Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle.

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.     

Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses.

The injection of plasmid DNA encoding hepatitis B virus (HBV) envelope proteins in mouse muscle leads to the induction of specific humoral and cellular immune responses. Most studies on DNA-based immunization have used viral promoters to drive antigen expression. In this study, we compared the efficiency of a muscle-specific promoter, the human desmin gene promoter, with the commonly used cytomegalovirus (CMV) early gene promoter. We showed that increased in vitro expression of HBV envelope proteins from the human desmin gene promoter has no effect on the in vivo immune response even after the injection of as little as 10 micrograms of DNA. The injection of vectors encoding HBV envelope proteins under the control of either the human desmin gene promoter or the CMV promoter induced humoral and cytotoxic immune responses at comparable levels and of the same duration. The recruitment of antigen-presenting cells to the DNA injection site by pretreatment of muscle with a necrotizing agent increases the precocity and the intensity of the responses, particularly when the nonspecific CMV vector was used.     

Plasmid DNA-based gene transfer with ultrasound and microbubbles

Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.     

包载治疗基因的聚合物纳米粒子:Ⅰ.纳米粒子制备及动物模型基因治疗实验研究

以可生物降解材料聚乳酸聚乙醇酸共聚物（Poly—dl—lactic—cp—glycolic，PLGA）为原料，采用多相乳化技术制备载VEGP纳米粒子。并对纳米粒子的粒径，VEGF含量，体外释放等进行了测定。VEGF纳米粒子和VEGF裸质粒被注射到兔下肢缺血模型的缺血部位，通过RT—PCR，免疫组化和血管造影等技术来验证基因治疗的效果，评价VEGF纳米粒子作为基因载体在动物模型基因治疗中的效率。制备的VEGF纳米粒子的平均粒径约为300nm，包埋效率在96％以上，纳米粒子中VEGF含量约4％。可在体外维持恒定释放约两周。两周基因注射结果表明VEGF-NP治疗组与裸质粒VEGF治疗组的毛细血管密度明显高于对照组，VEGF纳米粒子组（81．22per mm^2），对照组（29．54mm^2），两者有显著性差异（P〈0．05）。RT—PCR结果显示VEGF纳米粒子组表达（31．79au＊mm）明显高于VEGF裸质粒组（9．15au＊mm）。在动物模型中VEGF纳米粒子是比裸质粒DNA更好的基因载体系统，结果显示了纳米粒子可望在人类基因治疗中得到很好的应用。     

包载治疗基因的聚合物纳米粒子:I.纳米粒子制备及动物模型基因治疗实验研究

以可生物降解材料聚乳酸聚乙醇酸共聚物(Poly-dl-lactic-co-glycolic,PLGA)为原料,采用多相乳化技术制备载VEGF纳米粒子。并对纳米粒子的粒径,VEGF含量,体外释放等进行了测定。VEGF纳米粒子和VEGF裸质粒被注射到兔下肢缺血模型的缺血部位,通过RT-PCR,免疫组化和血管造影等技术来验证基因治疗的效果,评价VEGF纳米粒子作为基因载体在动物模型基因治疗中的效率。制备的VEGF纳米粒子的平均粒径约为300nm,包埋效率在96%以上,纳米粒子中VEGF含量约4%。可在体外维持恒定释放约两周。两周基因注射结果表明VEGF-NP治疗组与裸质粒VEGF治疗组的毛细血管密度明显高于对照组,VEGF纳米粒子组(81.22permm2),对照组(29.54mm2),两者有显著性差异(P<0.05)。RT-PCR结果显示VEGF纳米粒子组表达(31.79au*mm)明显高于VEGF裸质粒组(9.15au*mm)。在动物模型中VEGF纳米粒子是比裸质粒DNA更好的基因载体系统,结果显示了纳米粒子可望在人类基因治疗中得到很好的应用。     

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.     

Electrotransfer into skeletal muscle for protein expression

An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as study of gene function and gene therapy applications. Among the different non-viral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple. This technique is a physical method of gene delivery consisting in local application of electric pulses after DNA injection. Although this technique can be applied to almost any tissue of a living animal, including tumors, skin, liver, kidney, artery, retina, cornea or even brain, this review will focus on electrotransfer of plasmid DNA into skeletal muscle and its possible uses in gene therapy, vaccination, or functional studies. Skeletal muscle is a good target for electrotransfer of DNA as it is: a large volume easily accessible, an endocrine organ capable of expressing several local and systemic factors, and muscle fibres as post-mitotic cells have a long lifespan that allows long-term gene expression. In this review, we describe the mechanism of DNA electrotransfer, we assess toxicity and safety considerations related to this technique, and we focus on important therapeutic applications of electrotransfer demonstrated in animal models in recent years.     

Plasmid DNA gene therapy by electroporation: principles and recent advances

Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.     

Sustained release of PEG-g-chitosan complexed DNA from poly(lactide-co-glycolide)

Chitosan and its derivatives have emerged as promising gene-delivery vehicles because of their capability to form polyplexes with plasmid DNA and enhance its transport across cellular membranes through endocytosis. Evidently, polyplexes of chitosan and DNA significantly improve transfection efficiency; however, these polyplexes are not capable of sustained DNA release and, thus, prolong gene transfer. In order to achieve prolonged delivery of DNA/chitosan polyplexes, we have formulated microspheres by physically combining poly(ethylene glycol)-grafted chitosan (PEG-g-CHN) with poly(lactide-co-glycolide) (PLGA) using a modified conventional in-emulsion solvent evaporation method. Electrophoretic analysis of materials released from these microspheres suggests the presence of PEG-g-CHN complexed DNA and these microspheres are capable of sustained release of DNA/PEG-g-CHN for at least 9 weeks. The rate of DNA release can be modulated by varying the amount of PEG-g-CHN. The release products from these microspheres are bioactive and show enhanced transfection in vitro over DNA released from conventional PLGA microspheres containing no PEG-g-CHN. In vivo experiments also show that these microspheres are capable of achieving gene transfer in a rat hind limb muscle model.     

Muscular gene transfer using nonviral vectors

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.     

Enhanced expressions and histological characteristics of intravenously administered plasmid DNA in rat lung

Cationic liposome-mediated gene transfection is a promising method for gene therapy. In this study, the transfection efficiency and histological patterns were evaluated in rat lung after intravenous administration via femoral vein of naked plasmid DNA, naked plasmid DNA with pretreatment of DOTAP, and DOTAP-cholesterol-plasmid DNA complex. Plasmid DNA encoding bacterial LacZ gene was used. For quantification of LacZ gene expression, beta-galactosidase assay was performed. For histologic examination, X-gal staining and immunohistochemical staining for transfected gene products were performed. Pretreatment of DOTAP prior to the infusion of naked plasmid DNA increased transfection efficiency up to a level comparable to DOTAP-cholesterol-plasmid DNA complex injection. Transfected genes were mainly expressed in type II pneumocytes and alveolar macrophages in all animals. We conclude that the high transfection efficiency is achievable by intravenous administration of naked plasmid DNA with pretreatment of DOTAP, to a level comparable to DOTAP-cholesterol-plasmid DNA complex. In this regard, naked plasmid DNA administration with pretreatment of DOTAP could be a more feasible option for intravenous gene transfer than DOTAP-cholesterol-plasmid DNA complex, in that the former is technically easier and more cost-effective than the latter with a comparable efficacy, in terms of intravenous gene delivery to the lung.     

Sustained release of PEG-g-chitosan complexed DNA from poly(lactide-co-glycolide)

Chitosan and its derivatives have emerged as promising gene-delivery vehicles because of their capability to form polyplexes with plasmid DNA and enhance its transport across cellular membranes through endocytosis. Evidently, polyplexes of chitosan and DNA significantly improve transfection efficiency; however, these polyplexes are not capable of sustained DNA release and, thus, prolong gene transfer. In order to achieve prolonged delivery of DNA/chitosan polyplexes, we have formulated microspheres by physically combining poly(ethylene glycol)-grafted chitosan (PEG-g-CHN) with poly(lactide-co-glycolide) (PLGA) using a modified conventional in-emulsion solvent evaporation method. Electrophoretic analysis of materials released from these microspheres suggests the presence of PEG-g-CHN complexed DNA and these microspheres are capable of sustained release of DNA/PEG-g-CHN for at least 9 weeks. The rate of DNA release can be modulated by varying the amount of PEG-g-CHN. The release products from these microspheres are bioactive and show enhanced transfection in vitro over DNA released from conventional PLGA microspheres containing no PEG-g-CHN. In vivo experiments also show that these microspheres are capable of achieving gene transfer in a rat hind limb muscle model.     

In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery

Electroporation is a widely used technique for enhancing the efficiency of DNA delivery into cells. Application of electric pulses after local injection of DNA temporarily opens cell membranes and facilitates DNA uptake. Delivery of plasmid DNA by electroporation to alter gene expression in tissue has also been explored in vivo. This approach may constitute an alternative to viral gene transfer, or to transgenic or knock-out animals. Among the most frequently electroporated target tissues are skin, muscle, eye, and tumors. Moreover, different regions in the central nervous system (CNS), including the developing neural tube and the spinal cord, as well as prenatal and postnatal brain have been successfully electroporated. Here, we present a comprehensive review of the literature describing electroporation of the CNS with a focus on the adult brain. In addition, the mechanism of electroporation, different ways of delivering the electric pulses, and the risk of damaging the target tissue are highlighted. Electroporation has been successfully used in humans to enhance gene transfer in vaccination or cancer therapy with several clinical trials currently ongoing. Improving the knowledge about in vivo electroporation will pave the way for electroporation-enhanced gene therapy to treat brain carcinomas, as well as CNS disorders such as Alzheimer's disease, Parkinson's disease, and depression.     

Electroporation for Gene Transfer to Skeletal Muscles

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.     

Applications of muscle electroporation gene therapy

Muscle is a convenient and accessible site for non-viral gene delivery, which can manufacture gene products and provide a long-duration of gene expression. The level of gene expression after administration of naked DNA plasmid or polymer-formulated DNA plasmid containing a reporter gene to muscle via syringe injection, however, is very low. As a result, no significant therapeutic effect can be detected after saline- or polymer-mediated gene delivery into muscle. In 1998, investigators published a striking new approach--electrotransfection--for intramuscular gene delivery (now commonly referred to as electroporation or electroinjection). Electroporation of a non-viral gene into the muscles of small animals has increased the level of gene expression by as much as two orders of magnitude, which is comparable to levels achieved with adenoviral gene delivery. Three years later, intramuscular electroporation gene delivery technology has blossomed. Treatments for different diseases using this approach in animal models have been reported. In this review, I discuss the applications of intramuscular electroporation gene therapy to treat malignancies, renal disease, and anemia, and to prevent drug toxicity to sensory nerves.     

血管内皮生长因子165真核基因转染载体转染的骨骼肌细胞

背景：血管内皮生长因子基因转染治疗组织损伤的研究倍受关注,构建稳定可靠的人血管内皮生长因子真核表达载体有重要意义。 目的：克隆人血管内皮生长因子基因血管内皮生长因子165片段,构建pcDNA4-HisMax-C/VEGF165真核表达质粒,并验证其转染大鼠骨骼肌细胞的可靠性。 方法：采用反转录-聚合酶链反应技术,从人卵巢癌患者外周血中提取并扩增出血管内皮生长因子165基因片段,通过DNA重组技术将该基因片段重组于pcDNA4-HisMax-C真核表达载体上,构建成pcDNA4-HisMax-C/VEGF165重组质粒,聚合酶链反应扩增,分别用酶切电泳分析和DNA测序的方法对提取和重组DNA 进行鉴定。pcDNA4-HisMax-C/VEGF165重组质粒转染骨骼肌细胞1周后反转录-聚合酶链反应提取血管内皮生长因子基因并酶切电泳鉴定。 结果与结论：构建的重组质粒目的基因片段为人血管内皮生长因子165 cDNA,对大鼠骨骼肌细胞转染后检测到血管内皮生长因子165基因片段。提示成功地克隆了血管内皮生长因子165基因并构建了其真核表达质粒,能以此为载体转染至骨骼肌细胞,并已整合到骨骼肌的基因组参与转录,证明了其转染的有效性。     

Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines

Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.     

人类心房利钠肽基因在自发性高血压大鼠体内表达及对血压和肾脏的组织学影响

目的研究人类心房利钠肽（hANP）基因在自发性高血压大鼠（SHR）体内表达、持续时间和对血压及肾脏的组织学影响。方法 12只8周龄雄性SHR,随机分为两组,于左腿股四头肌注射pc DNA3.1-h ANP质粒的为实验组,在同一部位注射pc DNA3.1空质粒的为对照组,每周测量大鼠尾动脉收缩压,及利用放射免疫方法监测血中h ANP水平;10周后处死动物,RT-PCR和Western印迹杂交技术检测h ANP基因表达情况;HE染色和Masson染色观察对肾脏的组织形态学的影响。结果转基因后第1周起与对照组比较,实验组血压开始下降,两组动物一直相差（12±3.1）mm Hg（P〈0.05）,其作用可持续10周;且放射免疫方法监测实验组血中hANP水平较高;RT-PCR和Western印迹杂交技术检测显示实验组hANP基因在肌肉组织中高效表达,对照组则未见表达;组织形态学分析结果显示实验组肾脏组织细胞形态基本正常,胶原沉积明显减少。结论肌肉注射法将hANP基因导入SHR,hANP基因可在肌肉组织中高效表达,且hANP可释放如血,降低SHR的血压并对高血压靶器官肾脏的损害起到了治疗及预防作用。显示了hANP基因对高血压患者治疗的可能性。     

DNA疫苗抗肿瘤研究进展

质粒ＤＮＡ或称裸ＤＮＡ疫苗直接肌肉注射后,能表达编码基因的蛋白,并诱导宿主免疫反应。这为肿瘤基因－免疫治疗提供了一种新的途径。近１０年的研究表明,ＤＮＡ疫苗表达质粒骨架中的免疫刺激序列具有佐剂效应和促有丝分裂作用,这与免疫反应的诱导有密切关系。注射部位的肌细胞起着抗原蛋白表达储池作用,通过骨髓来源的抗原呈递细胞,最终诱发宿主体内ＣＴＬ和抗体反应。ＤＮＡ疫苗抗肿瘤的靶向性是由插入表达质粒的编码基因所     

Improvement of nonviral gene therapy by Epstein-Barr virus (EBV)-based plasmid vectors

The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.