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.     

Beta-amino ester polymers facilitate in vivo DNA transfection and adjuvant plasmid DNA immunization.

Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candidates identified in this screen, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] enhanced plasmid DNA transgene expression by sevenfold (P=0.0001) and its immunogenicity by 70% (P=0.03). We found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo. We also observed a log-linear correlation (R2=0.93) between peak cellular immune responses and transgene activity in all evaluated polymer-plasmid DNA formulations, clarifying the relationship between immunogenicity and the quantity of expressed antigen.     

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo

One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of the transgene, involves utilization of mechanical forces to facilitate transduction of DNA into cells. In this study, we evaluated the feasibility of mechanical insonation in gene transfers with naked DNA plasmid loading both in vitro and in vivo. We used an ultrasound probe, which can focus the ultrasonic beam in the exit zone of the probe. The reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and the neomycin 3'-phosphotransferase gene (neo), was used for evaluation of transfer efficiency. Expression of beta-gal in MC38 murine colon carcinoma cells was measured after insonation of 20 W/cm2 with continuous 1.0-MHz wave exposure. In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene. After cells were treated with geneticin, we also observed a difference in colonogenicity between noninsonated and insonated groups. When MC38 cells were implanted in syngeneic mice and plasmid was injected, the insonation that followed facilitated beta-galactosidase expression. These results indicate that insonation represents a potential approach for gene therapy when combined with naked DNA plasmid injection.     

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.

Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and beta-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 microg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 microg of plasmid DNA into a mouse. Histochemical analysis using beta-galactosidase gene as a reporter reveals that approximately 40percent of hepatocytes express the transgene. The time-response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.     

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.

Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter. Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.     

Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo.     

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound

Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55- (Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application. Because ultrasound waves can be focused on different anatomical locations in the human body without significant adverse effects, the control of DNA transfer by focused ultrasound is a promising in vivo method for spatial regulation of gene-based medical treatments.     

Ligand-mediated retargeting of recombinant adenovirus for gene transfer in vivo

The development of efficient and safe methods for in vivo gene transfer is central to the success of gene therapy. Recombinant adenoviral vectors, although highly efficient, are limited by the host immune response, potential safety hazards due to obligatory cotransfer of viral proteins, and their broad tissue tropism. Here, we demonstrate in an animal model that host range and tissue tropism of a recombinant adenovirus from a distant species can be modified by complexing adenovirus with a cell-specific ligand. Thus, a replication-deficient lacZ recombinant human adenovirus, which naturally does not infect avian cells, allowed highly efficient and specific gene transfer to the liver of ducks in vivo when complexed with N-acetylglucosamine, a ligand for the chicken hepatic lectin. This combination of ligand-mediated receptor targeting with adenoviral uptake and intracellular processing of a given gene represents a novel approach to gene therapy of inherited and acquired liver diseases.     

Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo

The study was to investigate the probability of increasing the transfection of the gene in tendons by ultrasound-targeted microbubble destruction (UTMD), and to search for the most suitable transfection conditions. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids was injected into rabbit Achilles tendons by different administration routes and the tendons were ultrasound pulse by different ultrasonic conditions in order to determine the most appropriate conditions. Then, the rabbits were divided into four groups: (1) ultrasound + microbubbles + plasmid; (2) ultrasound+ plasmid; (3) microbubble + plasmid; (4) plasmid only. EGFP expression in the tendons and other tissues, and the damage to tendon and paratenon were all observed. The results showed that EGFP expression in the tendon was higher by ultrasound pulse with 2 W cm(-2) of output intensity and a 20% duty cycle for 10 min. Local injection was determined to be the better administration route. Among the four groups, EGFP expression in Group 1 was higher than that in other groups. EGFP expression was highest on seventh day, then it gradually decrease over time, and lasted more than 56 days. EGFP expression was not found in other tissues. There was no obvious injury caused by UTMD. Under suitable conditions, it is feasible to use UTMD as a safe and effective gene transfection therapy for tendon injuries.     

The influence of blood on in vivo adenovirus bio-distribution and transduction.

Intravascular delivery of adenovirus (Ad) vectors is being developed for liver-directed gene therapy for targeting disseminated disease in cancer therapeutics and for targeting non-hepatic tissues and organs through vector engineering strategies. The utility of Ad vectors is not limited to serotype 5 (Ad5), and many alternate human serotypes and non-human serotypes of Ad are currently being investigated. Critical to intravascular delivery of Ad is the interaction of the virus with host blood cells and plasma proteins, because immediate contact is observed following injection. Although incompletely understood, recent studies suggest that these interactions are critical in dictating the particle bio-distribution and resulting transduction properties of Ad in vivo. For example, plasma proteins-in particular, vitamin K-dependent coagulation zymogens-are able to directly bind to Ad, and "bridge" the virus to receptors in the liver. Unraveling and characterizing these mechanisms will be of fundamental importance both for understanding basic Ad biology in vivo and for refinement and optimization of Ad vectors for human gene therapy.     

Highly efficient in vivo gene transfection by plasmid/PEI complexes coated by anionic PEG derivatives bearing carboxyl groups and RGD peptide.

A new class of an anionic poly (ethylene glycol) derivative, PEG-Suc, bearing 17.7 pairs of carboxylic acid-side chains was synthesized. PEG-Suc deposited onto the DNA/polyethyleneimine complexes without destroying them even at high dose ratio. Coating of the DNA complexes by PEG-Suc recharged their surface to negative, and effectively protected them from the albumin-induced aggregation. Paired carboxyl groups in the side chains showed higher proton sponge effect. Negatively charged surface would diminish the electrostatic binding of the complexes to the cells, and the transfection efficiency on the cultured cells was not high. RGD peptide side chain as a ligand to malignant cell surfaces was then introduced to compensate the reduced electrical adhesion. RGD-PEG-Suc-coated plasmid/PEI complex brought about more than 3 times higher reporter protein activity on the cultured B16 cells. Those bio-compatible DNA complexes with ligand attained very high gene expression in tumor, lung, and liver after injection into mouse tail vein.     

Ultrasound-mediated transfection of canine myocardium by intravenous administration of cationic microbubble-linked plasmid DNA.

We tested the hypothesis that targeted disruption of cationic microbubble-linked plasmid DNA, using diagnostic ultrasound, may aid transfection of large animal myocardium. Plasmid DNA encoding for CAT (pCAT, chloramphenicol acetyltransferase) was bound to a novel cationic microbubble containing MRX-225 for intravenous administration, and 16 dogs in 4 groups variously received this conjugate or plasmid only, or were exposed to ultrasound. Histochemical staining and enzyme-linked immunosorbent assay analysis showed CAT activity in the myocardium of only those animals that received microbubble-linked DNA and were exposed to ultrasound. Thus, disruption of cationic-linked, low-dose plasmid systems by diagnostic ultrasound may facilitate transfection of large animal hearts.     

Adenoviral-mediated gene transfer to fetal pulmonary epithelia in vitro and in vivo.

Vector-mediated gene transfer offers a direct method of correcting genetic pulmonary diseases and might also be used to correct temporary abnormalities associated with acquired, nongenetic disorders. Because the fetus or newborn may be a more immune tolerant host for gene transfer using viral vectors, we used replication defective recombinant adenoviral vectors to test the feasibility of gene transfer to the fetal pulmonary epithelium in vitro and in vivo. Both proximal and distal epithelial cells in cultured fetal lung tissues from rodents and humans diffusely expressed the lacZ transgene 3 d after viral infection. In vivo gene delivery experiments were performed in fetal mice and lambs. Delivery of Ad2/CMV-beta Gal to the amniotic fluid in mice produced intense transgene expression in the fetal epidermis and amniotic membranes, some gastrointestinal expression, but no significant airway epithelial expression. When we introduced the adenoviral vector directly into the trachea of fetal lambs, the lacZ gene was expressed in the tracheal, bronchial, and distal pulmonary epithelial cells 3 d after viral infection. Unexpectedly, reactive hyperplasia and squamous metaplasia were noted in epithelia expressing lacZ in the trachea, but not in the distal lung of fetal lambs. 1 wk after infection, adenovirus-treated fetuses developed inflammatory cell infiltrates in the lung tissue with CD4, CD8, IgM, and granulocyte/macrophage positive immune effector cells. Transgene expression faded coincident with inflammation and serologic evidence of antiadenoviral antibody production. While these studies document the feasibility of viral-mediated gene transfer in the prenatal lung, they indicate that immunologic responses to E1-deleted recombinant adenoviruses limit the duration of transgene expression.     

In vivo release and gene expression of plasmid DNA by hydrogels of gelatin with different cationization extents.

The objective of this paper is to investigate the in vivo release and gene expression of lacZ plasmid DNA (pSV-lacZ) by the hydrogels of cationized gelatin. Gelatin with different cationization extents was prepared by changing the amount of ethylenediamine added to aminize the carboxyl groups of gelatin with a water-soluble carbodiimide. The cationized gelatin prepared was crosslinked by various concentrations of glutaraldehyde (GA) to obtain cationized gelatin hydrogels with different cationization extents as the release carrier of plasmid DNA. When the cationized gelatin hydrogels incorporating 125I-labeled pSV-lacZ were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period was prolonged with an increase in the concentration of GA used for hydrogel preparation. In vivo experiments with 125I-labeled cationized gelatin hydrogels revealed that the higher the GA concentration, the longer the in vivo retention period of radioactivity remaining for every cationized gelatin hydrogel. Only for the hydrogels prepared from gelatin with the aminized percentages of 29.7, 41.6, and 47.8 mol.%, the time profile of pSV-lacZ retention correlated well with that of hydrogel retention. The gene expression by the cationized gelatin hydrogels incorporating pSV-lacZ depended on the aminized percentage of gelatin and was significant at the percentage of 41.6 mol.% or higher. It is possible that the pSV-lacZ was complexed with the degraded fragments of cationized gelatin and released with a positive charge, resulting in enhanced gene expression. We conclude that gelatin with a cationization extent of at least 41.6 mol.% is needed for the enhanced in vivo gene expression of plasmid DNA by the hydrogel release system.     

脂氟显微泡及质粒浓度对基因转染效率影响的研究

目的 研究脂氟显微泡及质粒浓度对pcDNA6.2-GW/EmGFP重组质粒转染效率及人肝癌HepG₂细胞死亡率的影响,初步探索转染最佳条件及shRNA重组质粒对靶基因survivin的抑制效果。方法 将重组质粒加入各组,设置6个质粒浓度梯度组。向各组加入脂氟显微泡混悬液,设置6个微泡浓度梯度组。对照组不加入微泡和质粒。根据前期优化结果,采用治疗性超声(1.2W/cm₂、占空比20%)辐照90s。48h后使用荧光显微镜及流式细胞仪检测细胞GFP表达效率。细胞死亡率通过台盼蓝染色计数获得。设立阳性质粒组P(+)和阴性质粒组P(－),western blot检测靶基因蛋白表达水平。 结果 当脂氟显微泡浓度小于等于 150μl/ml 时,转染率随微泡浓度增加而增加;当大于 150μl/ml 时,转染率却随微泡浓度增加而降低(p<0.05)。HepG₂细胞死亡率随微泡浓度增高,呈上升趋势(p<0.05)。在一定范围(20μl/ml)内,增加质粒浓度可以提高转染效率(p<0.05);但继续增加质粒浓度对基因转染率无明显影响(p>0.05)。质粒浓度的改变对细胞死亡率无明显影响。Western blot结果表明, P(+)组survivin基因表达受到明显抑制。结论 超声联合微泡可以有效介导基因转染,当微泡浓度为 150μl/ml,质粒浓度为 20μl/ml时,基因转染率最高,细胞死亡率较低。阳性重组质粒P(+)可下调靶基因表达水平,为后续基因转染的相关研究奠定基础。     

Selective in vivo transfection of murine biliary epithelia using polycation-enhanced adenovirus

We have investigated the use of polycations to increase adenovirus-mediated expression of transgenic protein to the biliary epithelia with a view to gene therapy for hepatobiliary disease in CF. We have shown that adenovirus carrying the beta-galactosidase transgene transfect both human and mouse biliary epithelia in primary culture and that in both instances adenovirus transfection can be significantly increased by co-complexing with polycation. In vivo administration of 1 x 109 p.f.u. adenovirus co-complexed with the polyamine polyethyenimine (PEI) into the mouse biliary duct leads to >80% positively stained biliary epithelia while adenovirus alone at the same titre infected <5% biliary epithelia. We suggest that the use of low titre polycation enhanced adenoviral delivery to the biliary tree of CF patients could be of therapeutic significance. As a prelude to an extensive in vivo functional investigation in CF null mice we have shown that Ad5/polycation complexes deliver functional CFTR to non-CFTR expressing cells in vitro more efficiently than Ad5 alone.     

EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo

Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.     

Enhanced nuclear import and transfection efficiency of plasmid DNA using streptavidin-fused importin-beta.

In order to enhance the nuclear import of exogenous genes, novel plasmid DNA/importin-beta conjugates, which consist of a biotinylated plasmid DNA and a recombinant streptavidin-fused importin-beta, were prepared. The spacer length between plasmid DNA and biotin and the number of introduced biotin were adjusted. The microinjection of plasmid DNA/importin-beta conjugates into the cytoplasm of NIH3T3 cells resulted in the nuclear localization of conjugates and the higher expression efficiency, compared to intact plasmid DNA alone. These results indicate that plasmid DNA/importin-beta conjugates would be an important tool to enhance the nuclear localization of exogenous DNA in non-viral gene delivery system.     

Hydrodynamics- and ultrasound-based transfection of heart with naked plasmid DNA

A novel, efficient transfection method, based on ultrasound and hydrodynamics, has been developed to transfect heart tissue with plasmid DNA. An ultrasound probe was aimed at the heart of anesthetized rats for 30 sec, at an intensity of 1 MHz and 2 W/cm2. The aorta was clamped and a phosphate-buffered saline (PBS) solution containing pSV-LacZ was quickly injected into the left ventricle. Each animal was maintained in this condition for 20 sec, and then the clamp was opened and the needle was removed. Electrocardiography, performed after 4 weeks, showed mild or no sign of ischemia in all groups. Visual evaluation of heart tissue samples from rats that received 100 microg of pSV-LacZ in 100 microl had only a few blue cells, indicating transfection, and those that received only PBS had no blue cells. However, all heart tissue samples from rats transfected with 100 to 500 microg of pSV-LacZ in 200 microl, or with 200 to 500 microg of pSV-LacZ in 100 micro had many blue cells. The base and epicardium of the heart tissue samples had many more blue cells than did the rest of the samples. Histological results, based on staining with hematoxylin and eosin, showed similar results between control and transfected groups. Therefore, we concluded that gene delivery by plasmid vector in association with ultrasound and hydrodynamics was highly effective in transfecting rat heart.