Injection Of Naked Plasmid DNA Research Articles

Intramuscular delivery of DNA releasing microspheres: Microsphere properties and transgene expression

Plasmid-loaded microspheres can provide localized and sustained release into the target tissue, and thus have the potential to enhance the efficiency of naked DNA at promoting transgene expression. In this report, microsphere design parameters are investigated by correlating the extent and duration of transgene expression intramuscularly to the polymer molecular weight and the mass of DNA delivered. Plasmid DNA was incorporated into poly (lactide- co-glycolide) microspheres using a cryogenic double emulsion process, and microspheres were injected intramuscularly. Bolus injection of naked plasmid was used for control, which exhibited transfection of muscle cells with transgene expression that gradually decreased over time. Microspheres fabricated from low molecular weight polymer had expression levels that increased from day 1 to day 92, which subsequently decreased through day 174. Decreasing the microsphere mass delivered resulted in steady expression during the same time. However, microspheres fabricated with high molecular weight polymer had expression for only 14 days. Intramuscular injection resulted in a foreign body response to the microspheres, and these infiltrating cells adjacent were primarily transfected. This understanding of microsphere properties that determine transgene expression and the distribution of transfected cells may facilitate their application to fields such as tissue engineering or DNA vaccines.

Tissue-Specific Characteristics of in Vivo Electric Gene: Transfer by Tissue and Intravenous Injection of Plasmid DNA

To evaluate the tissue-specific characteristics of electric gene transfer after tissue and intravenous injection of naked plasmid DNA (pDNA). pDNA encoding firefly luciferase was injected directly into the liver, kidney, spleen, skin and muscle, or into the tail vein of mice, and electric pulses were then applied to one of these organs. The distribution of transgene expressing cells was evaluated using pDNA encoding beta-galactosidase. Tissue injection of pDNA produced a significant degree of transgene expression in any tissue with the greatest amount in the liver, followed by kidney and spleen. The expression in these organs decreased quickly with time, and muscle showed the greatest expression at 7 days. Electroporation significantly increased the expression, and the expression level was comparable among the organs. Intravenous injection of pDNA followed by electroporation resulted in a significant expression in the liver, spleen, and kidney but not in the skin or muscle. Electric gene transfer to the liver, kidney, and spleen can be an effective approach to obtain significant amounts of transgene expression by either tissue or intravenous injection of pDNA, whereas it is only effective after tissue injection as far as skin- or muscle-targeted gene transfer is concerned.

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.

Topical application of plasmid DNA to mouse and human skin

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.

Hepatocyte growth factor as potential cardiovascular therapy

Hepatocyte growth factor is a mesenchyme-derived pleiotropic factor that regulates the growth, motility and morphogenesis of various types of cells, and is also a member of the angiogenic growth factors. Hepatocyte growth factor is secreted by vascular endothelial cells and smooth muscle cells, and the hepatocyte growth factor receptor, c-met, was also observed in these vascular cells. Treatment of human aortic endothelial cells with recombinant hepatocyte growth factor resulted in a significant increase in cell proliferation, accompanied by mitogen-activated protein kinase and Akt/protein kinase B phosphorylation. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As preclinical study of gene therapy using hepatocyte growth factor to treat peripheral arterial disease, naked hepatocyte growth factor plasmid was intramuscularly injected into the ischemic hind limb of rabbits in order to evaluate its angiogenic activity. Intramuscular injection of hepatocyte growth factor plasmid once on day 10 following surgery, produced significant augmentation of collateral vessel development in the ischemic limb on day 30. In the clinical setting, the authors further investigated the safety and efficacy of hepatocyte growth factor plasmid DNA in patients with critical limb ischemia, in a prospective open-labeled trial. Intramuscular injection of naked plasmid DNA was performed in the ischemic limbs of six patients with critical limb ischemia with arteriosclerosis obliterans (n = 3) or Buerger disease (n = 3) graded as Fontaine III or IV. In the efficacy evaluation, a reduction of pain scale of more than 1 cm on a visual analog pain scale was observed in five out of six patients. An increase in ankle pressure index of more than 0.1 was observed in five out of five patients. The long diameter of eight out of 11 ischemic ulcers in four patients was reduced by more than 25%. Intramuscular injection of naked hepatocyte growth factor plasmid is safe, feasible and can achieve successful improvement of ischemic limbs. Although the present data were obtained to demonstrate safety in a Phase I/early Phase II trial, the initial clinical outcome with hepatocyte growth factor gene transfer seems to indicate its usefulness as sole therapy for critical limb ischemia. Randomized placebo-controlled clinical trials of alternative dosing regimens of gene therapy will be required to define the efficiency of this therapy.

Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation

To increase transgene expression in the liver, electric pulses were applied to the left lateral lobe after intravenous injection of naked plasmid DNA (pDNA) or pDNA/liver targeting vector complex prepared with galactosylated poly(L-lysine) or galactosylated polyethyleneimine. Electroporation (250 V/cm, 5 ms/pulse, 12 pulses, 4 Hz) after naked pDNA injection dramatically increased the expression up to 200,000-fold; the expression level obtained was significantly greater than that achieved by the combination of pDNA/vector complex and electroporation. We clearly demonstrated that the expression was dependent on the plasma concentration of pDNA at the time when the electric pulses were applied. Separation of liver cells revealed that the distribution of naked pDNA as well as transgene expression was largely selective to hepatocytes in the electroporated lobe. The number of cells expressing transgene product using vascularly administered naked pDNA followed by electroporation was significantly (P<0.01) greater and more widespread than that obtained by local injection of naked pDNA. These results indicate that the application of in vivo electroporation to vascularly administered naked pDNA is a useful gene transfer approach to a large number of hepatocytes.

Sind die Gen- oder Stammzelltherapie eine therapeutische Option?

The aim of cardiovascular gene and stem cell therapy concepts is regeneration of vascular and myocardial cells in ischaemically damaged areas. Despite the wealth of more or less valid study results, two main directions for clinical investigation can be distinguished. Gene therapy with angiogenic growth factors in the form of repetitive intramuscular injections of naked plasmid DNA (pDNA) is simple to perform and is principally applied in clinical investigations in the field of peripheral arterial occlusive disease (“therapeutic angiogenesis”). Anumber of parallel phase II studies with nonviral pDNA for fibroblast growth factor-1 (FGF-1) are ongoing in the USA and Europe (TALISMAN studies). In ischaemic heart disease stem cell-based concepts are showing first clinical results. Adult stem cells are obtained by bone marrow puncture or ex-vivo expansion in blood of circulating stem cells also deriving from the patient’s bone marrow. After intracoronary administration what is hoped for is intensified neovascularisation of ischaemic myocardium through release of angiogenic growth factors from mononuclear bone marrow cells, and regeneration of infarcted myocardium by transdifferentiation of mesenchymal stem cells into myocardial cells. In this context the German multicentre study Transplantation Of adult Progenitor Cells in Ischaemic Heart Disease (TOPCARE-AMI study) and Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST study) will shortly widen our knowledge still further.

Single injection of naked plasmid encoding α-melanocyte-stimulating hormone protects against thioacetamide-induced acute liver failure in mice

Oxidative stress has been implicated in the propagation of acute liver injury. The aim of our study was to investigate whether gene transfer of α-melanocyte-stimulating hormone (α-MSH), a potent anti-inflammatory peptide, could prevent fulminant hepatic failure in mice. Acute liver damage was induced by intraperitoneal administration of thioacetamide. Hydrodynamics-based gene transfection with α-MSH expression plasmid via rapid tail vein injection was initiated 1 day prior to intoxication. The mortality in the α-MSH-treated mice was significantly lower compared to the vehicle group 3 days after injury. Liver histology significantly improved and TUNEL-positive hepatocytes decreased in the treated mice. The degradation of IκBα, endogenous inhibitor of nuclear factor κB, and upregulation of inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels were prevented in the α-MSH-treated group, indicating decreased oxidative stress and inflammation. These results suggest α-MSH gene therapy might protect against acute hepatic necroinflammatory damage with further potential applications.

A Facile Nonviral Method for Delivering Genes and siRNAs to Skeletal Muscle of Mammalian Limbs

Delivery is increasingly being recognized as the critical hurdle holding back the tremendous promise of nucleic acid-based therapies that include gene therapy and more recently siRNA-based therapeutics. While numerous candidate genes (and siRNA sequences) with therapeutic potential have been identified, their utility has not yet been realized because of inefficient and/or unsafe delivery technologies. We now describe an intravascular, nonviral methodology that enables efficient and repeatable delivery of nucleic acids to muscle cells (myofibers) throughout the limb muscles of mammals. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of a limb that is transiently isolated by a tourniquet or blood pressure cuff. Nucleic acid delivery to myofibers is facilitated by its rapid injection in sufficient volume to enable extravasation of the nucleic acid solution into muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. The simplicity, effectiveness, and safety of the procedure make this methodology well suited to limb muscle gene therapy applications.

Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound.

Although gene therapy might become a promising approach for central nervous system diseases, the safety issue is a serious consideration in human gene therapy. To overcome this problem, we developed an efficient gene transfer method into the adult rat brain based on plasmid DNA using a microbubble-enhanced ultrasound method, since microbubble-enhanced ultrasound has shown promise for transfecting genes into other tissues such as blood vessels. Using the microbubble-enhanced ultrasound method, luciferase expression was increased approximately 10-fold as compared to injection of naked plasmid DNA alone. Interestingly, the site of gene expression was limited to the site of insonation with intracisternal injection, in contrast to previous studies using viruses. Expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection, and in glial cells with intrastriatal injection. There was no obvious evidence of tissue damage by microbubble-enhanced ultrasound. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into the central nervous system, providing a new option for treating various diseases such as tumors.

IN VIVO DIFFERENTIATION OF TOLEROGENIC DENDRITIC CELLS IN THE LIVER, AN INSIGHT INTO HEPATIC TOLERANCE

P908 Aims: Discovery of liver transplant tolerance has led to an enthusiasm of investigating liver dendritic cells (DC) which play a key role in determining immunity and tolerance. Recent in vivo demonstration of myeloid CD11c+ hepatic DC with strong capacity to stimulate T cells certainly supports the notion that hepatic DC generate immunity. There is however, lacking of evidence of existence of liver tolerogenic DC in vivo. Methods and Results: We adopted, in this study, an aaproach to transfect gene(s) in the liver by tail vein rapid injection of naked plasmid carrying genes encoding IL-3, CD40L or GM-CSF, (GFP as reporter) into B6 (H-2b) mice, which enabled significant levels of transgene expression in the liver in 8h, reached the peak in 24 hr, decreased thereafter, and lasted for 5-7 days, as demonstrated either by detection of GFP with fluorescent microscopy or by serum levels of IL-3 with ELISA. While we confirmed the expansion of mixed population in the group of transfection with GM-CSF, most of them morphologically resembled myeloid DC, and located in portal and periportal areas. Another more homogeneous population was identified following transfection with IL-3 plus CD40L, not IL-3 alone, which is morphologically distinctive from myeloid DC and form big aggregates in expanded lobules as well as portal and periportal areas. FACS analysis revealed that in addition to common DC surface markers (MHC class II, CD80, CD86, and CD40), they were B220+, CD205+, but CD11b- and CD11c-, and expressed high TLR-9, but low TLR-4. We examined their response to bacterial stimuli. Not surprising, after LPS stimulation, the co-stimulatory molecule expression was upregulated, similar to myeloid DC, but what differ are their cytokine profiles. Demonstrated by RNAse protection assay, B220+/CD205+ DC expressed defective IL-12 mRNA with only p35 but not p40 mRNA, and up-regulated IL-10 but down-regulated IL-1 expression. In contrast, myeloid hepatic DC showed cross-board upregulation of inflammatory cytokine, including IL-1α, β, IL-6 and IL-12 (both p35 and p40). We studied in vitro DC-T cell interaction in an antigen-specific manner. Using both CD4 (DO10.11) and CD8 (Des) TCR transgenic T cells. In comparison to myeloid DC, B220+/CD205+ DC induced hyporesponsiveness of both CD4 and CD8 T cells measured by thymidine uptake with repressed levels of IL-2 (1.0 ng/ml vs 2.6 ng/ml), but similar level of IFN-γ (8.3 ng/ml vs 8.2 ng/ml). This hyporesponsiveness is not due to failure of T cell activation since CFSE-labeling showed T cell dividing similar to culture with myeloid DC. It appeared to result from shortened T cell survival. In contrst to myeloid DC administration of which exacerbated B6 cardiac allograft rejection in C3H (H-2k) recipients, B220+/CD205+ DC prolonged allograft survival. Conclusion: A tolerogenic DC population in the liver was differentiated in vivo, which may play an important role in balancing hepatic T cell immunity and tolerance.

190. Myocardial Gene Therapy by Naked Angiopoietin-1 Plasmid Injection Efficiently Induces Angiogenesis and Prevents the Remodeling Following Acute Myocardial Infarction

Background: Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. Previously, we reported that adenoviral vector mediated Ang1 gene therapy clearly promoted myocardial angiogenesis and reduced infarct size in a rat acute myocardial infarction (AMI) model. However, the alternative gene delivery method must explore for clinical application of myocardial Ang1 gene therapy, because of toxicity and inflammatory nature of adenoviral vector. Naked plasmid injection is the safest and convenient method for cardiovascular gene therapy and we have reported that naked CA (CMV enhancer/Chicken β-actin promoter)-promoter can bring remarkable myocardial transgene expression following naked CA-plasmid injection. Therefore, we attempted naked Ang1 plasmid gene therapy for rat AMI model and evaluated myocardial angiogenesis and effects for the remodeling following AMI.

1036. Effective Acceleration of Wound Healing by Simultaneous Transfestion of Hepatocyte Growth Factor Gene and Prostacyclin Synthase Gene Using Shima Jet

Background: Gene delivery into skin may be useful for treating skin diseases. We tried to evaluate the effectiveness of intradermal injection of naked plasmid into the skin by spring powered jet injector, Shima Jet, that was originally developed as non-needle jet injector of insulin for diabetic patients. Recently, there have been a lot of interests in the role of Hepatocyte Growth Factor (HGF) for wound healing. Re-epithelialization and granulation formation induced by HGF would promote the wound closure. On the other hand, the role of Prostagrandin I2 (Prostacyclin) in the vasolidation of blood vessels has been focused on and the effctiveness of Prostagrandin I2 Synthetase (PGIS) gene therapy for promoting blood flow has been reported. In this study we evaluated the effectiveness of gene therapy for healing of impaired wound by transfecting these genes together to the skin by Shima Jet.

Mechanism of Liver Gene Transfer by Mechanical Massage

Many metabolic diseases are caused by defects in the metabolic pathways in the liver. Others result from the absence of specific proteins normally produced and secreted by the liver. Because these metabolic disorders are usually caused by single gene defect, they are ideal candidates for gene therapy. We have previously shown that mouse liver can be transfected by mechanically massaging the liver (MML) after intravenous injection of naked plasmid DNA. We now show a significant linear relationship between the level of liver gene expression and the venous blood pressure, supporting the idea that gene transfer by MML is due, at least in part, to pressure-mediated effect. Liver transfection could not be blocked by co-injection of excess irrelevant DNA or poly I, suggesting that there is no involvement of receptors, including the scavenger receptor, in MML. Moreover, the level of gene expression could be further enhanced by a combination of MML and an increase in DNA retention-time in the liver. Persistence of gene expression could be significantly improved using an EBV-based plasmid vector. Our data suggest the mechanical massage produces transient membrane defects through which naked DNA can enter into the liver cells by simple diffusion.

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery.

The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.

Single intravenous injection of naked plasmid DNA encoding erythropoietin provides neuroprotection in hypoxia–ischemia rats

Hypoxic–ischemic (H–I) encephalopathy is a major contributor to morbidity and mortality in infants and children. To delineate the nature and mechanism(s) of neuroprotection via erythropoietin (EPO) gene therapy, we evaluated the effects of single intravenous injection of naked plasmid DNA encoding EPO in H–I infant rats. Single administration of naked plasmid containing EPO cDNA driven under cytomegalovirus promoter (pCMV-EPO) by rapid injection via the tail vein produced a remarkable level of human EPO protein in the circulation, peaking at one day and lasting for 14 days after injection. There were significant improvements of water maze task in H–I rats after EPO gene therapy. Our data showed that the mechanisms of EPO gene therapy were rescue of CA1 neurons from lethal H–I injury, prevention of neuronal apoptosis in CA1 region, and decrease of glial activation in corpus callosum. This could be the first report of successful treatment of H–I injury by a single intravenous infusion of EPO gene.

Kidney-targeted naked DNA transfer by retrograde injection into the renal vein in mice

We recently developed a novel kidney-targeted gene transfer technique in rats, using the retrograde renal vein injection of naked plasmid DNA. Many animal disease models are created in mice by transgenic or knockout technologies. However, it is much harder to perform renal vein injection in mice than in rats because they have a thin and short vein. Here we transferred the mouse interleukin (IL)-10 gene into mice by retrograde renal vein injection, using an IL-10 and immunoglobulin fusion protein (IL-10/Fc) (96-kDa) expression plasmid, pCAGGS-IL10/Fc. We observed a dose–response relationship between serum IL-10 levels and the amount of injected DNA. The serum IL-10 levels peaked at day 1 and then were sustained for at least 2 weeks. These results demonstrate that the kidney-targeted naked plasmid DNA transfer of mice by retrograde renal vein injection can be achieved, and the kidney serves as a depot organ for the production of large proteins.

Diverse efficacy of vaccination therapy using the α-fetoprotein gene against mouse hepatocellular carcinoma

Antitumor vaccination therapy approaches using naked plasmid DNA or recombinant viruses encoding tumor-associated antigens are currently in development. In the present study, we examined the therapeutic efficacy of vaccination using the mouse alpha-fetoprotein (AFP) gene in mouse hepatocellular carcinoma (HCC) cells. C57L/J or C3H/HeN mice were primed with an injection of naked plasmid DNA expressing mouse AFP followed by a booster of replication-defective adenovirus expressing mouse AFP (plasmid-AFP prime/adenovirus-AFP booster vaccination). The mice were then challenged with high AFP-producing Hepa1-6 cells or low AFP-producing MH134 cells, respectively, and the tumor growth rate was monitored. Plasmid-AFP prime/adenovirus-AFP booster vaccination promoted protective immunity against Hepa1-6 cells, and significantly increased the number of interferon-gamma-producing splenic cells in C57L/J mice. In addition, this vaccination protocol repressed the growth of pre-established Hepa1-6 tumors in C57L/J mice. However, plasmid-AFP prime/adenovirus-AFP booster vaccination did not induce protective immunity against MH134 cells in C3H/HeN mice. These results suggest that vaccination with the AFP gene is a promising strategy to treat HCC, but its outcome may be affected by the level of AFP expression in HCC or by the immunological response of the host.

Electroporation for gene transfer to skeletal muscles: current status.

Naked plasmid DNA can be used to introduce genetic material into a variety of cell types in vivo. However, such gene transfer and expression is generally very low compared with that achieved with viral vectors and so is unsuitable for clinical therapeutic application in most cases. This difference in efficiency has been substantially reduced by the introduction of in vivo electroporation to enhance plasmid delivery to a wide range of tissues including muscle, skin, liver, lung, artery, kidney, retina, cornea, spinal cord, brain, synovium, and tumors. The precise mechanism of in vivo electroporation is uncertain, but appears to involve both electropore formation and an electrophoretic movement of the plasmid DNA. Skeletal muscle is a favored target tissue for three reasons: there is a pressing need to develop effective therapies for muscular dystrophies; skeletal muscle can act as an effective platform for the long-term secretion of therapeutic proteins for systemic distribution; and introduction of DNA vaccines into skeletal muscle promotes strong humoral and cellular immune responses. All of these applications are significantly improved by the application of in vivo electroporation. Importantly, the increased efficiency of plasmid delivery following electroporation is seen in larger species as well as rodents, in contrast to the decreasing efficiencies with increasing body size for simple intramuscular injection of naked plasmid DNA. As this electroporation-enhanced non-viral gene delivery system works well in larger species and avoids the vector-specific immune responses associated with recombinant viruses, the prospects for clinical application are promising.

Gene therapy in cardiac surgery: intramyocardial injection of naked plasmid DNA for chronic myocardial ischemia.

Growth factor gene therapy represents one current approach in the therapy of myocardial ischemia. We assessed the in vitro and in vivo expression of naked plasmid DNA aiming at preservation of function in a chronically ischemic myocardial model. In vitro: Primary cardiac fibroblasts were transfected with plasmids encoding enhanced green fluorescent protein, human VEGF(121), human FGF-2, or porcine MCP-1. Protein synthesis was assessed microscopically, by ELISA, Western blotting, or intracellular immunofluorescence. In vivo: A LAD stenosis was created in healthy pigs. One week later, segmental myocardial shortening (SMS) and systemic hemodynamics (left ventricular stroke work index, LVSWI, time derivative of left ventricular pressure, dp/dt(max)) were assessed at baseline. Afterwards, the ischemic area received either intramyocardial injections of naked cytokine plasmid DNA or vector only, or was left untreated. One myocardial sample taken 1 h after plasmid injection was subjected to RT-PCR and PCR. After 3 months, cardiac function was re-examined. In vitro: Transfection of cardiac fibroblasts resulted in high gene expression for several days. In vivo: Plasmid-specific DNA and mRNA were found 1 h after plasmid injection (n=1). After 3 months, VEGF, FGF-2, and vector rendered better results of regional contractility at rest and of LVSWI. However, only VEGF and FGF-2 were effective with regard to regional contractility under dobutamine stress and to left ventricular contractility. In conclusion, intramyocardial injection of naked plasmid DNA encoding VEGF(121) or FGF-2 improved myocardial function in chronic ischemia in more aspects than vector only and was superior to untreated ischemia or MCP-1. This strategy can be considered a successful tool for growth factor stimulated preservation of function in chronic myocardial ischemia.