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Ann Thorac Surg 1995;60:721-728
© 1995 The Society of Thoracic Surgeons

Current Review

Potential Gene Therapy Strategies in the Treatment of Cardiovascular Disease

Robert T. Rowland, MD, Joseph C. Cleveland, Jr, MD, Xianzhong Meng, MD, Alden H. Harken, MD, James M. Brown, MD

Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
Gene therapy is the introduction of new genetic material into somatic cells to synthesize missing or defective proteins. Efficient methods for the introduction of genetic material into cells are available, both in vitro and in vivo. These strategies involve chemical, physical, and viral-mediated mechanisms of gene transfer. Application of these gene transfer techniques has led to the development of potential gene-based treatment strategies that could combat vascular and myocardial disease. Gene therapy in the treatment of cardiovascular disease promises to alter atherosclerotic risk factors, prevent vascular thrombotic disease, retard progression of disease in the peripheral vasculature, provide drug delivery systems, and prevent myocardial infarction in patients with coronary artery disease. This exciting technology will eventually become the ultimate intervention in the treatment of cardiovascular disease.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
Gene therapy is the introduction of normal genes into the somatic cells of patients to correct an inherited or acquired disorder through the synthesis of missing or defective gene products in vivo [1]. There has been intense investigation in cardiovascular medicine involving genetic manipulation and gene therapy. Cardiovascular disease is the leading cause of mortality in the United States, and although current surgical and interventional therapies are efficacious in many clinical settings, they do not attack the basic cause of the disease [2]. Gene therapy has progressed in many areas of cardiovascular medicine including hypercholesterolemia, atherosclerosis, and peripheral vascular and coronary artery disease such that practical intervention is now a realistic possibility.

For editorial comment, see 497

The first successful clinical gene transfer into a human took place in 1989, when cancer patients were transfused with genetically marked tumor infiltrating lymphocytes [3]. The lymphocytes were marked with a neomycin resistance gene from Escherichia coli, demonstrating that an exogenous gene could be safely transferred into a human. The therapeutic efficacy of gene therapy was subsequently revealed in patients with adenosine deaminase deficient severe combined immunodeficiency disease [4]. Lymphocytes were isolated, transfected with the adenosine deaminase deficient gene ex vivo, and returned to the patient, resulting in restoration of adenosine deaminase deficient activity and clinical improvement. Gene therapy has also been evaluated in the treatment of cancer. Tumor necrosis factor was inserted into the neomycin-resistant gene construct described above, resulting in more aggressive tumor infiltrating lymphocytes against advanced malignant melanoma cells [5]. Somatic cell gene therapy has since become a reality with more than 43 currently approved clinical protocols in the United States [6], providing genetic technology that promises to change the way cardiovascular disease is treated. The purposes of this review are (1) to describe currently available techniques permitting the introduction of potentially therapeutic genetic material into mammalian cells, (2) to examine the success of gene therapy in the treatment of hypercholesterolemia and eventually atherosclerosis, (3) to examine the success of gene transfer into endothelial and vascular smooth muscle cells, and (4) to explore the potential of gene transfer and genetic manipulation of coronary artery endothelium and myocardium.


   Recombinant DNA and the Transfection Process
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
Before transferring DNA into a host cell, a number of steps must take place to isolate a desired segment of DNA encoding a single protein (gene). These steps involve removing DNA from a cell and combining it with other DNA that will allow replication and expression of the genetic material [7–9]. Extraction of nucleic acids is accomplished by both cell lysis using detergents and digestion of proteins using nonspecific proteases. The extracted DNA is then cleaved into fragments at specific sites using a restriction endonuclease, an enzyme that cuts the DNA at specific base-pair sequences. After restriction enzyme digestion, gel electrophoresis is used to separate the DNA fragments. The migration of these fragments through the gel is governed by their length, whereby larger fragments move slower and travel less distance, allowing separation from smaller DNA segments.

For DNA to replicate, a sequence must be present that initiates replication. This initiation sequence is known as a replicon. Fragments of DNA do not typically contain replicons, and in the absence of replication they will be diluted out of host cells. Furthermore, even if the DNA fragment contains an initiation sequence, it may not function in a foreign host cell [8]. The nucleic acid fragment must therefore be attached to a suitable initiation sequence that permits expression of the DNA. This initiation sequence is known as a vector and represents a genetic construct used to propagate foreign DNA in a host cell. The vector DNA is isolated, purified, and cut using a restriction endonuclease that opens the vector and allows for the insertion of foreign nucleic acid. The DNA fragment is then inserted into the open vector after incubation with DNA ligase (Fig 1Go). The combination of foreign DNA inserts with vector DNA produces a chimera called a recombinant DNA molecule or artificial recombinant [9]. The recombinant DNA is then introduced into a host cell, where replication and expression of the genetic material takes place, resulting in modification of the cell phenotype.



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  		Fig 1. . Schematic diagram representing the construction of a recombinant DNA molecule. The vector DNA is opened with a specific restriction endonuclease and following incubation with the DNA fragment and the enzyme DNA ligase, a new DNA molecule is created containing the nucleic acid fragment and the gene of interest.

 
Gene modification of eukaryotic cells can be performed using three different approaches: gene replacement, gene correction, and gene augmentation [1, 10]. Gene replacement and gene correction involve altering host genes either through removing abnormal sequences and replacing them with functional genetic material or correcting the defective portion of a mutant gene, returning it to normal function. Gene augmentation introduces foreign genetic sequences into the host genome without altering defective host genes. Augmentation is the basis of current gene transfer systems, and the introduction of genes into somatic cells includes two distinct strategies: ex vivo/indirect transfer and in vivo/direct gene transfer [11, 12]. The ex vivo technique requires removal and culture of host cells in vitro. A therapeutic gene is then inserted into the cultured cells, and the genetically modified cells are returned to the host. In vivo gene transfer does not require ex vivo manipulation but instead involves direct in situ introduction of therapeutic genes into specific host tissues. In vivo transfer is technically easier and is accomplished in a single procedure, whereas the ex vivo approach allows targeting of specific cells and monitoring of potentially unwanted characteristics.

Once the strategy for introducing genetic material into the cells has been selected, a number of methods are available for transferring DNA into mammalian cells. The insertion of exogenous genes into cells is known as transfection or transduction and uses both viral and nonviral methods. Transfection of cells involves the delivery of DNA through chemical, physical, or virus-dependent mechanisms, whereas transduction refers to the transfer of genetic material using retroviral vectors [13]. Chemical and physical gene transfer methods allow for transient expression of DNA in host cells. Viral vectors have been developed and have improved the efficiency of gene delivery into cells. Retroviruses and adenoviruses are viral vectors currently in use, but successful transfection has also been accomplished with other DNA viruses [14, 15].

Although each method of delivery has its own advantages and disadvantages, they must share similar characteristics to be effective. Gene delivery systems must be able to efficiently transfect a large number of cells, provide stable replication of foreign DNA, have appropriate and regulated expression in the target tissue, and be adequately safe over the time of transfer and the life of the host [1]. Table 1Go describes some of the major advantages and disadvantages of each of these vector systems [1, 10, 11, 13, 16, 17], and although a detailed description of each delivery method is beyond the scope of this review, a brief overview is listed below.


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  		Table 1. . Advantages and Disadvantages of Gene Delivery Methods Into Mammalian Cells
 
Chemical Methods of Gene Delivery
Chemical methods of gene transfer include calcium phosphate co-precipitation, DEAE-dextran, and liposome-mediated transfer. Calcium phosphate transfection is one of the most commonly used in vitro gene transfer techniques [9]. Recombinant DNA is co-precipitated with calcium phosphate and added to cell culture, where it enters the cells by endocytosis. Sufficient quantities of DNA enter the nucleus, allowing genetic and phenotypic transformation. Although commonly used in vitro, the calcium phosphate method is limited by a low rate of cell transfection [18]. Recombinant DNA can also be complexed with polycations such as DEAE-dextran, which promotes incorporation of foreign DNA into cells in vitro. Although transfection is more efficient relative to calcium phosphate co-precipitation, the transferred DNA is less stable and is degraded more rapidly [16].

Liposomes are cationic lipid carriers of pharmacologically active ingredients, and DNA incorporated into liposomes provides another means of transfection. Liposomes enter cells by fusion, endocytosis, or local plasma membrane destabilization. Once intracellular, the DNA reaches the nucleus resulting in gene expression (Fig 2Go). Liposomes provide efficient transfection and stable replication in vivo [19]. Chemical gene delivery provides stable in vitro gene expression, but liposomes have extended this advantage into the in vivo environment. However, one disadvantage all three chemical gene delivery methods have in common is the lack of cell specificity such that DNA is introduced into all cells exposed to the delivery vehicle [13]. Nontargeted delivery is a major reason why these methods are mainly used in the in vitro setting. To focus transfection to specific cell types in vivo, these cells must be anatomically isolated such that only desired cells are exposed to the chemical vector.



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  		Fig 2. . Retroviral, adenoviral and liposome-mediated gene transfer into the mammalian cell: 1 = integration into host cell chromosomal DNA, 2 = transcription, 3 = translation, and 4 = reverse transcriptase. (A) Gene transfer using a replication-deficient retrovirus. The virus binds to specific cell surface receptors and fuses with the plasma membrane, whereby the viral genetic material is released into the cytoplasm. The single-stranded viral RNA then undergoes reverse transcription to form double-stranded DNA that is transported to the nucleus and integrated into the host cell chromosomal DNA. The new gene is transcribed with the creation of a messenger RNA, which is translated into the protein product of interest. (B) Gene transfer using a replication-deficient adenovirus. The adenovirus binds to the cell surface and is taken into the cell by an endosome. The adenovirus then disrupts the endosome and the viral DNA is released into the cell cytoplasm. The DNA is transported into the nucleus where it undergoes RNA transcription. Translation of the messenger RNA then leads to expression of the gene of interest. (C) Gene transfer using liposomes. The DNA is contained within a cationic-lipid liposome and subsequently enters the cell by local membrane destabilization or via an endocytotic mechanism. The DNA then becomes localized in the nucleus, where expression of the desired gene takes place.

 
Physical Methods of Gene Transfer
Physical methods of gene transfer include microinjection and electroporation. Direct microinjection of plasmid or ``naked'' DNA directly into the cell nucleus can be achieved using special equipment under microscopic control [20]. The DNA bypasses the cytoplasm and thus avoids degradation in cellular compartments. This eliminates the use of a carrier system and provides stable expression but has little practical application because microinjection cannot be applied to a large scale [13]. Electroporation is another physical method of gene transfer whereby DNA is incorporated into cells after disruption of the plasma membrane using high electrical voltage. The brief electrical pulses produce nanometer-sized pores allowing for passage of the DNA vector. This method is commonly used for cell transfection in vitro and appears to have a high transfer efficiency in all cell types. In addition, closure of electrically induced pores is delayed at lower temperatures, thus increasing the probability that the DNA of interest will enter the cell [16, 21].

Viral Methods of Gene Delivery
As an alternative to chemical and physical means, viral-dependent methods of gene transfer have been developed that improve the efficiency of delivery into mammalian cells. Retroviral and adenoviral vectors are currently the most extensively used viral delivery systems, and both have been used for indirect and direct gene transfer. The retroviruses are useful because they have a known genetic structure and efficiently infect cells of multiple species. These RNA viruses contain reverse transcriptase that allows replication through a double-stranded DNA intermediate form. The DNA is then transported to the nucleus and is integrated at a random site into the host DNA. Retroviral-mediated gene transfer requires that replication-deficient viral particles be made that carry therapeutic genes [11]. A retroviral vector is constructed by deleting the structural genes required for viral replication (the gag gene encoding specific antigens, the pol gene encoding reverse transcriptase, and the env gene encoding the envelope protein) [22]. The gene of interest is then inserted into the deleted genome of the virus, downstream from a promoter sequence that allows expression of the gene. The retrovirus cannot replicate once it infects a cell but the replicated double-stranded DNA is integrated and subsequently translated, producing the protein product of interest without translation of viral proteins (see Fig 2Go). The integrated DNA is maintained in the host genome after cell division, allowing for gene expression over an extended period of time. Limitations of retroviral vectors include the necessity of target cell replication for integration of the recombinant viral genome as well as the potential to generate replication-competent virus [23]. Despite these limitations, replication-deficient retroviral vectors have been used extensively in cardiovascular gene therapy research.

The adenovirus, a large DNA virus, has also been modified to deliver genes into mammalian cells [24]. Portions of the E1 region of the viral genome that contain information necessary for viral replication are deleted creating a replication-incompetent virus. A gene of interest is then inserted into this genome, producing a vector for gene transfer. Once the adenovirus binds to the cell membrane and is endocytosed, the virus can disrupt the endosome leading to DNA release. The DNA then enters the nucleus, where it can only undergo transcription into messenger RNA (due to deletion of viral replication genes) and subsequent translation of desired proteins (see Fig 2Go). In contrast to the retrovirus, the adenovirus does not require host cell replication for gene expression because integration of the viral genome is not a component of the adenoviral life cycle. The adenovirus efficiently infects nondividing cells and expresses large amounts of gene products. However, this life cycle creates a disadvantage because the gene of interest cannot replicate with the cell and is lost during cell division. Furthermore, viral structural proteins are synthesized that are potentially hazardous to host cells and there is a tendency for adenoviral vectors to generate replication-competent virus that may infect and lyse host cells. Herpesvirus, adeno-associated virus, vaccinia virus, and influenza virus have also been engineered to deliver genes into mammalian cells but disadvantages such as host cell cytotoxicity, chromosomal rearrangements, or significant immune responses have limited their use [13–15].

In summary, different methods of gene transfer are available and certain advantages and disadvantages exist for each. This technology has been applied to animal models to create potential strategies to treat diseases of the cardiac and vascular systems. The following sections describe potential gene-based strategies to treat cardiovascular disease, specifically hypercholesterolemia and atherosclerosis, peripheral vascular disease, and ischemic heart disease.


   Gene Therapy of Hypercholesterolemia/ Atherosclerosis
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
Familial hypercholesterolemia is an inherited disorder caused by a deficiency of low-density lipoprotein receptors (LDLR) [25]. Individuals with familial hypercholesterolemia often have development of premature coronary artery disease and peripheral vascular disease. Potential new treatments for familial hypercholesterolemia have been developed through the use of molecular techniques and the use of Watanabe heritable hyperlipidemic rabbits [26]. This animal model is deficient in functional LDLR activity due to a deletion in a portion of the LDLR gene [25, 27], and provides an excellent model of familial hypercholesterolemia. Watanabe heritable hyperlipidemic fibroblasts [28] and hepatocytes [29], both deficient in LDLR, have been transduced with human complementary DNA for the LDLR in vitro using retrovirus-mediated gene transfer. Chowdhury and associates [29], using the rabbit model of hypercholesterolemia, transplanted autologous hepatocytes transfected with the LDLR gene (transduced ex vivo with recombinant retroviruses) into the spleen of the animal from which the cells were originally derived. Greater than 95% of the cells entered the portal circulation and seeded the liver, resulting in a 30% to 50% decrease in serum cholesterol lasting more than 120 days. Although this study used recombinant retroviruses, a limitation of these viral vectors arises from the requirement of cell replication for gene expression and the low rate of hepatoycte proliferation. To circumvent this problem, adenoviral vectors, which do not require host cell replication, have been used [24] and extensively reviewed [30]. In an in vivo rat model, intraportal administration of recombinant adenoviruses containing the E coli lacZ gene or the human {alpha}1-antitrypsin gene was followed by hepatocyte expression of the gene products for 3 days to 4 weeks, respectively [31]. Expression of the transfected genes demonstrated that adenoviral vectors may be applicable for the in vivo introduction of genes encoding the LDLR. Subsequently, a recombinant adenovirus carrying the human LDLR gene has been used in a mouse model [32]. The virus has intravenously injected and seeded the liver with approximately 90% of hepatocytes expressing the virally transferred gene. Plasma cholesterol decreased and an increased rate of iodine-125--labeled low-density lipoprotein clearance was documented in the treated mice. Using the techniques of homologous recombination, mice have been produced that lack functional LDLR and develop a marked elevation in plasma intermediate-density lipoproteins and low-density lipoprotein levels [33]. After intravenous injection of a recombinant adenovirus encoding the human LDLR, elevated intermediate-density and low-density lipoprotein levels were reduced to normal. Injection led to high-level expression of the intact receptor in the liver of the LDLR-deficient mouse, acutely reversing the hypercholesterolemic effects of LDLR deficiency. Furthermore, adenovirus-mediated apolipoprotein A1 gene transfer in mice has been shown to increase circulating high-density lipoprotein levels [34]. These examples of hepatocyte gene augmentation suggest a gene-based therapeutic strategy to treat familial hypercholesterolemia that could attenuate the premature development of atherosclerosis and alter risk of cardiovascular disease.


   Genetic Manipulation of Diseased Peripheral Vasculature
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
The endothelium and vascular smooth muscle cells, being in direct contact with the blood, provide a target for viral or nonviral transfection. Efficient gene transfer and expression has been demonstrated using rabbit aortic endothelial cells in vitro [35]. Genes for nonsecreted proteins and a growth hormone have been successfully expressed in these cells, and these findings suggest that endothelial cells may serve as vehicles for functioning recombinant genes in vivo. Two strategies exist to introduce exogenous genes into endothelial cells in vivo. First, the gene may be transferred into the endothelial cell in vitro followed by reintroduction of the cell into the host. Second, the gene may be transferred directly into the endothelial cell in vivo within a localized arterial segment. Dichek and others [36] demonstrated the use of genetically engineered endothelial cells in vitro using intravascular stents. Steel vascular stents were covered with sheep endothelial cells transduced in vitro with genes encoding either bacterial beta-galactosidase or human tissue-type plasminogen activator. Expression of intracellular beta-galactosidase and high-level secretion of tissue-type plasminogen activator from the endothelial cells was shown both before and after seeding of the stents. Endothelial cells also remained in place after balloon inflation [36, 37] and after exposure to pulsatile flow [37] indicating that the cells may remain viable even in a high-shear in vivo setting. The use of intravascular stents is complicated by local thrombosis and restenosis secondary to intimal proliferation [38–40]. Expression of the tissue-type plasminogen activator in vivo favors a thrombolytic environment on the endothelial surface that could prevent thrombosis in a localized segment of vessel.

Similar work has been done using vascular grafts. Wilson and associates [41] used canine jugular vein endothelial cells infected with a retroviral vector expressing the enzyme beta-galactosidase. Dacron grafts, seeded with the genetically modified cells, were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Five weeks later the grafts were harvested and analyzed. Viable endothelial cells were found lining patent grafts and expression of beta-galactosidase was observed within these cells. Considering the feasibility of this in vivo technique, it is speculated that this technology could be used in the treatment of atherosclerosis, in the prevention of stenosis or occlusion in vascular stents and prostheses, or as a drug delivery system [1, 41].

In addition to the use of vascular stents or grafts, the delivery of genetically modified endothelial cells to specific anatomic sites has been developed. Nabel and others [42] evaluated whether endothelial cells genetically modified in vitro could be implanted onto an arterial segment in vivo. Porcine endothelial cells were infected in vitro with a retroviral vector expressing the enzyme beta-galactosidase. The endothelial cells were then introduced into denuded iliofemoral artery segments using a double-balloon catheter system (Fig 3Go). Segments were removed 2 to 4 weeks later and beta-galactosidase staining was observed in endothelial cells within the intimal layer, indicating that these cells were successfully implanted on the vessel wall. Using similar methods, vascular smooth muscle cells infected with a retroviral vector in vitro have been introduced into the intima and media of denuded iliofemoral artery segments [43]. Following these developments, Nabel and associates [44] hypothesized that vascular cells could be directly infected in vivo. Using the same double-balloon catheter technique, beta-galactosidase expressing retroviral vectors were directly instilled into porcine iliofemoral artery segments. Arterial segments were infected in vivo and expressed beta-galactosidase for 5 months. Furthermore, the intima, media, and adventitia were all infected by the retrovirus and no evidence of recombinant beta-galactosidase activity was detected outside the focal arterial segments.



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  		Fig 3. . The double-balloon catheter and the modified perfusion balloon catheter used for intravascular and intracoronary delivery of genetic material respectively. (A) The double-balloon catheter has proximal and distal balloons, which create a central space upon inflation. The DNA and vector are instilled through small pores into a discrete segment of the vessel. (B) The modified perfusion balloon contains six laser-created pores in the balloon component of the catheter. The balloon is inflated and contacts the vessel wall circumferentially. The DNA and vector are then delivered via the balloon port, whereby the transfection solution escapes through the pores.

 
Replication-deficient adenoviral vectors have also been used for in vivo peripheral artery gene transfer. Lemarchand and others [45] injected adenoviral vectors, containing the E coli lacZ gene or the human {alpha}1-antitrypsin gene, into sheep jugular vein and carotid artery segments [45]. Using the adenoviral vectors, gene expression was detectable within endothelial cells 1 to 14 days after injection. Willard and associates [46] also used a recombinant adenoviral vector for in vivo gene transfer. Endothelial cells and vascular smooth muscle cells in rabbit jugular vein and carotid artery segments were successfully transfected, further demonstrating the capability of adenoviral vectors to transfer exogenous genes into arterial walls. Without the use of viral vectors, iliofemoral arteries have also been directly transfected with liposomes containing beta-galactosidase DNA [44] or luciferase DNA [47]. Both endothelial cells and smooth muscle cells expressed beta-galactosidase for up to 6 months [44], and luciferase activity was detected in endothelial cells for 72 hours after transfection [47]. Other investigators have efficiently introduced plasmid DNA alone into dog femoral arteries without the use of a transfection vehicle [48]. These studies demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo. In addition, viral vectors may not be the only means of transferring genes into vascular cells, and other methods may be equally efficacious. Retroviral vectors, adenoviral vectors, and liposome-DNA complexes all represent attractive delivery vehicles for the introduction of genes into endothelial cells and vascular smooth muscle cells. These techniques have potential application in the treatment of many cardiovascular diseases such as atherosclerosis and, again, may lead to the design of drug delivery systems.


   Gene Transfer Into the Myocardium and Coronary Vasculature
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
Conclusion
 Acknowledgments
 References
 
Somatic gene therapy also has potential for the treatment of diseases involving either the myocardium or coronary vasculature. The ability to program recombinant gene expression in these tissues requires a vector with enhanced activity in cardiac myocytes and endothelial cells and a method for in vivo introduction of such a vector into these cells. Multiple studies in animal models have demonstrated this method.

The adenovirus is a useful vector for expressing recombinant genes in the myocardium because of the nonreplicative nature of cardiac myocytes. Kirshenbaum and associates [49] have shown that the adenovirus is a highly efficient vector of gene transfer into rat myocytes in vitro. Long-term in vivo gene transfer throughout mouse cardiac muscle after intravenous administration of a recombinant (E coli beta-galactosidase) adenovirus has also been reported [50]. Expression of the enzyme was observed for a 12-month period in both neonatal and adult animals, providing a potential route of gene therapy for cardiac diseases diagnosed in both immature and mature myocardium. Adenoviral vectors have also been introduced into the coronary sinus and coronary arteries of mice and rabbits using percutaneous coronary gene transfer [51, 52]. Recombinant protein expression was observed both in the coronary vessels and in the surrounding myocardium with no activity found in hearts injected with a control adenovirus preparation.

The lack of cardiac myocyte replication may not limit transfection solely to adenoviral vectors. Lin and others [53] directly introduced a recombinant beta-galactosidase gene into the left ventricular wall in vivo by the injection of purified plasmid DNA. The beta-galactosidase gene was subsequently expressed in cardiac myocytes for up to 4 weeks. Myocardial gene expression after injection of plasmid DNA has also been developed in canine myocardium [54], and the efficiency of expression of gene constructs compared favorably with other transfection methods. The results of these studies demonstrate that mitosis is not necessary for successful transfection of cardiac myocytes, and infectious viral vectors may not be required in this setting.

These techniques may also be used to stimulate the formation of collateral vessels in areas of ischemic myocardium using recombinant genes expressing angiogenic growth factors [49]. Coronary collateral flow has been increased in ischemic myocardium after intracoronary infusion of vascular endothelial growth factor [55] and intracoronary [56] or periadventitial [57] administration of basic fibroblast growth factor. Vascular endothelial growth factor and basic fibroblast growth factor are known potent angiogenic proteins both in the in vitro and in vivo environments [58–61]. The augmented collateral flow was associated with improved cardiac systolic function [56] and reduced infarct size [56, 57]. Gene transfer of angiogenic growth factors into cardiac myocytes may provide prolonged expression of the gene product such that intracoronary infusion may be unnecessary. Induction of angiogenesis has subsequently been shown after in vivo gene transfer directly into the myocardium [62]. Plasmids containing human fibroblast growth factor were injected into the left ventricular wall of rats and 21 days later capillary density increased 32% in the injected regions of the myocardium. Although increased capillary density was demonstrated, no correlation with myocardial infarct size was evaluated.

Methods to genetically alter hepatocytes, peripheral artery endothelial cells, smooth muscle cells, or cardiac myocytes have also been used to manipulate the coronary vasculature. Lim and others [47] used a luciferase reporter gene construct for direct lipid-mediated gene transfer into coronary arteries of dogs. Luciferase activity was detected 72 hours later in the coronary arteries and revealed that reporter genes could be introduced into coronary arteries in vivo with successful recombinant protein expression. The luciferase gene has also been transfected into coronary arteries using a porous perfusion balloon system (see Fig 3Go) [46]. Furthermore, replication-deficient adenoviral vectors have been used to introduce exogenous genes into the coronary vasculature of rabbits via percutaneous coronary gene transfer [54] and via perforated balloon catheters in swine [63]. Markedly higher levels of recombinant gene expression were obtained using adenoviral vectors than liposome-mediated gene transfer [63].

Both viral and nonviral vectors are capable of transferring recombinant genes into cardiac myocytes and coronary vasculature with successful gene product expression. The clinical utility of transfecting human myocardium or coronary endothelial cells with growth factors to promote angiogenesis has potential application in the treatment of ischemic heart disease. In addition, delivery systems for gene transfer are also being developed that could easily use current cardiac catheterization techniques.


   Conclusion
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
Acknowledgments
 References
 
Human gene therapy has become a reality in a short period of time with approved protocols in existence to treat noncardiovascular disorders. Highly efficient methods of gene transfer are available allowing the expression of recombinant genes in many different mammalian cell types. Molecular biology has introduced multiple potential treatment strategies, in animal models, that could be applied to humans to combat diseases of the cardiac and vascular systems. Gene transfer into the human cardiovascular system has not been carried out, but likely will be in the near future with promising application. Eventually gene therapy will be the ultimate intervention for cardiovascular pathology, attacking the basic cause of the disease and supplementing current surgical and interventional therapies.


   Acknowledgments
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
References
 
This work was supported in part by National Institutes of Health grants HL44186, GM49222, and GM08315A.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 
Address reprint requests to Dr Rowland, Department of Surgery, University of Colorado Health Sciences Center, 4200 E Ninth Ave (C305), Denver CO 80262.


   References
Top
 Footnotes
 Abstract
 Introduction
 Recombinant DNA and the...
 Gene Therapy of...
 Genetic Manipulation of Diseased...
 Gene Transfer Into the...
 Conclusion
 Acknowledgments
 References
 

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