Am J Physiol Heart Circ Physiol 303: H629 –H638, 2012. First published July 20, 2012; doi:10.1152/ajpheart.00126.2012. Review Nonviral gene therapy targeting cardiovascular system Cheng-Huang Su,1,2 Yih-Jer Wu,1,3 Hsueh-Hsiao Wang,3 and Hung-I Yeh1,3 1 Departments of Internal Medicine and Medical Research, Mackay Memorial Hospital, New Taipei City, Taiwan; 2Mackay Medicine, Nursing and Management College, New Taipei City, Taiwan; 3Mackay Medical College, New Taipei City, Taiwan Submitted 15 February 2012; accepted in final form 17 July 2012 gene therapy; vector; ultrasound; cavitation; microbubble is part of a collection on Physiological Basis of Cardiovascular and Gene Therapies. Other articles appearing in this collection, as well as a full archive of all Review collections, can be found online at http://ajpheart. physiology.org/. THIS REVIEW ARTICLE Overview of Applications of Gene Therapy Gene therapy is a novel approach that can be applied through a variety of clinical therapeutic procedures in which nucleic acids are introduced into human cells for the purpose of treating, curing, or ultimately preventing disease. The development of gene vectors for effectively carrying genes into cells has made a great deal of progress in recent years. Nonviral vectors should have a better chance to circumvent some of the problems possibly occurring with viral vectors, such as unwanted immune response and oncogenesis. In general, although nonviral vectors, such as plasmid DNA, allow remarkable organ specificity, they are often limited by low transfection efficiency (TE). In contrast, virus-based vectors deliver the transgene more efficiently, but organ specificity may be reduced and immunogenic properties can limit their practical Address for reprint requests and other correspondence: H.-I Yeh, Depts. of Internal Medicine and Medical Research, Mackay Memorial Hospital; Mackay Medical College, No. 46, Sec. 3, Jhong-Jheng Rd, San-Jhih District, New Taipei City 252, Taiwan (e-mail: [email protected]). http://www.ajpheart.org applications. Both systems need dose optimization and development of strategies to improve vector technology. Thus far, the main categories of methods that have been used to deliver genes into cells or tissues in gene therapy protocols are as follows: viral vectors (68.8%), nonviral chemical vectors (6.4%), naked/ plasmid DNA (18.6%), and physical delivery systems (0.3%; data adapted from http://www.wiley.com//legacy/wileychi/genmed/ clinical/, clinical trials website of The Journal of Gene Medicine, updated June 2011). In the present review, we summarized the progress made in the past few years regarding techniques developed in the following two areas: 1) chemical carrier-mediated gene delivery, such as cationic polymer and lipid, and 2) naked DNA transfection by a physical method, such as ultrasound (US) along with microbubbles. In addition, the nonviral carrier and technology of cardiovascular gene delivery are briefly overviewed, while the applications to the biomedical researches including regenerative medicine are introduced. Candidate Diseases for Gene Therapy There are several promising areas for gene therapy in genetic and acquired diseases. Thus far, more than 1,500 clinical trials have taken place worldwide. The diseases most commonly treated with gene therapy are cancer (64.6%), cardiovascular diseases (8.5%), monogenic diseases (8.3%), and infectious 0363-6135/12 Copyright © 2012 the American Physiological Society H629 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 Su C, Wu Y, Wang H, Yeh H. Nonviral gene therapy targeting cardiovascular system. Am J Physiol Heart Circ Physiol 303: H629 –H638, 2012. First published July 20, 2012; doi:10.1152/ajpheart.00126.2012.—The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual’s cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer. Review H630 CARDIOVASCULAR NONVIRAL GENE THERAPY Among the target genes that encode angiogenic factors used in clinical trials for CAD and PAD, VEGF (121 and 165 aminoacid isoforms of VEGF1 and VEGF2) and fibroblast growth factor (FGF, FGF1, and FGF4) families are relatively well investigated. VEGF and FGF in gene delivery have been shown to induce functionally significant angiogenesis in preclinical (19, 41) and clinical studies (53, 79, 91) for ischemic diseases. Although some studies showed no benefit or inconsistent results of growth factor gene therapy, designs may need to be further optimized with proper patient selection and growth factor delivery (40, 93). Another strategy investigated in angiogenic therapy is based on the transcription factor hypoxiainducible factor-1␣ (82) by which the expression of several angiogenic genes, including VEGF and the VEGF receptors KDR/FLT-1, are regulated. Additionally, a factor associated with regulation of angiogenesis is the peptide PR-39, which is a proline- and arginine-rich peptide and increases the cellular levels of hypoxia-inducible factor-1␣ by inhibiting its proteosomal degradation (2). Although it is possible that nonspecific action is related to charge (55), PR-39 has been shown to enhance the expression of VEGF, its receptors KDR and FLT-1, and the FGF receptor 1. For treatment of cardiovascular diseases, in addition to angiogenic genes, there are important nonangiogenic genes or nucleotides related to cell survival, such as genes associated with reduction-oxidation (redox) reactions and microRNAs (miRNAs). Redox regulation is an essential physiological process in the cell survival of almost all types of cells, including cardiomyocytes. Loss of cellular redox balance results in the development of oxidative stress in the cells, causing cellular dysfunction (12). There are critical antioxidant systems in cardiomyocytes to balance the oxidative stress: thioredoxin (Trx), glutaredoxin (Grx), and peroxiredoxin (Prx), all of which participate in redox regulation to protect the cells from oxidative stress and to antagonize apoptotic signaling (6). Overexpression of mitochondrial Prx-3 in mouse model showed resistance to ventricular remodeling and failure after myocardial infarction (68). Therapeutic strategies designed to attenuate mitochondrial oxidative stress such as enhanced expression of the antioxidant Prx-3 might be beneficial in preventing cardiac failure. Overexpression of both Grx-1 and Grx-2 rendered the heart resistant to ischemia-reperfusion injury and implicates a role in cardioprotection and redox signaling of the ischemic myocardium (66, 75). The above findings may suggest that Trx, Grx, and Prx are potential treatment targets for myocardial salvage in conditions associated with overwhelming oxidative stress. miRNAs consist of noncoding single-stranded RNAs of ⬃22 nucleotides that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. They have been implicated in the regulation of a diverse spectrum of cardiac functions, including cardiovascular cell differentiation, growth, proliferation, and apoptosis (50, 101). Among human miRNA genes, miRNA-21 was reported to be a mediator of extracellular signal-regulated kinase/mitogen-activated protein kinase signaling and is crucial for fibroblast survival and activation (43, 102). Further analysis showed that miRNA-21 is predominantly overexpressed in cardiac fibroblasts, especially those existing in the failing heart (83, 102). Moreover, in 2011, functional recovery and engraftment of transplanted cardiac progenitor cells that were transduced with lentivirus carrying a miRNA prosurvival cocktail (miRNA-21, AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 diseases (8.1%) (data adapted from clinical trials website of The Journal of Gene Medicine). Cardiovascular disease is among the leading cause of morbidity and mortality in the developed world. The first investigation of vascular gene transfer was demonstrated in 1989 by Nabel et al. (73), who transfected porcine endothelial cells ex vivo with a retrovirus carrying the -galactosidase gene and reintroduced the cells onto the denuded iliofemoral artery of a syngeneic pig. Since then, recombinant viruses have been used for gene transfer in most clinical studies. However, the death of a 18-year-old boy (54), who died of complications from a massive inflammatory response shortly after receiving a dose of adenovirus carrying a corrective gene in a phase I trial in 1999, illustrates the challenge in gene therapy. The mechanisms for this catastrophic reaction have never been completely understood, but a dose-specific adverse effect or a previous immunization due to a viral infection has been hypothesized as a potential cause. The future choice of viral or nonviral vectors will critically depend on specific purposes in gene therapy, potential adverse effects, and further improvements in vector design, though this review will focus on nonviral methods by which clinical or preclinical trials have shown feasibility into cardiovascular diseases. Among those diseases related to aging process, gene therapy may provide an alternative treatment option in peripheral artery disease (PAD) and coronary artery disease (CAD). Neovascularization that plays a critical role in PAD and CAD involves complex processes that include the coordination of cytokines to induce new conduits of blood supply. Among these events, angiogenesis involves enhanced vascular permeability, vascular cell migration and proliferation, and collateral vessel formation (34). In patients with ischemic cardiovascular diseases, progressive occlusion of arteries often results in the formation of collateral vessels that supply the ischemic tissues. However, the natural compensatory development of neovascularization is often incomplete partly due to insufficient availability of angiogenic factors. Therapeutic angiogenesis is a method based on direct administration of angiogenic agents such as recombinant protein, indirect delivery of target genes that encode relevant proteins, or use of stem cells that have been shown to secret angiogenic factors and enhance angiogenesis in preclinical and clinical studies (26, 37). Clinical trials of the vascular endothelial growth factor (VEGF) gene was started in 1994 by Isner et al. (37). Since then, several angiogenic cytokines have been investigated in clinical trials. Administration of proper angiogenic proteins with controllable therapeutic levels and pharmacokinetics are required in this regard. Since the limited half-life of angiogenic proteins restricts the action duration, development of slow-release delivery systems to prolong the action may be more effective. In addition, multifactor therapy may be necessary to achieve adequate angiogenesis and provide functionally significant improvements in human tissue perfusion because of the sophisticated angiogenic process (103). Regarding angiogenic gene therapy, induction of inflammatory and immune responses, low efficiency of gene expression, and nonspecific gene transfer to other cell types with uncontrolled levels of growth factor are limiting factors (51). Advantages of gene therapy in angiogenesis included persistent expression of the angiogenic factor with prolonged, local action. So far, several genes of growth factors and transcription factors have been tried to achieve therapeutic angiogenesis. Review CARDIOVASCULAR NONVIRAL GENE THERAPY -24, and -221) can be improved in a murine model of myocardial infarction (36). Therefore, miRNAs may be a gene therapy target to promote cell survival in heart failure by regulating other genes. Current Nonviral Gene-transfer Systems their key mechanisms, diseases/status, limitations, and advantages. Nonviral vectors. The safety concerns associated with viral vectors have encouraged the development of nonviral vectors. pDNA delivered by nonviral methods is maintained in an extrachromosomal site, not integrated into the cellular genome. The most popular materials used in current nonviral applications include purified pDNA, chemical vectors such as lipids and synthetic polymers. Nonviral methods of gene delivery used in current clinical trials include pDNA and liposomal complexes. DIRECT GENE TRANSFER USING PLASMID DNA. The simplest and safest nonviral gene delivery system currently in use in vivo is the direct gene transfer with naked pDNA. However, as the rapid degradation by nucleases and the clearance by the mononuclear phagocyte system in the systemic circulation, expression levels after the injection of naked DNA are generally limited. In 1990, Wolff et al. (108) first reported that intramuscularly injected naked pDNA can be expressed in myofibers. One of the promising approaches in this field is the combined use of naked DNA and physical approaches (such as electroporation) to enhance plasmid-mediated gene expression in muscle (20, 87). In the past 10 years, intramuscular injection of pDNA has been applied in a strategy of therapeutic angiogenesis (96). Isner and colleagues have used VEGF to enhance the formation of collateral blood vessels around occluded native arteries. Two catheter-based trials of myocardial gene delivery using pDNA encoding VEGF-2 have been reported (63, 105). In 2011, an open-label clinical study investigated the efficacy and safety of intramuscular injection of naked pDNA encoding the human hepatocyte growth factor (HGF) gene in Japanese patients with Buerger’s disease (89). The size of ischemic ulcers decreased in 6/9 (66.7%) patients, and the ulcers healed completely in 5/9 (55.6%) patients after gene therapy. The investigators concluded that HGF gene therapy is safe and effective for treating critical limb ischemia. LIPID-BASED GENE DELIVERY SYSTEMS. First reported by Felgner et al. in 1987 (23), lipid-based gene delivery is still one of the major systems to increase the TE of naked DNA (15). Liposomes or lipoplexes are formed by DNA with positively charged lipids and detergents (16, 60). In lipoplex structure, DNA backbones are surrounded with positively charged lipids to form condensed complexes that possibly protect them Fig. 1. Entry of plasmid into nucleus using nonviral gene delivery can be achieved either by physical methods, such as ultrasound (US) or electroporation, to increase cell permeability or via chemical vectors through endocytosis. Main steps impeding the nuclear accumulation of DNAvector complexes that need to be overcome include 1) cell binding, 2) cell entry, 3) endosomal escape, 4) degradation by endonucleases, 5) nuclear entry, and 6) decomplexation. In contrast, entry of plasmid DNA into nucleus by physical methods mainly encounters steps 2, 4, and 5. Note that both approaches required cytoplasmic transport between steps 2 and 5. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 Extra- and intracellular barriers for a successful gene delivery. A feasible gene therapy vector needs to meet three criteria: safety, adequate gene transfer efficiency, as well as stable expression of the transgene for a duration appropriate for treating the disease. Nonviral methods may only cause transient transfection and lack the ability to integrate into the genome that limit their use in conditions requiring long-term gene expression such as chronic heart failure. However, it is possible that transient expression of therapeutic genes can be favorable in certain applications. One such example is in bone healing, in which bone morphogenetic protein-2 just needs to express only for a few days because it triggers a cascade of activity that does not end even if it is withdrawn (56, 57). Moreover, nonviral vectors, due to anatomical and cellular barriers, limit the overall efficiency in gene therapy. There are biological obstacles impeding the nuclear accumulation of pDNA or DNA-vector complexes that need to be overcome: cell binding, cell entry/endocytosis, endosome escape, cytoplasmic transport, and nuclear entry (Fig. 1). Most of the chemical vectors compact DNA into particles by neutralizing the anionic DNA phosphate backbone and balancing charge repulsion force from the anionic cell surface (67). Considering a normal intracellular trafficking, after DNA-vector complexes interact with the cell membrane, they are endocytosed (97). This step may be nonspecific (mediated via electrostatic interactions) or may involve a specific target (using an antibody conjugates) (98). The vector should be designed to allow the DNA-vector complex to effectively escape from the endosome into the cytoplasm to protect it from the subsequent lysosomal degradation, and the vector should be capable of transporting its cargo to the nucleus. Additionally, there are many factors influencing the efficacy and safety of nonviral vector-mediated gene transfer, such as the charge of the vector, formulation of DNA/vector complexes (42), and interaction with serum and blood cells (85). Unfortunately, the ideal gene delivery systems are still under investigation. The potential nonviral vectors and physical approaches are summarized in Table 1 in terms of H631 Review H632 CARDIOVASCULAR NONVIRAL GENE THERAPY Table 1. A summary of nonviral gene delivery systems for treatment of cardiovascular diseases Delivery Methods Naked plasmid (VEGF, HGF, FGF) Chemical vectors Central Mechanism Advantages Limitations PAD and CAD (phase I/II)* Safety and simplicity Low transfection efficiency Liposomes PAD and CAD (phase I/II)* Safety Low to medium high-efficiency, immunogenicity Polymers Dendrimers In vitro and in vivo Low cytotoxicity Low immunogenicity Fair efficiency Complement activation, low efficiency Low efficiency Cytotoxicity Safety and flexibility Low efficiency Good efficiency and reproducibility Tissue damage, limited accessibility of electrodes to internal organs Shallow penetration of DNA into target tissues PLL PEI Physical methods Ultrasound ⫾ microbubble In vitro and in vivo Enhancement of cell membrane permeability Enhancement of cell membrane permeability Gene gun High-pressure helium stream Magnetofection Magnetic force plus endocytosis Good efficiency, transfer genes to nondividing cells Flexibility and low cytotoxicity Transient transfection See text for abbreviations. *Clinical trials completed or currently conducted. against extracellular or intracellular nucleases. The resulting net-positive charge of lipid-DNA complexes may facilitate fusion with the negatively charged molecules of the cell membrane (glycoproteins and proteoglycans) that may subsequently facilitate their cellular uptake by endocytosis (112). Many cationic molecules cannot form liposomes alone and are normally accompanied by a neutral lipid (colipid or helper lipids), such as dioleoylphosphatidylethanolamine (DOPE) (22). DOPE promotes hexagonal-phase lipid polymorphism that is related to membrane fusion and increase in TE in vitro (47). In addition, most of the cationic lipids are more or less toxic to cells, and the presence of DOPE could lead to reduced charge ratio and less toxicity. Cationic lipids generally have the advantages of being inexpensive and can be engineered to have targeted specificity. However, because of the excessive surface charge, the half-life of cationic lipids in circulation is very short. Elimination of cationic lipids in systemic circulation occurs upon the formation of larger aggregates because of their interactions with the negatively charged serum molecules. Lipoplexes tend to initially accumulate in the pulmonary vasculature because of the first passage effect (58). As a result, cells that are transfected are mainly pulmonary vascular and endothelial cells. The drawback of a fast clearance of cationic lipids from the circulation limits their use to transfer gene to cells located beyond vascular endothelial cells. Surface shielding or modification of DNA-vector complex through the use of hydrophilic and charge neutral polymers such as polyethylene glycol (PEG) to reduce excessive charge-charge interaction appears very effective in prolonging the circulation half-life of lipoplexes (32). However, it is noted that the presence of the PEG moiety on the surface of lipoplexes reduces an interaction between lipoplexes and cell membrane and, as a result, reduces the TE. Several strategies have been used to make the PEG shielding conditional and nonpermanent, such as PEG-lipid conjugates. Other drawbacks of lipoplex-mediated gene delivery are short duration of gene expression and acute toxicity. When combined with unmethylated cytosine-phosphate guanosine-containing DNA, cat- ionic lipids can stimulate potent inflammatory response in the hosts. Surface shielding with PEG-lipid and systematic deletions and mutations of cytosine-phosphate-guanosine-containing DNA sequences from plasmid sequences have resulted in some promising data in suppressing cytokine production (8). Delivery of growth factors by liposomes has been reported to be effective in animal models of angiogenesis (3, 65). In 2009, Hedman et al. (33) studied the long-term effects (a total follow-up time of 8.1 years) and safety of the local VEGF-A catheter-based gene transfer in 103 patients with CAD between three groups (adenoviral vector, liposome vector, or control) (33). The results showed that local intracoronary VEGF gene transfer is safe and does not cause an increased risk of major adverse cardiovascular events, cancer, or diabetes. More recently, in 2011, Muona et al. (71) examined the effects of VEGF on 25 patients having received VEGF-A gene transfer for the treatment of symptomatic PAD (71). There were no significant differences between study groups in the causes of death or in the incidence of cancer or diabetic retinopathy. Polymer-based gene delivery systems. The principle of cationic polymers as nonviral DNA vehicles is based on the concept of forming condensed DNA particles by complex formation with cationic polymers-polyplexes. The use of polycationic polymers leads to electrostatic neutralization of anionic charges of DNA and condenses the polynucleotide structure of DNA into nanosized complexes, thereby protecting it from nuclease digestion and facilitating the cellular uptake through endocytosis. Many polycationic molecules are used, including poly-Llysine (PLL), dendrimers, polyornithine, and polyethyleneimine (PEI). PEI and PLL are the commonest and most important ones used as nonviral vectors. PLL has been used to condense pDNA under various salt conditions (59). The PLLDNA particles have been shown to be protected against DNA degradation (28). Electron microscopic studies had demonstrated that PLL-DNA complexes assumes a rod-like appearance with a diameter of 15 nm and a length of 109 ⫾ 36 nm, AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 Electroporation Endocytosis Endocytosis DNA condensation Protein sponge effect Diseases/Status Review CARDIOVASCULAR NONVIRAL GENE THERAPY DNA-gold beads are cheap and easy to prepare. The gene gun delivery into the skin is a promising alternative to the injection of naked pDNA into muscle for genetic vaccinations (13). Several animal studies have reported that gene gun transfection of skin tissues can elicit immune responses to protect the animals from infection (27, 64). These immune responses have also been employed as a therapeutic approach for the treatment of cancer in human clinical trials (107). As a method to treat other disorders, Goudy et al. (30) have shown that vaccination in an animal model of type 1 diabetes with glutamic acid decarboxylase gene has induced type 2 immunity that resulted in blocking -cell autoimmunity (30). ELECTROPORATION. The application of controlled electric fields to facilitate cell permeabilization (electroporation) was first described in the 1960s (14). Extracellular molecules can pass through the cell membrane via the pores transiently generated by the electrical pulses. Since 1982, the use of electric pulses for cell electroporation has been used to introduce foreign DNA into cells in vitro and in vivo (77, 104). Skeletal muscle is a good candidate for electroporation (7). In general, DNA injection followed by electroporation showed 10- to 1,000-fold enhancements of expression compared with naked DNA injection alone but are accompanied by an elevation of serum creatine kinase levels (31). Indeed, one key limitation of electroporation is that a substantial procedurerelated damage may occur and, as a result, may limit the efficiency of transfection and its use in cardiovascular tissues (18, 69). In addition to electroporation followed by local injection, in vivo electroporation performed in a localized manner after a systemic injection of pDNA has been reported (84). Although in vivo electroporation technique is generally efficient when parameters are optimized and can produce good reproducibility, the key limitation is the accessibility of the electrodes to the internal organs and its clinical applications need further optimization. US-BASED TECHNOLOGY IN GENE DELIVERY. US waves are defined as mechanical sound waves that have a frequency above the audible sound of humans, generally about 20 kHz. Bioeffects of US. Among the physical effects of US, acoustic cavitation (4) is thought to be the most important bioeffect. Briefly, as the US waves propagate through the medium, the characteristic compression (Fig. 2) and rarefaction (Fig. 2) cause microscopic gas bubbles in the tissue fluid to contract and expand. Two types of cavitation are recognized. Gas body activation (61) or stable cavitation is the term used to describe bubbles that oscillate in diameter with the passing pressure Fig. 2. Dynamic size change of microbubbles (inertial cavitation) within an acoustic field of a continuouswave US. The rapid growth in size (rarefaction cycle) and the collapse of microbubbles (compression cycle) lead to the implosion of the microbubbles and local generation of heat, shock wave, free radical formation, and light, some of which provide the energy for sonoporation of cell membrane. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 much smaller than lipoplexes. The poor circulatory half-lives of PLL-DNA complexes, typically shorter than 3 min, also limit their use in vivo (110). In the recent years, PLL-PEG conjugates with defined chemical composition have been shown to improve delivery to different organs (113) and have been applied in phase I/II clinical trials for treatment of ocular degenerative diseases and cystic fibrosis (48). Generally, PLL or PLL-DNA complexes were reported to have low immunogenicity (92). Among cationic polymers, PEI, which is used in both linear and branched forms with molecular masses (MMs) between 0.7 and 800 kDa (29), has been one of the most effective polymer-based transfection agents. The polycationic PEI is receiving much attention because of its characteristic of condensing DNA with an intrinsic endosomolytic activity and has the ability to capture protons that are pumped into endolysosomes—“proton sponge” effect (9). This permits the escape of endocytosed PEI-DNA complexes. However, it is highly cytotoxic. Factors influencing cytotoxicity, TE, and the use for in vivo transfection include MM, incubation time, and the charge ratio of polymer to DNA used (25, 39). In general, high MM PEI (⬎25 kDa) is toxic to cells, whereas polymers with medium to low MM (5–25 kDa) are more efficient and less toxic (25). The toxic effect of PEI on cells can be partially reduced by conjugation with other polymers such as PEG (80). Upon systemic administration, these polyplexes of small particle size tend to aggregate to form larger complexes and accumulate in major tissues including lung and liver. So far, there have been no major clinical trials of cardiovascular diseases conducted by using cationic polymers alone. Physical approaches. To date, there are three major physical approaches of gene delivery-gene gun, electroporation and US by creating transient membrane holes using mechanical forces. Additionally, magnetofection, which uses magnetic force to concentrate particles containing nucleic acid into target cells, has been recently developed as a feasible nonviral method to combine current chemical and physical methods in gene delivery (35, 76, 81). DIRECT GENE TRANSFER BY GENE GUN. Gene gun (111) uses a high-pressure helium stream to deliver DNA, coated onto gold particles of varying sizes (1–3 m), directly into the cytoplasm. The efficiency of the gene gun is variable, and the duration of the expression is transient. A drawback of this tool is the shallow penetration of DNA into target tissue. The advantages of the gene gun, relative to some viral vectors, are that it can be used to transfer genes to nondividing cells and the H633 Review H634 CARDIOVASCULAR NONVIRAL GENE THERAPY transfection up to 18-fold and the combination of US and PEI synergistically increased transfection up to 200-fold, resulting in TF 34%. In 2010, our results showed that ultrasonic microbubble transfection causes phenotypic changes of primary vascular endothelial cells by enhancing proliferation and upregulating kinase insert domain-containing receptor and finslike tyrosine kinase-1, while possesses no obvious adverse effect on viable transfected cells (94). Although these are promising in vitro findings, US-based gene delivery is still in its infancy. Since 1996, there have been several in vivo investigations concerning US-assisted gene delivery. Most importantly, in 2002, Taniyama et al. (100), by using intramuscular injection of rat hindlimbs, reported a 10-fold increase in luciferase activity after injection of plasmid and Optison, followed by topical external US compared with plasmid alone. Subsequently, using HGF plasmid in a rabbit ischemic hindlimb model, they demonstrated that transfection with plasmid and Optison followed by US resulted in increased capillary formation in the treatment group after 5 wk. Therefore, ultrasonic microbubble-mediated HGF plasmid delivery could be feasible for safe clinical gene therapy to treat PAD without a viral vector system. Using intravenously infusion of microbubbles loaded with plasmids encoding the human VEGF165 gene into rat myocardial microcirculation, Korpanty et al. (49) performed US exposure along with microbubbles (49). The data showed that the capillary density increased to 18% at 5 days and 33% at 10 days before returning to control levels at 30 days. Theoretically, it is feasible to apply the US energy to deliver the growth factor gene into the ischemic tissue in clinical setting. The molecular mechanism of USassisted gene delivery in therapeutic angiogenesis remains to be determined. In 2011, Chen et al. (10), by using tail vein injections in mice, investigated the efficiency of in vivo gene transfer to the myocardium mediated by US-targeted liposome microbubble destruction accompanied by PEI. The significantly enhanced transgene expression was noted related to US-targeted microbubble destruction in the presence of PEI, and the results showed the feasibility of US-based method for primary myocardial disease. At present, there are two main hurdles as to why gene therapy has not globally succeeded in the clinical setting: first, inefficient delivery of gene of interest to their correct sites of action and, second, safety concern of some viral-based vectors that are one to three orders of magnitude more efficient than nonviral techniques in gene delivery in vivo. Many gene delivery methods are much less efficient in vivo than in vitro (such as liposome-mediated transfection). US is a versatile modality and has several potential advantages over other techniques, especially that it can be focused and in turn targeted to specific and, if necessary, to deep locations within the body along with its high safety profile, low costs, and repeatable applicability. Last but not least, in efforts to further improve the level of transgene expression, targeted gene delivery may be one of the promising methods that will work through US. In this regard, it would be feasible to design a target-specific microbubble that can selectively bind to the areas of interest in the tissue/body. These active targeting strategies can be achieved by the development of targeted microbubbles by attaching antibodies or peptides to microbubble shells (45, 95). Therefore, the targeted microbubbles with a specific ligand to the target receptors that are expressed in the diseased area can AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 variations of the sound wave. As the ultrasonic intensity increases, inertial cavitation (70) (Fig. 2) occurs when the amplitude of bubble oscillations enhances to a point where the inward moving wall of fluid has sufficient inertia that it cannot reverse direction when the acoustic pressure reverses and subsequently persists to compress the gas in the bubble. Finally, the bubble implodes violently, producing pressure discontinuities (shock waves), free radicals, microjets, extremely high-localized temperatures (at least 5,000 K), pressures (up to 1,200 bars), and light (Fig. 2). Formation of shock waves, bubble wall motion, and microjets may affect membrane permeability (62). Both stable and inertial cavitation may induce membrane permeabilization (100). Applications of US in gene delivery. US-mediated transient pore formation in the cell membranes (sonoporation) can be regarded to be the same as the promotion of membrane permeability induced by acoustic cavitation. Generally, the efficiency of sonoporation-mediated gene delivery depends on DNA concentration, the frequency and intensity of US exposure, the duration of exposure, and the presence of contrast agent. Most gene delivery studies use the therapeutic US at driving frequency of 1–3 MHz with an intensity of 0.5–2.5 W/cm2 (44). Microbubble echo contrast agents and their applications in gene delivery. The concept of US contrast imaging was introduced in the 1960s, and gene delivery using microbubbles has been intensively studied since 2000 (90). The ideal microbubble contrast agents should be nontoxic, injectable intravenously, capable of crossing the pulmonary capillary bed after a peripheral injection, and stable enough to achieve enhancement for the duration of the examination. They are typically gas-encapsulated microbubbles around 1–10 m in diameter (1). Contrast agents have a gas core that is filled with air or a higher MM substance such as perfluoropropane with lower aqueous solubility. The surrounding shell can be stiff (e.g., denatured albumin) or more flexible (lipid or phospholipids), and the shell thickness can vary from 10 to 200 nm. A critical improvement in US-assisted gene delivery was the combination of US irradiation with microbubbles. Most importantly, microbubbles have been shown to lower the energy threshold for cavitation by US energy and to have the potential of enhancing cavitation (24). When US interacts with the microbubbles leading to cavitation, pDNA are driven across cell membranes into the target cells (106). US-assisted gene delivery in vitro and in vivo. Naked pDNA is the simplest nonviral vector. However, disadvantages in systemic gene delivery with naked DNA have also been found, since pDNA vector can be rapidly degraded and neutralized by endogenous DNases. Therefore, it is reasonable to combine naked pDNA delivery with another method to improve the transgene efficiency. In 1987, Fechheimer et al. (21) first demonstrated that US had potential as a tool of pDNA delivery into murine fibroblasts. The first major research in this field came in 1996. Kim et al. (44) investigated the potential use of US exposure as a novel transfection method for experimental use. In 2000, Lawrie et al. (52), sonicating vascular smooth muscle cells in the presence of Optison and naked luciferase pGL3 plasmid (52), achieved transgene expression levels 300fold higher than with naked pDNA alone. In 2007, Deshpande and Prausnitz (17) performed US-assisted in vitro reporter gene transfection and found that US sonication alone increased Review CARDIOVASCULAR NONVIRAL GENE THERAPY H635 be applied either for the purpose of attaining US imaging or for a potentially therapeutic purpose via US-induced cavitation. Furthermore, since most of the major progress in US-based gene delivery involves microbubble cavitation by which a synergistic effect in enhancing US-assisted gene transfer occurs, further understanding of cavitation physics and US-microbubble-cell interactions cannot be overemphasized. Integrated Nonviral Gene Delivery into Cell Therapy: Cell-Based Gene Delivery Future Perspectives Gene therapy by growth factor gene delivery in therapeutic angiogenesis or nonangiogenic gene delivery for myocardial salvage is emerging as a potential strategy for the treatment of Fig. 3. Schematic presentation of the application of cell-based nonviral gene delivery technology in regenerative medicine. Therapeutic angiogenesis is achieved via paracrine effects and/or incorporation of transfected cells into tissues by implanting the cells using different methods. cardiovascular diseases. Viral-based gene delivery is efficient but safety aspects have usually limited its clinical applications (78). Various nonviral transfer approaches were developed to facilitate the delivery of transgenes into target cells without concern of insertion mutation, though dose optimization of nonviral vectors needs to be performed because of potential cytotoxicity. In the past few years, the work continued in improving delivery modalities with increased local distribution and reduced systemic circulation of chemical vectors. Naked DNA remains the preferred method of DNA transfer to cardiovascular ischemic tissues and has been explored extensively in clinical trials because of its simplicity and safety. However, insufficient gene delivery is still the major hurdle of plasmidmediated gene therapy. Improving plasmid-mediated gene transfer either by physical methods or chemical carriers to enhance cellular uptake and reduce susceptibility to circulating nucleases has shown successful applications for in vivo gene delivery. Further improvements to increase the efficiency and reduce the cytoxicity of nonviral vectors are required before their clinical implication. The potential interest will rely on developing new vector system that are more target-specific and a better understanding of the limiting steps that nonviral vector must overcome. Last but not least, although only short- and mid-term follow-up data are available, the methods and tech- AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 Gene transfer can be performed either by isolating stem cells from the patient, delivering the gene(s) into the cells in vitro and then transplanting the genetically modified stem cells back into the patient, or by directly introducing the delivery vector into the target sites in vivo. The in vivo approach is limited by nonspecific cellular targeting and inefficient gene delivery. This may explain that several clinical trials failed to show the benefit of in vivo growth factor gene therapy. With the recent rapid progress in molecular biology together with the steady advance of genome projects, gene and cell therapy has become an exciting new hope for the treatment of cardiovascular diseases. Cell-based gene delivery is a novel ex vivo strategy that uses autologous cells (such as endogenous stem cells and progenitor cells) as natural carrier vehicles of gene to the target site after in vitro transfection with a functional gene of interest (11) (Fig. 3). The use of adult progenitor cells, for example, endothelial progenitor cells (EPCs), as angiogenic substrate that is capable of synthesizing multiple cytokines is an attractive strategy for neovascularization of ischemic cardiovascular diseases (86, 99, 109). Genetic manipulation of EPCs to overexpress angiogenic genes may enhance their biological functions, such as migration, proliferation, and secretion of angiogenic growth factors (5, 46). The advantage of the ex vivo approach is to provide specific genetically modified cells that may directly participate in tissue regeneration and, most importantly, allow for both paracrine (mainly) and autocrine effects from the expressed cytokine (72, 88). In cell-based gene delivery, the EPCs serve not only as a vehicle for gene delivery to target sites but also as a tissueengineering tool in restoring normal tissues (74). By means of delivering autologous cells, cell-based gene delivery is capable of circumventing the unwanted inflammatory response, eliminating the possibility of insertional mutagenesis of other genes caused by viral vectors, and possibly achieving prolonged expression by transfection concomitantly using various nonviral methods including ultrasonic microbubble transfection, electroporation, and in vitro chemical vector transfection. However, in the long term, this approach needs to develop further steps to avoid contamination, to isolate, manipulate, and expand cells, as well as to develop less invasive strategies to enhance endogenous stem cell function and survival ex vivo. In the recent years, insights have been focused on gene transfer by means of cellular transplantation to induce neovascularization in ischemic tissue (38, 46). Genetically manipulated stem cells, in which a VEGF gene can be transferred by US or other physical methods ex vivo, may yield a promising future in therapeutic angiogenesis. Review H636 CARDIOVASCULAR NONVIRAL GENE THERAPY nology that merge nonviral gene and cell therapy such as delivery of proangiogenic plasmids with EPCs may give rise to several attractive opportunities to achieve more efficient and nontoxic strategies for gene therapy in regenerative medicine. GRANTS This study was supported by National Science Council (Taiwan) Grant 99-2314-B-195-010-MY2 and Medical Research Department of the Mackay Memorial Hospital (Taiwan) Grant MMH-E 100003. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS REFERENCES 1. Allen JS, May DJ, Ferrara KW. Dynamics of therapeutic ultrasound contrast agents. Ultrasound Med Biol 28: 805–816, 2002. 2. Anbanandam A, Albarado DC, Tirziu DC, Simons M, Veeraraghavan S. Molecular basis for proline- and arginine-rich peptide inhibition of proteasome. J Mol Biol 384: 219 –227, 2008. 3. Aoki M, Morishita R, Taniyama Y, Kida I, Moriguchi A, Matsumoto K, Nakamura T, Kaneda Y, Higaki J, Ogihara T. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets. Gene Ther 7: 417–427, 2000. 4. Apfel RE. Acoustic cavitation: a possible consequence of biomedical uses of ultrasound. Br J Cancer Suppl 45: 140 –146, 1982. 5. Asahara T, Kalka C, Isner JM. Stem cell therapy and gene transfer for regeneration. Gene Ther 7: 451–457, 2000. 6. Berndt C, Lillig CH, Holmgren A. Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system. Am J Physiol Heart Circ Physiol 292: H1227– H1236, 2007. 7. Bettan M, Emmanuel F, Darteil R, Caillaud JM, Soubrier F, Delaere P, Branelec D, Mahfoudi A, Duverger N, Scherman D. High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. Mol Ther 2: 204 –210, 2000. 8. Blume G, Cevc G. Liposomes for the sustained drug release in vivo. Biochim Biophys Acta 1029: 91–97, 1990. 9. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92: 7297–7301, 1995. 10. Chen ZY, Liang K, Qiu RX, Luo LP. Ultrasound- and liposome microbubble-mediated targeted gene transfer to cardiomyocytes in vivo accompanied by polyethylenimine. J Ultrasound Med 30: 1247–1258, 2011. 11. Cho HC, Marban E. Biological therapies for cardiac arrhythmias: can genes and cells replace drugs and devices? Circ Res 106: 674 –685, 2010. 12. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 48: 749 –762, 2010. 13. Condon C, Watkins SC, Celluzzi CM, Thompson K, Falo LD Jr. DNA-based immunization by in vivo transfection of dendritic cells. Nat Med 2: 1122–1128, 1996. 14. Coster HG. A quantitative analysis of the voltage-current relationships of fixed charge membranes and the associated property of “punchthrough”. Biophys J 5: 669 –686, 1965. 15. Dass CR. Lipoplex-mediated delivery of nucleic acids: factors affecting in vivo transfection. J Mol Med (Berl) 82: 579 –591, 2004. 16. Dauty E, Remy JS, Blessing T, Behr JP. Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in culture. J Am Chem Soc 123: 9227–9234, 2001. 17. Deshpande MC, Prausnitz MR. Synergistic effect of ultrasound and PEI on DNA transfection in vitro. J Control Release 118: 126 –135, 2007. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 C.-H.S. and H.-I.Y. conception and design of research; C.-H.S. performed experiments; C.-H.S., Y.-J.W., H.-H.W., and H.-I.Y. interpreted results of experiments; C.-H.S. drafted manuscript; C.-H.S. and H.-I.Y. edited and revised manuscript; H.-I.Y. approved final version of manuscript. 18. Durieux AC, Bonnefoy R, Busso T, Freyssenet D. In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage. J Gene Med 6: 809 –816, 2004. 19. Faries PL, Pomposelli FB Jr, Quist WC, LoGerfo FW. Assessing the role of gene therapy in the treatment of vascular disease. Ann Vasc Surg 14: 181–188, 2000. 20. Fattori E, La Monica N, Ciliberto G, Toniatti C. Electro-genetransfer: a new approach for muscle gene delivery. Somat Cell Mol Genet 27: 75–83, 2002. 21. Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG. Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 84: 8463–8467, 1987. 22. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269: 2550 –2561, 1994. 23. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84: 7413–7417, 1987. 24. Feril LB Jr, Kondo T, Zhao QL, Ogawa R, Tachibana K, Kudo N, Fujimoto S, Nakamura S. Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents. Ultrasound Med Biol 29: 331–337, 2003. 25. Fischer D, Bieber T, Li Y, Elsasser HP, Kissel T. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res 16: 1273–1279, 1999. 26. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 333: 1757–1763, 1995. 27. Fuller DH, Murphey-Corb M, Clements J, Barnett S, Haynes JR. Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination. J Med Primatol 25: 236 –241, 1996. 28. Gao X, Huang L. Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry (Mosc) 35: 1027–1036, 1996. 29. Godbey WT, Wu KK, Mikos AG. Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J Biomed Mater Res 45: 268 –275, 1999. 30. Goudy KS, Wang B, Tisch R. Gene gun-mediated DNA vaccination enhances antigen-specific immunotherapy at a late preclinical stage of type 1 diabetes in nonobese diabetic mice. Clin Immunol 129: 49 –57, 2008. 31. Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M, Nishioka WK, Wheeler CJ, Manthorp M, Sawdey M. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188. Mol Ther 4: 407–415, 2001. 32. Harvie P, Wong FM, Bally MB. Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles. J Pharm Sci 89: 652–663, 2000. 33. Hedman M, Muona K, Hedman A, Kivela A, Syvanne M, Eranen J, Rantala A, Stjernvall J, Nieminen MS, Hartikainen J, Yla-Herttuala S. Eight-year safety follow-up of coronary artery disease patients after local intracoronary VEGF gene transfer. Gene Ther 16: 629 –634, 2009. 34. Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. J Cell Mol Med 10: 45–55, 2006. 35. Holzbach T, Vlaskou D, Neshkova I, Konerding MA, Wortler K, Mykhaylyk O, Gansbacher B, Machens HG, Plank C, Giunta RE. Non-viral VEGF(165) gene therapy—magnetofection of acoustically active magnetic lipospheres (’magnetobubbles’) increases tissue survival in an oversized skin flap model. J Cell Mol Med 14: 587–599, 2010. 36. Hu S, Huang M, Nguyen PK, Gong Y, Li Z, Jia F, Lan F, Liu J, Nag D, Robbins RC, Wu JC. Novel microRNA prosurvival cocktail for improving engraftment and function of cardiac progenitor cell transplantation. Circulation 124: S27–S34, 2011. 37. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 348: 370 –374, 1996. Review CARDIOVASCULAR NONVIRAL GENE THERAPY 59. Liu G, Molas M, Grossmann GA, Pasumarthy M, Perales JC, Cooper MJ, Hanson RW. Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation. J Biol Chem 276: 34379 –34387, 2001. 60. Lleres D, Dauty E, Behr JP, Mely Y, Duportail G. DNA condensation by an oxidizable cationic detergent. Interactions with lipid vesicles. Chem Phys Lipids 111: 59 –71, 2001. 61. Lohse D. Sonoluminescence: cavitation hots up. Nature 434: 33–34, 2005. 62. Lokhandwalla M, McAteer JA, Williams JC Jr, Sturtevant B. Mechanical haemolysis in shock wave lithotripsy (SWL): II. In vitro cell lysis due to shear. Phys Med Biol 46: 1245–1264, 2001. 63. Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation 105: 2012–2018, 2002. 64. Lunn DP, Soboll G, Schram BR, Quass J, McGregor MW, Drape RJ, Macklin MD, McCabe DE, Swain WF, Olsen CW. Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene. Vaccine 17: 2245–2258, 1999. 65. Makinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava E, Yla-Herttuala S. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 6: 127–133, 2002. 66. Malik G, Nagy N, Ho YS, Maulik N, Das DK. Role of glutaredoxin-1 in cardioprotection: an insight with Glrx1 transgenic and knockout animals. J Mol Cell Cardiol 44: 261–269, 2008. 67. Manning GS. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q Rev Biophys 11: 179 –246, 1978. 68. Matsushima S, Ide T, Yamato M, Matsusaka H, Hattori F, Ikeuchi M, Kubota T, Sunagawa K, Hasegawa Y, Kurihara T, Oikawa S, Kinugawa S, Tsutsui H. Overexpression of mitochondrial peroxiredoxin-3 prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation 113: 1779 –1786, 2006. 69. McMahon JM, Signori E, Wells KE, Fazio VM, Wells DJ. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase—increased expression with reduced muscle damage. Gene Ther 8: 1264 –1270, 2001. 70. Miller MW, Miller DL, Brayman AA. A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 22: 1131–1154, 1996. 71. Muona K, Makinen K, Hedman M, Manninen H, Yla-Herttuala. 10-year safety follow-up in patients with local VEGF gene transfer to ischemic lower limb. Gene Ther 19: 392–395, 2012. 72. Murayama T, Tepper OM, Silver M, Ma H, Losordo DW, Isner JM, Asahara T, Kalka C. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo. Exp Hematol 30: 967–972, 2002. 73. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ. Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 244: 1342–1344, 1989. 74. Nagaya N, Kangawa K, Kanda M, Uematsu M, Horio T, Fukuyama N, Hino J, Harada-Shiba M, Okumura H, Tabata Y, Mochizuki N, Chiba Y, Nishioka K, Miyatake K, Asahara T, Hara H, Mori H. Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 108: 889 – 895, 2003. 75. Nagy N, Malik G, Tosaki A, Ho YS, Maulik N, Das DK. Overexpression of glutaredoxin-2 reduces myocardial cell death by preventing both apoptosis and necrosis. J Mol Cell Cardiol 44: 252–260, 2008. 76. Namgung R, Singha K, Yu MK, Jon S, Kim YS, Ahn Y, Park IK, Kim WJ. Hybrid superparamagnetic iron oxide nanoparticle-branched polyethylenimine magnetoplexes for gene transfection of vascular endothelial cells. Biomaterials 31: 4204 –4213, 2010. 77. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1: 841–845, 1982. 78. Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, Libby P, Dichek DA. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 38. Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM, Asahara T. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 105: 732–738, 2002. 39. Jeong JH, Song SH, Lim DW, Lee H, Park TG. DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline). J Control Release 73: 391–399, 2001. 40. Kastrup J, Jorgensen E, Ruck A, Tagil K, Glogar D, Ruzyllo W, Botker HE, Dudek D, Drvota V, Hesse B, Thuesen L, Blomberg P, Gyongyosi M, Sylven C. Direct intramyocardial plasmid vascular endothelial growth factor-A165 gene therapy in patients with stable severe angina pectoris A randomized double-blind placebo-controlled study: the Euroinject One trial. J Am Coll Cardiol 45: 982–988, 2005. 41. Khan TA, Sellke FW, Laham RJ. Therapeutic angiogenesis for coronary artery disease. Curr Treat Options Cardiovasc Med 4: 65–74, 2002. 42. Kichler A, Zauner W, Ogris M, Wagner E. Influence of the DNA complexation medium on the transfection efficiency of lipospermine/ DNA particles. Gene Ther 5: 855–860, 1998. 43. Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 1802: 396 –405, 2010. 44. Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME. Ultrasound-mediated transfection of mammalian cells. Hum Gene Ther 7: 1339 –1346, 1996. 45. Klibanov AL. Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem 16: 9 –17, 2005. 46. Kobayashi K, Kondo T, Inoue N, Aoki M, Mizuno M, Komori K, Yoshida J, Murohara T. Combination of in vivo angiopoietin-1 gene transfer and autologous bone marrow cell implantation for functional therapeutic angiogenesis. Arterioscler Thromb Vasc Biol 26: 1465–1472, 2006. 47. Koltover I, Salditt T, Radler JO, Safinya CR. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science 281: 78 –81, 1998. 48. Konstan MW, Davis PB, Wagener JS, Hilliard KA, Stern RC, Milgram LJ, Kowalczyk TH, Hyatt SL, Fink TL, Gedeon CR, Oette SM, Payne JM, Muhammad O, Ziady AG, Moen RC, Cooper MJ. Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution. Hum Gene Ther 15: 1255–1269, 2004. 49. Korpanty G, Chen S, Shohet RV, Ding J, Yang B, Frenkel PA, Grayburn PA. Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles. Gene Ther 12: 1305–1312, 2005. 50. Kumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and disease. RNA Biol 8: 706 –713, 2011. 51. Laham RJ, Simons M, Sellke F. Gene transfer for angiogenesis in coronary artery disease. Annu Rev Med 52: 485–502, 2001. 52. Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, Newman CM. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 7: 2023–2027, 2000. 53. Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia AN, Hermiller JB, Hillegass WB, Rocha-Singh K, Moon TE, Whitehouse MJ, Annex BH. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet 359: 2053–2058, 2002. 54. Lehrman S. Virus treatment questioned after gene therapy death. Nature 401: 517–518, 1999. 55. Li J, Post M, Volk R, Gao Y, Li M, Metais C, Sato K, Tsai J, Aird W, Rosenberg RD, Hampton TG, Sellke F, Carmeliet P, Simons M. PR39, a peptide regulator of angiogenesis. Nat Med 6: 49 –55, 2000. 56. Lin CY, Chang YH, Kao CY, Lu CH, Sung LY, Yen TC, Lin KJ, Hu YC. Augmented healing of critical-size calvarial defects by baculovirusengineered MSCs that persistently express growth factors. Biomaterials 33: 3682–3692, 2012. 57. Lin CY, Lin KJ, Kao CY, Chen MC, Lo WH, Yen TC, Chang YH, Hu YC. The role of adipose-derived stem cells engineered with the persistently expressing hybrid baculovirus in the healing of massive bone defects. Biomaterials 32: 6505–6514, 2011. 58. Liu F, Qi H, Huang L, Liu D. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther 4: 517–523, 1997. H637 Review H638 79. 80. 81. 82. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. inflammation, and neointimal hyperplasia. J Clin Invest 96: 2955–2965, 1995. Nikol S, Baumgartner I, Van Belle E, Diehm C, Visona A, Capogrossi MC, Ferreira-Maldent N, Gallino A, Wyatt MG, Wijesinghe LD, Fusari M, Stephan D, Emmerich J, Pompilio G, Vermassen F, Pham E, Grek V, Coleman M, Meyer F. Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia. Mol Ther 16: 972–978, 2008. Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6: 595–605, 1999. Plank C, Zelphati O, Mykhaylyk O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Adv Drug Deliv Rev 63: 1300 –1331, 2011. Rajagopalan S, Olin J, Deitcher S, Pieczek A, Laird J, Grossman PM, Goldman CK, McEllin K, Kelly R, Chronos N. Use of a constitutively active hypoxia-inducible factor-1alpha transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase I dose-escalation experience. Circulation 115: 1234 –1243, 2007. Roy S, Khanna S, Hussain SR, Biswas S, Azad A, Rink C, Gnyawali S, Shilo S, Nuovo GJ, Sen CK. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue. Cardiovasc Res 82: 21–29, 2009. Sakai M, Nishikawa M, Thanaketpaisarn O, Yamashita F, Hashida M. Hepatocyte-targeted gene transfer by combination of vascularly delivered plasmid DNA and in vivo electroporation. Gene Ther 12: 607–616, 2005. Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, Hashida M. Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid. Gene Ther 8: 677–686, 2001. Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Dimmeler S, Zeiher AM. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 44: 1690 –1699, 2004. Scherman D, Bigey P, Bureau MF. Applications of plasmid electrotransfer. Technol Cancer Res Treat 1: 351–354, 2002. Shi Q, Wu MH, Fujita Y, Ishida A, Wijelath ES, Hammond WP, Wechezak AR, Yu C, Storb RF, Sauvage LR. Genetic tracing of arterial graft flow surface endothelialization in allogeneic marrow transplanted dogs. Cardiovasc Surg 7: 98 –105, 1999. Shigematsu H, Yasuda K, Sasajima T, Takano T, Miyata T, Ohta T, Tanemoto K, Obitsu Y, Iwai T, Ozaki S, Ogihara T, Morishita R. Transfection of human HGF plasmid DNA improves limb salvage in Buerger’s disease patients with critical limb ischemia. Int Angiol 30: 140 –149, 2011. Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH, Grayburn PA. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 101: 2554 –2556, 2000. Shyu KG, Chang H, Wang BW, Kuan P. Intramuscular vascular endothelial growth factor gene therapy in patients with chronic critical leg ischemia. Am J Med 114: 85–92, 2003. Stankovics J, Crane AM, Andrews E, Wu CH, Wu GY, Ledley FD. Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/polylysine/DNA complexes. Hum Gene Ther 5: 1095–1104, 1994. Stewart DJ, Kutryk MJ, Fitchett D, Freeman M, Camack N, Su Y, Della Siega A, Bilodeau L, Burton JR, Proulx G, Radhakrishnan S. VEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial. Mol Ther 17: 1109 –1115, 2009. Su CH, Chang CY, Wang HH, Wu YJ, Bettinger T, Tsai CH, Yeh HI. Ultrasonic microbubble-mediated gene delivery causes phenotypic 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. changes of human aortic endothelial cells. Ultrasound Med Biol 36: 449 –458, 2010. Su CH, Yeh HI, Hou JY, Tsai CH. Nonviral technologies for gene therapy in cardiovascular research. Int J Gerontol 2: 35–47, 2008. Takeshita S, Isner JM. Peripheral angiogenesis: therapeutic angiogenesis for peripheral vascular occlusive disease. Curr Interv Cardiol Rep 1: 147–156, 1999. Tan PH, Chan CL, George AJ. Strategies to improve non-viral vectors—potential applications in clinical transplantation. Expert Opin Biol Ther 6: 619 –630, 2006. Tan PH, Manunta M, Ardjomand N, Xue SA, Larkin DF, Haskard DO, Taylor KM, George AJ. Antibody targeted gene transfer to endothelium. J Gene Med 5: 311–323, 2003. Tanaka K, Sata M. Therapeutic application of bone marrow-derived progenitor cells for vascular diseases: magic bullets having the good without the bad? Int J Gerontol 1: 10 –21, 2007. Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9: 372–380, 2002. Thum T, Catalucci D, Bauersachs J. MicroRNAs: novel regulators in cardiac development and disease. Cardiovasc Res 79: 562–570, 2008. Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M, Galuppo P, Just S, Rottbauer W, Frantz S, Castoldi M, Soutschek J, Koteliansky V, Rosenwald A, Basson MA, Licht JD, Pena JT, Rouhanifard SH, Muckenthaler MU, Tuschl T, Martin GR, Bauersachs J, Engelhardt S. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature 456: 980 –984, 2008. Tirziu D, Simons M. Angiogenesis in the human heart: gene and cell therapy. Angiogenesis 8: 241–251, 2005. Titomirov AV, Sukharev S, Kistanova E. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1088: 131–134, 1991. Vale PR, Losordo DW, Milliken CE, McDonald MC, Gravelin LM, Curry CM, Esakof DD, Maysky M, Symes JF, Isner JM. Randomized, single-blind, placebo-controlled pilot study of catheter-based myocardial gene transfer for therapeutic angiogenesis using left ventricular electromechanical mapping in patients with chronic myocardial ischemia. Circulation 103: 2138 –2143, 2001. Vannan M, McCreery T, Li P, Han Z, Unger E, Kuersten B, Nabel E, Rajagopalan S. Ultrasound-mediated transfection of canine myocardium by intravenous administration of cationic microbubble-linked plasmid DNA. J Am Soc Echocardiogr 15: 214 –218, 2002. Weiner DB, Kennedy RC. Genetic vaccines. Sci Am 281: 50 –57, 1999. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transfer into mouse muscle in vivo. Science 247: 1465–1468, 1990. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364: 141–148, 2004. Xu B, Wiehle S, Roth JA, Cristiano RJ. The contribution of poly-Llysine, epidermal growth factor and streptavidin to EGF/PLL/DNA polyplex formation. Gene Ther 5: 1235–1243, 1998. Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 87: 9568 –9572, 1990. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 270: 18997–19007, 1995. Ziady AG, Gedeon CR, Miller T, Quan W, Payne JM, Hyatt SL, Fink TL, Muhammad O, Oette S, Kowalczyk T, Pasumarthy MK, Moen RC, Cooper MJ, Davis PB. Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo. Mol Ther 8: 936 –947, 2003. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00126.2012 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 15, 2017 83. CARDIOVASCULAR NONVIRAL GENE THERAPY
© Copyright 2024 Paperzz