormone with no mammalian homologs, the fusion protein dimerizes, binds to the response element, and induces expression up to 1,000-fold (91). One advantage to this system is that the synthetic receptor protein and response element bind to no other mammalian hormones or transcription factors, respectively, resulting in very low background expression in the absence of drug. Thus expression can be very tightly controlled.
Finally, the Geneswitch uses a mifepristone-binding progesterone receptor fused to the DNA binding domain of the yeast GAL4 protein and the transactivation domain from the NF-B p65 subunit (12). This fusion protein in turn binds to a GAL4 upstream activating sequence placed upstream of a minimal promoter to regulate gene expression. When the hormone is added, gene expression is turned on, leading to an up to 50,000-fold induction of transgene (12, 123). When the drug is removed, expression returns to baseline within 5 days in vivo. This system has been used effectively to turn expression on and off repeatedly in the same animals, with no loss of induction with subsequent drug administrations.
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APPROACHES TO DOWNREGULATING GENE EXPRESSION |
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DNAzymes are catalytic oligonucleotides that selectively bind to an RNA substrate by Watson-Crick base pairing and cleave phosphodiester bonds, resulting in decreased target mRNA levels and subsequent protein production (57, 132). These reagents have been used effectively to downregulate a number of genes in cultured cells and, more recently, in animal models. Several studies have targeted early growth response factor 1 (EGR-1) in the vasculature to reduce intimal hyperplasia in injured vessels with liposomes (16, 17, 102). Although the DNAzymes effectively reduced EGR-1 mRNA and protein levels in the intima and elicited a therapeutic effect, again, as seen for liposomal delivery of DNA, transfer of the nucleic acid to the smooth muscle required endothelial cell injury.
Our lab (92) has used electroporation to deliver DNAzymes to vascular smooth muscle in uninjured, intact vessels in the rat. In this study, we targeted the PKC gene, because of its implicated role in regulating agonist-induced vascular smooth muscle contraction (13, 80). With a spoonlike electrode designed for plasmid transfer to the vasculature, 31-mer single-stranded DNAzymes were transferred to the mesenteric vessels of rats in a field strength- and dose-dependent manner. As for plasmids, optimal DNAzyme delivery (as determined by DNAzyme-mediated reduction of PKC
mRNA and protein levels) was achieved with eight 10-ms pulses at 200 V/cm and a 100 µM dose of oligonucleotide. Under these conditions, a 60% reduction in PKC
mRNA and protein was detected in SMCs in vivo. As for plasmid delivery, the advantage to this approach is that electroporation mediated delivery to smooth muscle without a requirement for endothelial cell damage. Thus, by choosing the desired gene product to be targeted, DNAzymes can be designed, synthesized, and delivered to tissues relatively easily to target various muscle types in vivo and effectively downregulate endogenous gene expression.
A second approach for reducing gene activity in vivo is through the use of dominant-negative mutants. Dominant-negative mutants are proteins that are not only catalytically inactive themselves, but also have the ability to inactivate wild-type versions of themselves in the cell. The easiest way to think about this is in terms of a protein that normally dimerizes in order to function. If a large amount in inactive monomer is added to the system, the active monomers are titrated out so that any dimers that form are either between two inactive mutants or an active monomer and an inactive mutant; in either case, the resulting dimers would be inactive. In one study, a dominant-negative PKC mutant gene was used in the vasculature to study vasoconstriction in a rat model (110), based on studies suggesting that PKC
may play a role in adrenoreceptor-mediated contraction of mesenteric arteries (13, 80). To examine the role of PKC
in vasoconstriction, a dominant-negative PKC
(PKC
-KN) (40) was transferred to vessels by electroporation from the adventitial surface with a spoonlike electrode, and 2 days later vessels were excised and mounted on a myograph for functional studies of phenylephrine-induced vasoconstriction (110). Not only did transfer of the dominant-negative PKC
mutant attenuate phenylephrine responses compared with control, nonelectroporated vessels, but the level of attenuation was indistinguishable from that achieved with the pharmacological isoform-nonspecific PKC inhibitor chelerythrine.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
2. Aihara H and Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16: 867870, 1998.[CrossRef][ISI][Medline]
3. Andre F and Mir LM. DNA electrotransfer: its principles and an updated review of its therapeutic applications. Gene Ther 11, Suppl 1: S33S42, 2004.[CrossRef]
4. Aoki M, Morishita R, Higaki J, Moriguchi A, Kida I, Hayashi S, Matsushita H, Kaneda Y, and Ogihara T. In vivo transfer efficiency of antisense oligonucleotides into the myocardium using HVJ-liposome method. Biochem Biophys Res Commun 231: 540545, 1997.[CrossRef][ISI][Medline]
5. Armeanu S, Pelisek J, Krausz E, Fuchs A, Groth D, Curth R, Keil O, Quilici J, Rolland PH, Reszka R, and Nikol S. Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo. Mol Ther 1: 366375, 2000.[CrossRef][ISI][Medline]
6. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Struhl K. Short Protocols in Molecular Biology (4th ed.). New York: Wiley, 1999.
7. Babiuk S, Baca-Estrada ME, Foldvari M, Middleton DM, Rabussay D, Widera G, and Babiuk LA. Increased gene expression and inflammatory cell infiltration caused by electroporation are both important for improving the efficacy of DNA vaccines. J Biotechnol 110: 110, 2004.[CrossRef][ISI][Medline]
8. Bertrand A, Ngo-Muller V, Hentzen D, Concordet JP, Daegelen D, and Tuil D. Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters. Am J Physiol Cell Physiol 285: C1071C1081, 2003.
9. Blomberg P, Eskandarpour M, Xia S, Sylven C, and Islam KB. Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle. Biochem Biophys Res Commun 298: 505510, 2002.[CrossRef][ISI][Medline]
10. Bolz SS and Pohl U. Highly effective non-viral gene transfer into vascular smooth muscle cells of cultured resistance arteries demonstrated by genetic inhibition of sphingosine-1-phosphate-induced vasoconstriction. J Vasc Res 40: 399405, 2003.[CrossRef][ISI][Medline]
11. Budker V, Zhang G, Danko I, Williams P, and Wolff J. The efficient expression of intravascularly delivered DNA in rat muscle. Gene Ther 5: 272276, 1998.[CrossRef][ISI][Medline]
12. Burcin MM, Schiedner G, Kochanek S, Tsai SY, and O'Malley BW. Adenovirus-mediated regulable target gene expression in vivo. Proc Natl Acad Sci USA 96: 355360, 1999.
13. Buus CL, Aalkjaer C, Nilsson H, Juul B, Moller JV, and Mulvany MJ. Mechanisms of Ca2+ sensitization of force production by noradrenaline in rat mesenteric small arteries. J Physiol 510: 577590, 1998.
14. Campeau P, Chapdelaine P, Seigneurin-Venin S, Massie B, and Tremblay JP. Transfection of large plasmids in primary human myoblasts. Gene Ther 8: 13871394, 2001.[CrossRef][ISI][Medline]
15. Caplen NJ. Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference. Gene Ther 11: 12411248, 2004.[CrossRef][ISI][Medline]
16. Cassidy KJ, Bull JL, Glucksberg MR, Dawson CA, Haworth ST, Hirschl R, Gavriely N, and Grotberg JB. A rat lung model of instilled liquid transport in the pulmonary airways. J Appl Physiol 90: 19551967, 2001.
17. Chen J, Kitchen CM, Streb JW, and Miano JM. Myocardin: a component of a molecular switch for smooth muscle differentiation. J Mol Cell Cardiol 34: 1345, 2002.[CrossRef][ISI][Medline]
18. Chen S, Shohet RV, Bekeredjian R, Frenkel P, and Grayburn PA. Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound-targeted microbubble destruction. J Am Coll Cardiol 42: 301308, 2003.
19. Danialou G, Comtois AS, Matecki S, Nalbantoglu J, Karpati G, Gilbert R, Geoffroy P, Gilligan S, Tanguay JF, and Petrof BJ. Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model. Mol Ther 11: 257266, 2005.[CrossRef][ISI][Medline]
20. Danko I, Fritz JD, Jiao S, Hogan K, Latendresse JS, and Wolff JA. Pharmacological enhancement of in vivo gene expression in muscle. Gene Ther 1: 114121, 1994.[ISI][Medline]
21. Dean DA. Electroporation of the vasculature and the lung. DNA Cell Biol 22: 797806, 2003.[CrossRef][ISI][Medline]
23. Dean DA, Machado-Aranda D, Blair-Parks K, Yeldandi AV, and Young JL. Electroporation as a method for high-level non-viral gene transfer to the lung. Gene Ther 10: 16081615, 2003.[CrossRef][ISI][Medline]
24. DeBruyne LA, Li K, Chan SY, Qin L, Bishop DK, and Bromberg JS. Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses. Gene Ther 5: 10791087, 1998.[CrossRef][ISI][Medline]
25. Dev NB, Hofmann GA, Dev SB, and Rabussay DP. Intravascular electroporation markedly attenuates neointima formation after balloon injury of the carotid artery in the rat. J Interventional Cardiol 13: 331338, 2000.
26. Dev NB, Preminger TJ, Hofmann GA, and Dev SB. Sustained local delivery of heparin to the rabbit arterial wall with an electroporation catheter. Cathet Cardiovasc Diagn 45: 337345, 1998.[CrossRef][ISI][Medline]
27. Dileo J, Miller TE Jr, Chesnoy S, and Huang L. Gene transfer to subdermal tissues via a new gene gun design. Hum Gene Ther 14: 7987, 2003.[CrossRef][ISI][Medline]
28. Doh SG, Vahlsing HL, Hartikka J, Liang X, and Manthorpe M. Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA. Gene Ther 4: 648663, 1997.[CrossRef][ISI][Medline]
29. Dona M, Sandri M, Rossini K, Dell'Aica I, Podhorska-Okolow M, and Carraro U. Functional in vivo gene transfer into the myofibers of adult skeletal muscle. Biochem Biophys Res Commun 312: 11321138, 2003.[CrossRef][ISI][Medline]
30. Draghia-Akli R, Ellis KM, Hill LA, Malone PB, and Fiorotto ML. High-efficiency growth hormone-releasing hormone plasmid vector administration into skeletal muscle mediated by electroporation in pigs. FASEB J 17: 526528, 2003.
31. Draghia-Akli R, Fiorotto ML, Hill LA, Malone PB, Deaver DR, and Schwartz RJ. Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs. Nat Biotechnol 17: 11791183, 1999.[CrossRef][ISI][Medline]
32. Ehsan A, Mann MJ, Dell'Acqua G, and Dzau VJ. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg 121: 714722, 2001.
33. Ellison KE, Bishopric NH, Webster KA, Morishita R, Gibbons GH, Kaneda Y, Sato B, and Dzau VJ. Fusigenic liposome-mediated DNA transfer into cardiac myocytes. J Mol Cell Cardiol 28: 13851399, 1996.[CrossRef][ISI][Medline]
34. Elmadbouh I, Rossignol P, Meilhac O, Vranckx R, Pichon C, Pouzet B, Midoux P, and Michel JB. Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications. J Gene Med 6: 11121124, 2004.[CrossRef][ISI][Medline]
35. Factor P, Dumasius V, Saldias F, and Sznajder JI. Adenoviral-mediated overexpression of the NA,K-ATPase 1 subunit gene increases lung edema clearance and improves survival during acute hyperoxic lung injury in rats. Chest 116: 24S25S, 1999.
36. Ferrer A, Foster H, Wells KE, Dickson G, and Wells DJ. Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins. Gene Ther 11: 884893, 2004.[CrossRef][ISI][Medline]
37. Fewell JG, MacLaughlin F, Mehta V, Gondo M, Nicol F, Wilson E, and Smith LC. Gene therapy for the treatment of hemophilia B using PINC-formulated plasmid delivered to muscle with electroporation. Mol Ther 3: 574583, 2001.[CrossRef][ISI][Medline]
38. Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, and Staus SE. Virology. New York: Raven, 1996.
39. Gehl J, Skovsgaard T, and Mir LM. Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochim Biophys Acta 1569: 5158, 2002.[ISI][Medline]
40. Genot EM, Parker PJ, and Cantrell DA. Analysis of the role of protein kinase C-alpha, -epsilon, and -zeta in T cell activation. J Biol Chem 270: 98339839, 1995.
41. Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH, and Hyde SC. Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1 promoter. Gene Ther 8: 15391546, 2001.[CrossRef][ISI][Medline]
42. Gollins H, McMahon J, Wells KE, and Wells DJ. High-efficiency plasmid gene transfer into dystrophic muscle. Gene Ther 10: 504512, 2003.[CrossRef][ISI][Medline]
43. Gossen M and Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89: 55475551, 1992.
44. Harada M, Toki Y, Numaguchi Y, Osanai H, Ito T, Okumura K, and Hayakawa T. Prostacyclin synthase gene transfer inhibits neointimal formation in rat balloon-injured arteries without bleeding complications. Cardiovasc Res 43: 481491, 1999.[CrossRef][ISI][Medline]
45. Harms JS and Splitter GA. Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum Gene Ther 6: 12911297, 1995.[ISI][Medline]
46. Harrison RL, Byrne BJ, and Tung L. Electroporation-mediated gene transfer in cardiac tissue. FEBS Lett 435: 15, 1998.[CrossRef][ISI][Medline]
47. Hartikka J, Sawdey M, Cornefert-Jensen F, Margalith M, Barnhart K, Nolasco M, Vahlsing HL, Meek J, Marquet M, Hobart P, Norman J, and Manthorpe M. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 7: 12051217, 1996.[ISI][Medline]
48. Helbling-Leclerc A, Scherman D, and Wils P. Cellular uptake of cationic lipid/DNA complexes by cultured myoblasts and myotubes. Biochim Biophys Acta 1418: 165175, 1999.[ISI][Medline]
49. Herweijer H, Zhang G, Subbotin VM, Budker V, Williams P, and Wolff JA. Time course of gene expression after plasmid DNA gene transfer to the liver. J Gene Med 3: 280291, 2001.[CrossRef][ISI][Medline]
50. Hong K, Sherley J, and Lauffenburger DA. Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector. Biomol Eng 18: 185192, 2001.[CrossRef][ISI][Medline]
51. Hoshina K, Koyama H, Miyata T, Shigematsu H, Takato T, Dalman RL, and Nagawa H. Aortic wall cell proliferation via basic fibroblast growth factor gene transfer limits progression of experimental abdominal aortic aneurysm. J Vasc Surg 40: 512518, 2004.[CrossRef][ISI][Medline]
52. Huber PE, Mann MJ, Melo LG, Ehsan A, Kong D, Zhang L, Rezvani M, Peschke P, Jolesz F, Dzau VJ, and Hynynen K. Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery. Gene Ther 10: 16001607, 2003.[CrossRef][ISI][Medline]
53. Iwashita H, Yoshida M, Nishi T, Otani M, and Ueda S. In vivo transfer of a neuronal nitric oxide synthase expression vector into the rat bladder by electroporation. BJU Int 93: 10981103, 2004.[CrossRef][ISI][Medline]
54. Kawabata K, Takakura Y, and Hashida M. The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 12: 825830, 1995.[CrossRef][ISI][Medline]
55. Keogh MC, Chen D, Lupu F, Shaper N, Schmitt JF, Kakkar VV, and Lemoine NR. High efficiency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipid-DNA complex. Gene Ther 4: 162171, 1997.[CrossRef][ISI][Medline]
56. Keogh MC, Chen D, Schmitt JF, Dennehy U, Kakkar VV, and Lemoine NR. Design of a muscle cell-specific expression vector utilising human vascular smooth muscle -actin regulatory elements. Gene Ther 6: 616628, 1999.[CrossRef][ISI][Medline]
57. Khachigian LM. Catalytic DNAs as potential therapeutic agents and sequence-specific molecular tools to dissect biological function. J Clin Invest 106: 11891195, 2000.
58. Kitsis RN, Buttrick PM, McNally EM, Kaplan ML, and Leinwand LA. Hormonal modulation of a gene injected into rat heart in vivo. Proc Natl Acad Sci USA 88: 41384142, 1991.
59. Klugherz BD, Jones PL, Cui X, Chen W, Meneveau NF, DeFelice S, Connolly J, Wilensky RL, and Levy RJ. Gene delivery from a DNA controlled-release stent in porcine coronary arteries. Nat Biotechnol 18: 11811184, 2000.[CrossRef][ISI][Medline]
60. Kondo I, Ohmori K, Oshita A, Takeuchi H, Fuke S, Shinomiya K, Noma T, Namba T, and Kohno M. Treatment of acute myocardial infarction by hepatocyte growth factor gene transfer: the first demonstration of myocardial transfer of a "functional" gene using ultrasonic microbubble destruction. J Am Coll Cardiol 44: 644653, 2004.
61. Latta-Mahieu M, Rolland M, Caillet C, Wang M, Kennel P, Mahfouz I, Loquet I, Dedieu JF, Mahfoudi A, Trannoy E, and Thuillier V. Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther 13: 16111620, 2002.[CrossRef][ISI][Medline]
62. Lauritzen HP, Reynet C, Schjerling P, Ralston E, Thomas S, Galbo H, and Ploug T. Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo. Pflügers Arch 444: 710721, 2002.[CrossRef][ISI][Medline]
63. Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, and Newman CM. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 7: 20232027, 2000.[CrossRef][ISI][Medline]
64. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW, Beatty B, Squire J, O'Brodovich H, and Lukacs GL. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther 6: 482497, 1999.[CrossRef][ISI][Medline]
65. Li H and Capetanaki Y. An E box in the desmin promoter cooperates with the E box and MEF-2 sites of a distal enhancer to direct muscle-specific transcription. EMBO J 13: 35803589, 1994.[Abstract]
66. Li S, MacLaughlin FC, Fewell JG, Gondo M, Wang J, Nicol F, Dean DA, and Smith LC. Muscle-specific enhancement of gene expression by incorporation of the SV40 enhancer in the expression plasmid. Gene Ther 8: 494497, 2001.[CrossRef][ISI][Medline]
67. Li X, Eastman EM, Schwartz RJ, and Draghia-Akli R. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat Biotechnol 17: 241245, 1999.[CrossRef][ISI][Medline]
68. Liang KW, Hoffman EP, and Huang L. Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid. Mol Ther 1: 236243, 2000.[CrossRef][ISI][Medline]
69. Liu F, Nishikawa M, Clemens PR, and Huang L. Transfer of full-length Dmd to the diaphragm muscle of Dmdmdx/mdx mice through systemic administration of plasmid DNA. Mol Ther 4: 4551, 2001.[CrossRef][ISI][Medline]
70. Liu F, Song Y, and Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6: 12581266, 1999.[CrossRef][ISI][Medline]
71. Liu MA, McClements W, Ulmer JB, Shiver J, and Donnelly J. Immunization of non-human primates with DNA vaccines. Vaccine 15: 909912, 1997.[CrossRef][ISI][Medline]
72. Lu QL, Bou-Gharios G, and Partridge TA. Non-viral gene delivery in skeletal muscle: a protein factory. Gene Ther 10: 131142, 2003.[CrossRef][ISI][Medline]
73. Lu QL, Liang HD, Partridge T, and Blomley MJ. Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Ther 10: 396405, 2003.[CrossRef][ISI][Medline]
74. Ma X, Glover C, Miller H, Goldstein J, and O'Brien E. Focal arterial transgene expression after local gene delivery. Can J Cardiol 17: 873883, 2001.[ISI][Medline]
75. Machado-Aranda D, Adir Y, Young JL, Briva A, Budinger GRS, Yeldandi A, Sznajder JI, and Dean DA. Gene transfer of the Na+,K+-ATPase 1 subunit using electroporation increases lung liquid clearance in rats. Am J Respir Crit Care Med 171: 204211, 2005.
77. Mann MJ, Gibbons GH, Hutchinson H, Poston RS, Hoyt EG, Robbins RC, and Dzau VJ. Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues. Proc Natl Acad Sci USA 96: 64116416, 1999.
78. Mann MJ, Whittemore AD, Donaldson MC, Belkin M, Conte MS, Polak JF, Orav EJ, Ehsan A, Dell'Acqua G, and Dzau VJ. Ex vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet 354: 14931498, 1999.[CrossRef][ISI][Medline]
79. Martin JB, Young JL, Benoit JN, and Dean DA. Gene transfer to intact mesenteric arteries by electroporation. J Vasc Res 37: 372380, 2000.[CrossRef][ISI][Medline]
80. Masuo M, Reardon S, Ikebe M, and Kitazawa T. A novel mechanism for the Ca2+-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. J Gen Physiol 104: 265286, 1994.[Abstract]
81. Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6: 508514, 1999.[CrossRef][ISI][Medline]
82. Matsumoto T, Komori K, Shoji T, Kuma S, Kume M, Yamaoka T, Mori E, Furuyama T, Yonemitsu Y, and Sugimachi K. Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse. Gene Ther 8: 11741179, 2001.[CrossRef][ISI][Medline]
83. McLean J, Thurston G, and McDonald D. Sites of uptake and expression of cationic liposome DNA complexes injected intravenously. In: Nonviral Vectors for Gene Therapy, edited by Huang L, Hung M-C, and Wagner E. San Diego, CA: Academic, 1999, p. 120139.
84. McMahon JM, Signori E, Wells KE, Fazio VM, and Wells DJ. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidaseincreased expression with reduced muscle damage. Gene Ther 8: 12641270, 2001.[CrossRef][ISI][Medline]
85. Mennuni C, Calvaruso F, Zampaglione I, Rizzuto G, Rinaudo D, Dammassa E, Ciliberto G, Fattori E, and La Monica N. Hyaluronidase increases electrogene transfer efficiency in skeletal muscle. Hum Gene Ther 13: 355365, 2002.[CrossRef][ISI][Medline]
86. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, and Scherman D. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96: 42624267, 1999.
87. Mir LM, Bureau MF, Rangara R, Schwartz B, and Scherman D. Long-term, high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle. C R Acad Sci III 321: 893899, 1998.[ISI][Medline]
88. Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, Orlopp K, Lochmuller H, Petrof BJ, Nalbantoglu J, and Karpati G. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther 10: 447455, 2004.[CrossRef][ISI][Medline]
89. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, and Dzau VJ. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 90: 84748478, 1993.
90. Murakami T, Nishi T, Kimura E, Goto T, Maeda Y, Ushio Y, Uchino M, and Sunada Y. Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation. Muscle Nerve 27: 237241, 2003.[CrossRef][ISI][Medline]
91. No D, Yao TP, and Evans RM. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 93: 33463351, 1996.
92. Nunamaker EA, Zhang HY, Shirasawa Y, Benoit JN, and Dean DA. Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression. Am J Physiol Heart Circ Physiol 285: H2240H2247, 2003.
93. Otten G, Schaefer M, Doe B, Liu H, Srivastava I, zur Megede J, O'Hagan D, Donnelly J, Widera G, Rabussay D, Lewis MG, Barnett S, and Ulmer JB. Enhancement of DNA vaccine potency in rhesus macaques by electroporation. Vaccine 22: 24892493, 2004.[CrossRef][ISI][Medline]
94. Panetta CJ, Miyauchi K, Berry D, Simari RD, Holmes DR, Schwartz RS, and Caplice NM. A tissue-engineered stent for cell-based vascular gene transfer. Hum Gene Ther 13: 433441, 2002.[CrossRef][ISI][Medline]
95. Park SW, Gwon HC, Jeong JO, Byun J, Kang HS, You JR, Cho SS, Lee MJ, Lee Y, Kim S, and Kim DK. Intracardiac echocardiographic guidance and monitoring during percutaneous endomyocardial gene injection in porcine heart. Hum Gene Ther 12: 893903, 2001.[CrossRef][ISI][Medline]
96. Payen E, Bettan M, Rouyer-Fessard P, Beuzard Y, and Scherman D. Improvement of mouse -thalassemia by electrotransfer of erythropoietin cDNA. Exp Hematol 29: 295300, 2001.[CrossRef][ISI][Medline]
97. Perlstein I, Connolly JM, Cui X, Song C, Li Q, Jones PL, Lu Z, DeFelice S, Klugherz B, Wilensky R, and Levy RJ. DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection. Gene Ther 10: 14201428, 2003.[CrossRef][ISI][Medline]
98. Pitard B, Pollard H, Agbulut O, Lambert O, Vilquin JT, Cherel Y, Abadie J, Samuel JL, Rigaud JL, Menoret S, Anegon I, and Escande D. A nonionic amphiphile agent promotes gene delivery in vivo to skeletal and cardiac muscles. Hum Gene Ther 13: 17671775, 2002.[CrossRef][ISI][Medline]
99. Potter H, Weir L, and Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 81: 71617165, 1984.
100. Ribault S, Neuville P, Mechine-Neuville A, Auge F, Parlakian A, Gabbiani G, Paulin D, and Calenda V. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res 88: 468475, 2001.
101. Samakoglu S, Fattori E, Lamartina S, Toniatti C, Stockholm D, Heard JM, and Bohl D. Minor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in
thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles. Blood 97: 22132220, 2001.
102. Santiago FS, Lowe HC, Kavurma MM, Chesterman CN, Baker A, Atkins DG, and Khachigian LM. New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med 5: 1438, 1999.[CrossRef]
103. Sarkar N, Blomberg P, Wardell E, Eskandarpour M, Sylven C, Drvota V, and Islam KB. Nonsurgical direct delivery of plasmid DNA into rat heart: time course, dose response, and the influence of different promoters on gene expression. J Cardiovasc Pharmacol 39: 215224, 2002.[CrossRef][ISI][Medline]
104. Satkauskas S, Bureau MF, Puc M, Mahfoudi A, Scherman D, Miklavcic D, and Mir LM. Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. Mol Ther 5: 133140, 2002.[CrossRef][ISI][Medline]
105. Sawa Y, Kaneda Y, Bai HZ, Suzuki K, Fujimoto J, Morishita R, and Matsuda H. Efficient transfer of oligonucleotides and plasmid DNA into the whole heart through the coronary artery. Gene Ther 5: 14721480, 1998.[CrossRef][ISI][Medline]
106. Schakman O, Gilson H, de Coninck V, Lause P, Verniers J, Havaux X, Ketelslegers JM, and Thissen JP. Insulin-like growth factor-I gene transfer by electroporation prevents skeletal muscle atrophy in glucocorticoid-treated rats. Endocrinology 146: 17891797, 2005.
107. Scherer LJ and Rossi JJ. Approaches for the sequence-specific knockdown of mRNA. Nat Biotechnol 21: 14571465, 2003.[CrossRef][ISI][Medline]
108. Sen L, Hong YS, Luo H, Cui G, and Laks H. Efficiency, efficacy, and adverse effects of adenovirus vs. liposome-mediated gene therapy in cardiac allografts. Am J Physiol Heart Circ Physiol 281: H1433H1441, 2001.
109. Shinmura K, Morishita R, Aoki M, Higaki J, Ogihara T, Kaneda Y, and Tani M. Catheter-delivered in vivo gene transfer into rat myocardium using the fusigenic liposomal mediated method. Jpn Heart J 41: 633647, 2000.[CrossRef][ISI][Medline]
110. Shirasawa Y, Rutland TJ, Young JL, Dean DA, and Benoit JN. Modulation of protein kinase C (PKC)-mediated contraction and the possible role of PKcepsilon in rat mesenteric arteries. Front Biosci 8: a133a138, 2003.[ISI][Medline]
111. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, and Malone RW. Theory and in vivo application of electroporative gene delivery. Mol Ther 2: 178187, 2000.[CrossRef][ISI][Medline]
112. Suzuki J, Morishita R, Amano J, Kaneda Y, and Isobe M. Decoy against nuclear factor-B attenuates myocardial cell infiltration and arterial neointimal formation in murine cardiac allografts. Gene Ther 7: 18471852, 2000.[CrossRef][ISI][Medline]
113. Takahashi A, Palmer-Opolski M, Smith RC, and Walsh K. Transgene delivery of plasmid DNA to smooth muscle cells and macrophages from a biostable polymer-coated stent. Gene Ther 10: 14711478, 2003.[CrossRef][ISI][Medline]
114. Takeshita S, Gal D, Leclerc G, Pickering JG, Riessen R, Weir L, and Isner JM. Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation. In vitro and in vivo findings in a rabbit model of vascular injury. J Clin Invest 93: 652661, 1994.[ISI][Medline]
115. Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, and Morishita R. Local delivery of plasmid DNA into rat carotid artery using ultrasound. Circulation 105: 12331239, 2002.
116. Tomiyasu K, Oda Y, Nomura M, Satoh E, Fushiki S, Imanishi J, Kondo M, and Mazda O. Direct intra-cardiomuscular transfer of 2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters. Gene Ther 7: 20872093, 2000.[CrossRef][ISI][Medline]
117. Tomiyasu K, Satoh E, Oda Y, Nishizaki K, Kondo M, Imanishi J, and Mazda O. Gene transfer in vitro and in vivo with Epstein-Barr virus-based episomal vector results in markedly high transient expression in rodent cells. Biochem Biophys Res Commun 253: 733738, 1998.[CrossRef][ISI][Medline]
118. Turunen MP, Hiltunen MO, Ruponen M, Virkamäki L, Szoka FC Jr, Urtti A, and Ylä-Herttuala S. Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes. Gene Ther 6: 611, 1999.[CrossRef][ISI][Medline]
119. Vacik J, Dean BS, Zimmer WE, and Dean DA. Cell-specific nuclear import of plasmid DNA. Gene Ther 6: 10061014, 1999.[CrossRef][ISI][Medline]
120. Vicat JM, Boisseau S, Jourdes P, Laine M, Wion D, Bouali-Benazzouz R, Benabid AL, and Berger F. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum Gene Ther 11: 909916, 2000.[CrossRef][ISI][Medline]
121. Von der Leyen HE, Braun-Dullaeus R, Mann MJ, Zhang L, Niebauer J, and Dzau VJ. A pressure-mediated nonviral method for efficient arterial gene and oligonucleotide transfer. Hum Gene Ther 10: 23552364, 1999.[CrossRef][ISI][Medline]
122. Wang J, Jiao S, Wolff JA, and Knechtle SJ. Gene transfer and expression in rat cardiac transplants. Transplantation 53: 703705, 1992.[ISI][Medline]
123. Wang XJ, Liefer KM, Tsai S, O'Malley BW, and Roop DR. Development of gene-switch transgenic mice that inducibly express transforming growth factor 1 in the epidermis. Proc Natl Acad Sci USA 96: 84838488, 1999.
124. Wang Y, Bai Y, Price C, Boros P, Qin L, Bielinska AU, Kukowska-Latallo JF, Baker JR Jr, and Bromberg JS. Combination of electroporation and DNA/dendrimer complexes enhances gene transfer into murine cardiac transplants. Am J Transplant 1: 334338, 2001.[CrossRef][ISI][Medline]
125. Wang Y, Boros P, Liu J, Qin L, Bai Y, Bielinska AU, Kukowska-Latallo JF, Baker JR Jr, and Bromberg JS. DNA/dendrimer complexes mediate gene transfer into murine cardiac transplants ex vivo. Mol Ther 2: 602608, 2000.[CrossRef][ISI][Medline]
126. Wolff JA, Ludtke JJ, Acsadi G, Williams P, and Jani A. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1: 363369, 1992.[Abstract]
127. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, and Felgner PL. Direct gene transfer into mouse muscle in vivo. Science 247: 14651468, 1990.[ISI][Medline]
128. Yew NS, Marshall J, Przybylska M, Wysokenski DM, Ziegler RJ, Rafter PW, Li C, Armentano D, and Cheng SH. Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3. Hum Gene Ther 10: 18331843, 1999.[CrossRef][ISI][Medline]
129. Yew NS, Przybylska M, Ziegler RJ, Liu D, and Cheng SH. High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter. Mol Ther 4: 7582, 2001.[CrossRef][ISI][Medline]
130. Yew NS, Zhao H, Przybylska M, Wu IH, Tousignant JD, Scheule RK, and Cheng SH. CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 5: 731738, 2002.[CrossRef][ISI][Medline]
131. Young JL, Benoit JN, and Dean DA. Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature. Gene Ther 10: 14651470, 2003.[CrossRef][ISI][Medline]
132. Young JL and Dean DA. Nonviral gene transfer strategies for the vasculature. Microcirculation 9: 3549, 2002.[CrossRef][ISI][Medline]
133. Zelenin AV, Kolesnikov VA, Tarasenko OA, Shafei RA, Zelenina IA, Mikhailov VV, Semenova ML, Kovalenko DV, Artemyeva OV, Ivaschenko TE, Evgrafov OV, Dickson G, and Baranovand VS. Bacterial -galactosidase and human dystrophin genes are expressed in mouse skeletal muscle fibers after ballistic transfection. FEBS Lett 414: 319322, 1997.[CrossRef][ISI][Medline]
134. Zhang G, Budker V, Williams P, Subbotin V, and Wolff JA. Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Hum Gene Ther 12: 427438, 2001.[CrossRef][ISI][Medline]
135. Zhang G, Gao X, Song YK, Vollmer R, Stolz DB, Gasiorowski JZ, Dean DA, and Liu D. Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther 11: 675682, 2004.[CrossRef][ISI][Medline]
136. Zhao H, Hemmi H, Akira S, Cheng SH, Scheule RK, and Yew NS. Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. Mol Ther 9: 241248, 2004.[ISI][Medline]
137. Zhou R, Geiger RC, and Dean DA. Intracellular trafficking of nucleic acids. Expert Opin Drug Deliv 1: 127140, 2004.[CrossRef]
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