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Cardiovascular Research 2007 73(3):453-462; doi:10.1016/j.cardiores.2006.09.021
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Copyright © 2006, European Society of Cardiology

Targeting the heart with gene therapy-optimized gene delivery methods

Oliver J. Müller*, Hugo A. Katus and Raffi Bekeredjian

Internal Medicine III, University of Heidelberg, Germany

* Corresponding author. Universitätsklinikum Heidelberg, Innere Medizin III, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Tel.: +49 6221 5639401; fax: +49 6221 565516. Email address: o.mueller{at}dkfz-heidelberg.de

Received 17 July 2006; revised 22 September 2006; accepted 26 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 
With evolving knowledge in molecular and cellular cardiology, cardiac gene therapy has already been investigated in clinical studies. Different vector systems for cardiac gene therapy have been developed in recent years. While non-viral vectors, such as plasmid DNA, allow remarkable organ specificity, they are often limited by low transfection efficiency and transient gene expression. In contrast, adenoviral or adeno-associated virus-based vectors transfer the transgene more efficiently, but organ specificity may be reduced and immunogenic properties can limit their applicability. Using advanced transcriptional and transductional targeting strategies, viral vectors have been improved in the last few years. Recently, more efficient serotypes of adeno-associated viruses have been identified that show increased transduction rates, thus reducing the necessity for high virus titers. Combination with specific application techniques, such as intramyocardial injection, catheter-based perfusion, ultrasound targeted microbubble destruction, or retroinfusion may further enhance vector efficiency. This review article will give a broad overview of different gene delivery strategies that have been applied in experimental and clinical studies targeting the heart.

KEYWORDS Gene therapy; Vector; Ultrasound; Angiogenesis; Virus


   1. Introduction
Top
 Abstract
 1. Introduction
2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 
In the last decades, potential therapeutic targets for cardiac gene therapy have been identified, due to impressive advances in molecular cardiology. While preclinical studies in animal models have shown promising results in the field of heart failure, arrhythmia, restenosis, acute and chronic ischemia, and storage diseases, only myocardial angiogenesis has been investigated in clinical trials [1,2]. Although experience in therapeutic angiogenesis has increased, a clear beneficial role in improving clinical outcome has not been shown in randomised multicenter trials yet. Low efficacy of myocardial gene transfer, limited duration of transgene expression and suboptimal choice of therapeutic genes have been held responsible for the lack of significant clinical success [1–3]. While the choice of therapeutic genes can be easily modified with evolving knowledge in molecular biology and physiology, improving efficient and long-term delivery of therapeutic genes seems to be the bottleneck in clinical gene therapy. Recent advances in the development of novel application systems on the one hand and improved viral- and non-viral vector systems on the other hand facilitated transduction or transfection of the heart in animal studies and have started to move gene therapy towards clinical applications. This review article will give an overview of different vector and delivery systems that have been tested in the heart, and will discuss chances, limitations and new developments in cardiac gene therapy.


   2. Delivery barriers of cardiac gene transfer
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 
In order to improve cardiac gene transfer, it is necessary to consider the existing delivery barriers in biodistribution, cellular uptake, and intracellular trafficking (Fig. 1). Depending on the type of vector, various limitations have to be overcome. For efficient gene transfer, viral vectors need to escape neutralizing antibodies in the circulation as well as transduction of antigen presenting cells, which may result in induction of a T-cell response against the vector or the transgene. On the other hand, naked DNA needs to be protected from serum DNases that would degrade DNA, before having a chance to transfect cells. Transduction of non-target organs, predominantly liver and spleen, should be avoided. If not directly injected into the myocardium, vectors also need to cross the endothelial barrier in the capillary wall. Spreading of the vector throughout the myocardium and binding to the cell surface are further crucial steps of transduction. When using naked DNA, passage through the cell membrane is another challenging event. Even successful uptake into the cardiomyocyte does not ensure efficient gene expression since the vector needs to escape lysosomal inactivation and mediate nuclear transfer of the vector DNA. Furthermore, the regulatory sequence should enable efficient cardiac transcription and be resistant to downregulation as it may occur with strong viral promoters [4,5]. Finally, there should be no immune response against the vector surface or the protein encoded by the transgene.


Figure 1
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  		Fig. 1 Schematic representation of barriers that have to be overcome in order to reach the nucleus of the target cell. The schematic demonstrates different strategies to pass these barriers: A. transvascular approach (intravenous, intraarterial, retroinfusion); B. intramyocardial injection; C. endothelial penetration facilitated by viral vector; D. ultrasound mediated vector delivery; E. receptor mediated viral uptake; F. DNA penetration of cell membrane by intramyocardial injection, lipsome or ultrasound.

 
Some of these obstacles can be overcome by developing appropriate techniques that enable targeted administration of the vector itself. Direct injection of the vector into the myocardium using injection catheters or surgical procedures bypass the endothelial barrier and result in high local concentrations of the vector [6]. Promising alternatives are approaches based on facilitated local transendothelial delivery such as retroinfusion and ultrasound mediated gene transfer [7,8]. Another step is transductional targeting of the vector by optimizing its surface to enable an efficient cellular uptake and delivery of packaged DNA into the nucleus [9]. Finally, expression of the delivered DNA can be restricted to the myocardium using transcriptional targeting with a cardiac-specific regulatory region [10–16].

Combination of well designed viral or non-viral vectors with a suitable delivery system should help to overcome the biological barriers and result in efficient gene transfer.


   3. Advances in vector development
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 
Vectors are gene delivery vehicles that are used to carry the genetic material. They may be based on plasmid DNA with or without complexing agents, or viral particles. Viral vectors devoid of genes responsible for immunogenic effects or replication take advantage of the efficient transduction mechanisms of the original virus.

Both categories of vectors have specific and inherent characteristics that shall be described in the following section. The future choice of viral or non-viral vectors will depend on specific aims in gene therapy, potential side effects and further improvements in vector design.

3.1. Non-viral vectors
Despite many disadvantages of non-viral vectors, such as low transfection efficiency and their inability to enter cells without help, some intriguing advantages remain, stimulating research in this field. Such advantages include low immunogenic properties, low costs of production, low toxicity and the option for very high organ specificity. A huge number of various non-viral gene transfer methods have been developed in the last decades. In this review we will only focus on those methods that have been shown to successfully transfect the heart.

The most simple and most widely used vector is naked DNA. DNA has various advantages. It can be produced by bacteria as plasmid DNA and is therefore cost efficient in production, it can be easily cleaned to GMP-grade quality, it posses little immunogenic activity, it has very low toxicity, it is water soluble, heat stable, and it can be degraded rapidly (the latter can be a disadvantage in certain applications). However, one major disadvantage remains – naked DNA cannot enter cells spontaneously with sufficient efficiency. In order to transfect the heart with naked DNA, direct intramyocardial injection has to be applied. This technique is the most advanced of all non-viral transfection methods, despite its obvious disadvantage of invasiveness and procedure related risks. Several experimental studies in animals have shown that direct injection of plasmid-DNA into the myocardium may result in organ specific and safe gene expression [17–19]. The most common genes used for those studies were angiogenic factors such as vascular endothelial growth factor (VEGF) [20–23], hepatocyte growth factor [24] and Sonic hedgehog [25]. Early encouraging results lead to the first clinical trials applying DNA injection for cardiac angiogenesis in patients with coronary artery disease (CAD). In an initial phase I study Losordo et al. demonstrated that direct intramyocardial injection of plasmid DNA encoding VEGF in five patients was safe and resulted in improved symptoms of CAD [26]. However, there was no control group. Subsequent studies using a placebo control group and catheter-based injection methods once again proved safety of this technique and showed some effects on secondary end-points, such as less symptoms and improved myocardial perfusion [23,27,28]. However, none of these studies were able to prove a significant effect on angiogenesis and myocardial viability. New insights in angiogenesis have shown that VEGF is not an optimal candidate gene for therapeutic angiogenesis [29]. Thus, future studies have to prove that combined angiogenic factors or transcription factors regulating angiogenic genes, such as hypoxia inducible factor 1 alpha, are superior to the earlier approaches.

As described above, direct intramyocardial injection of plasmid DNA is feasible but has still a low efficiency. To improve transfection efficiency various compounds have been developed to form complexes with DNA. Substances that successfully transfect the heart include lipopolymers [30], liposomes [31], gelatin complexes [32], poloxamine nanospheres [33] and lipoproteins [34]. Of all these complexes, liposomes have the additional advantage to serve as an intravenous gene therapy vector. Unfortunately, intravenous administration of DNA carrying liposomes will lead to transfection of many organs, with a particularly high transfection rate of the lung [35–37].

An entirely different technique that may achieve organ specific and non-invasive gene delivery with non-viral vectors is the more recently developed method of ultrasound targeted microbubble destruction (UTMD). This technique is based upon physical properties of second generation ultrasound contrast agents. These gas filled microbubbles oscillate when sonified by ultrasound. At high ultrasound energies, oscillations lead to their destruction. Microbubbles can be loaded with plasmid DNA, infused intravenously and finally destroyed in the heart by ultrasound, thus transfecting the target organ. Passage of cell membranes is facilitated by secondary mechanical effects, such as high velocity fluid microjets [38,39]. It is unclear however, if transfection mainly affects endothelial cells or if myocytes can be transfected, too. Since a spatial and temporal proximity of microbubbles, ultrasound and DNA is necessary for successful transfection, remarkable organ specificity can be achieved [8,40]. Despite initial successful studies with therapeutic genes [41,42] and satisfactory safety of this technique [43], transfection efficiency still remains low and needs further improvement.

A number of experimental transfection methods used for cardiac gene transfer are not yet ready for clinical applications. These include electroporation [44], gene gun [45] or DNA coating on surgical suture [46]. Some of these methods could be used to pre-treat cardiac transplants with genes that may reduce the risk for rejection, as shown by several preclinical studies transfecting hearts ex vivo before transplantation [47–49].

Although specificity of non-viral vector approaches may be increased by choosing a suitable mode of delivery such as direct intramyocardial injection or ultrasound mediated gene transfer, the major limitation of non-viral vectors remains the low transfection efficiency due to poor cellular uptake. Despite all improvements, non-viral techniques may only lead to transient transfection, lacking the ability to integrate into the genome or persist in an episomal form. This limits their applicability in conditions requiring long-term gene expression such as heart failure or hereditary cardiac diseases such as cardiomyopathies. However, it is conceivable that transient expression of therapeutic genes can be favorable in certain applications, such as angiogenesis.

3.2. Viral vectors 
The major advantage of viral vectors is the high transduction rate. Virus-mediated gene delivery resulted in 30- to 360-fold higher levels of cardiac transduction with adeno-associated viral or adenoviral vectors after direct intramyocardial injections in rabbits compared to plasmid approaches (uncomplexed and complexed) [50]. Depending on the vector concentration, transduction efficiencies were up to 75% of cardiomyocytes around the needle track after direct injection of adenoviral vectors in adult pigs [51]. The superior transduction efficiency of viral vectors is due to a more efficient cellular uptake and efficient intracellular transport of packaged DNA to the nucleus. In contrast to non-viral vectors, viruses are taken up upon binding to specific surface receptors and are able to escape from degradation in lysosomes [52–55].

Adenoviral vectors are the most frequently used system in experimental and clinical gene transfer studies targeting the heart since they enable highly efficient cardiac gene delivery and can be produced in sufficient quantities. However, adenoviral vectors are limited by only transient gene expression caused by immune response against viral gene products resulting in the clearance of transduced cells [56–58]. Non-sustained gene transfer was initially considered favourable for safety reasons in several clinical phase II/III trials having used adenoviral vectors for cardiac gene transfer of angiogenic factors [1]. Since clinical effects in those angiogenesis trials were below expectations, the short duration of gene transfer was discussed as a potential limitation [2].

Third generation (so called "gutless" or "high capacity") adenoviral vectors have been developed that lack the complete viral genome except for the packaging sequence [59]. These vectors have an increased packaging capacity (up to 28–34 kb) and induce less tissue inflammation since they do not encode any viral proteins [60]. Prolonged gene expression was observed upon transfer of a lacZ reporter gene with high capacity adenoviral vectors only in skeletal muscle of lacZ transgenic, but not wild type mice [61]. Similarly, a side by side comparison with first generation adenoviral vectors upon intramyocardial injection in adult rats did not demonstrate significant increase in expression of a GFP reporter gene despite of reduced myocardial inflammation [62]. The transient expression in those experiments was explained by considerable immune response against the transgene itself. Such immune response might have been caused by transduction of dendritic cells and subsequent gene expression in these cells that play a key role in triggering cytotoxic immune response [57]. Using a tissue-specific promoter, formation of antibodies against the transgene could be prevented in a mouse model [63]. Therefore, future strategies for sustained cardiac gene transfer by high capacity adenoviral vectors should prefer transgenes that do not elicit an immune response, should use a cardiac-specific promoter sequence to prevent expression in antigen-presenting cells or should modify the adenoviral tropism in order to avoid transduction of these cells. The latter would require a transductional targeting strategy based on identification of cardiac targeting motives which may be incorporated in existing vectors with ablated natural tropism [60,64].

Adeno-associated virus (AAV) is increasingly recognized as a promising alternative to adenovirus because of its safety profile: AAV is a non-pathogenic parvovirus that cannot be amplified without co-infection with a helper virus. AAV vectors transduce the myocardium as efficiently as adenoviral vectors and – in contrast to shorter expression with adenoviral vectors – allow stable expression of transgenes over several months [65,66].

AAV-2 vectors have been successfully used in several experimental therapeutic approaches such as protection from ischemia/reperfusion injury in a rat model [67] or inotropic therapy in cardiomyopathic hamsters [68,69] and revealed beneficial effects on neoangiogenesis, infarct-size, and cardiac function in a murine model of myocardial ischemia [70]. To achieve highly selective transduction of myocardial tissue, AAV vectors were administered via intramyocardial injection or perfusions of coronary arteries [67–70].

Recent advances in AAV vector development allowed – at least in rodents – an efficient gene transfer via a transvascular route. These so called pseudotyped vectors take advantage of the ability of certain AAV serotypes to efficiently cross the blood vessel barrier. Systemic application of pseudotyped AAV-6 or-8 vectors resulted in uniform and extensive transfer of a lacZ reporter gene in adult mice [71,72]. Intravenous injection of AAV-8 vectors enabled reconstitution of {delta}-sarkoglycan in {delta}-sarkoglycan-deficient TO-2 hamsters [73] and extended their lifespan by preventing heart failure in this animal model of dilated cardiomyopathy. Systemic transfer of AAV-7 pseudotyped vectors overexpressing acid {alpha}-glucosidase has successfully reduced cardiac glycogen content in a murine model of glycogen storage disease type II (Pompe disease) that causes death in infancy from cardiorespiratory failure [74]. These promising results underline the principal suitability of AAV serotype vectors for systemic gene transfer in mice and hamsters. Identification of further AAV serotypes, such as AAV-9 and studies in larger animals may lead to suitable AAV vectors for human gene therapy. However, potent pseudotyped AAV vectors such as AAV-6 may also show higher transfer efficiencies in non-cardiac organs [16].

Since wild type AAV vectors are not cardiac specific, modification of the natural tropism of an AAV serotype may be a promising approach to decrease potential side effects due to extracardiac gene transfer. One approach to reduce undesired liver transduction is to use vectors packaged into mutant AAV-2 (R484E; R585E) capsids, resulting in 100-fold increased ratio of cardiac to hepatic reporter activity [16]. The AAV2 (R484E; R585E) mutant capsids do not bind to the AAV-2 primary receptor heparin sulfate proteoglycane due to an inactivation of the heparin-binding motif at R484 and R585 [75]. These vectors are characterized by markedly reduced infection of murine livers in vivo without affecting transduction of the heart where binding to heparin sulfate proteogylcane plays no role. Such data driven approaches are limited by the number of unknown variables determining successful transduction such as penetration of the endothelial cell layer, physical barriers of the target tissue such as the extracellular matrix, or the intracellular fate of vectors. Combinatorial approaches displaying a library of randomized peptide motifs on the AAV surface [76,77] may allow identification of AAV-vectors targeted to the myocardium.


   4. Regulatory sequences for efficient and specific cardiac gene expression
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 
In addition to transductional targeting strategies that are based on vector surface modification, transcriptional targeting can be used to improve transgene expression and cell specificity. Transcriptional targeting takes advantage of cardiac-specific promoters that regulate expression of myocardial proteins. This approach will not prevent uptake of vector particles in extracardiac tissues, but may restrict gene expression to the myocardium. Originally, regulatory sequences of genes predominantly expressed in the myocardium have been analyzed in transgenic animal models. This led to a better understanding of regulatory mechanisms involved in cardiac gene expression. Thus, suitable promoter sequences could induce a cardiac phenotype in transgenic animal models [78]. Further studies using viral vectors showed that the regulatory sequences of genes encoding {alpha}-myosin heavy chain or myosin light chain-2v could also be used to transcriptionally target gene expression to the heart despite presence of vector genomes in extracardiac tissues [12–16,70,79,80] (Table 1). However, expression levels in adult hearts were rather weak, most probably due to developmental downregulation of promoter activity [81]. Since most preclinical studies aim to show a strong effect of a distinct therapeutic approach, the strong and ubiquitous active CMV-promoter has been used most frequently. Another similarly potent, but rather unspecific promoter is the β-actin hybrid promoter that leads to early and widespread cardiac transduction after intramyocardial injection of AAV-6 vectors [82]. In order to overcome the low expression levels of tissue-specific regulatory sequences, fusion with strong viral enhancers or hypoxia-regulatory elements, as shown for the (MLC) 2v-promoter, has been promoted to increase transduction levels in rodents [16,70,83]. An alternative approach of increasing promoter activity may be a random assembly of regulatory elements into synthetic promoter libraries, and screening of individual clones for transcriptional activity in vitro and in vivo. This has been shown for an artificial skeletal muscle promoter whose transcriptional potency greatly exceeds those of natural myogenic and viral promoters [84]. Further improvements in transcriptional targeting will be important for the evolution from intracardiac applications to systemic applications of gene therapy vectors. Table 1 lists regulatory sequences that have demonstrated successful cardiac gene expression using adenoviral and AAV vectors.


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  		Table 1 Regulatory sequences driving cardiac gene expression in vivo

 
Promoter elements not only determine tissue-specificity and expression levels, but also duration of transgene expression. The use of tissue-specific regulatory sequences results in markedly prolonged gene expression by prevention of promoter downregulation on the one hand [4,5] and transgene expression in antigen presenting cells on the other hand [63,85].


   5. Advances in delivery systems
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
6. Clinical Prospects
 Acknowledgments
 References
 
As discussed in the section for non viral vectors, naked DNA was predominantly applied by direct intramyocardial injection. With viral vectors, most experimental studies in animals used a similar approach. Myocardial injection led to efficient local gene delivery, but was often limited by a patchy pattern of vector transfer and procedure related risks [50,51,65,86]. Injection of viral vector into the pericardial sack has been described as another alternative. However, this technique did not demonstrate efficient transmural gene expression [87,88]. In order to achieve more homogeneous myocardial gene transfer in a defined target area, infusion of adenoviral vector into coronary arteries by a percutaneous transluminal approach was analyzed which resulted in low transfer efficiency [89]. An explanation might be the temporary exposure of the vector to the coronary endothelium and subsequent fast systemic distribution. Variables that can determine efficiency of viral gene delivery via the coronary circulation are coronary flow, vector concentration, and endothelial permeability, as systematically analyzed in isolated hearts [90,91].

Novel approaches in viral gene transfer focus on prolonging the exposure time of the vector with the cardiac endothelium and increasing endothelial permeability. This was successfully demonstrated in a rat model by simultaneous clamping of the aorta and pulmonary artery for a brief period of time resulting in repeated re-circulation of the vector in the coronary arteries [92]. A longer crossclamping time could be obtained by hypothermia or cardiac arrest in small animals [94–97] and cardiopulmonary bypass in large animals [98]. Approaches to increase endothelial permeability are high intravascular pressure, ultrasound [8,99], or capillary-modulating substances, such as serotonin, histamine and VEGF [71,91,100]. Vascular pressure can be increased either by crossclamping the aorta [79,92,94] or percutaneous occlusion with a balloon catheter [95,99]. Coronary venous blockade during antegrade intracoronary viral gene delivery was shown to be feasible in a porcine model [93]. A different method takes advantage of selective retroinfusion through the coronary sinus to achieve highly efficient adenoviral gene transfer targeted to the ischemic myocardium with limited gene expression in control organs in a porcine model [7,103]. Finally, ultrasound targeted microbubble destruction that was described in the section for non-viral vectors, was successfully used to augment adenoviral uptake into the heart. By loading viral particles on microbubbles, it is possible to achieve high local concentrations of viral vectors in the cardiac capillaries after ultrasound mediated destruction of such microbubbles [98,104]. In addition, microbubble destruction can increase capillary permeability and thus facilitate viral passage into the myocardium. Recently alternative percutaneous techniques have been pursued. The NOGA system, a catheter-based electro-mechanical mapping and injection system, allowed efficient intramyocardial injection of plasmid DNA and adenoviral vectors in a porcine model [24,101,102]. This approach has also been used for percutaneous myocardial gene transfer in clinical studies [23].

Ideally, the most desirable cardiac gene therapy vector could be infused intravenously and would show efficient and organ specific gene transfer in the heart. Some AAV serotypes show such characteristic in rodents, such as AAV serotype-6,-7,-and 8 [16,71,74,79,105]. When using VEGF to increase vascular permeability even lower vector titers of AAV-6 could be used [71]. However, the amount of virus necessary for larger animals or patients would clearly exceed current production limits. Furthermore, there is little experience on potential side effects when administering such large amounts of viral particles in humans systemically. Nevertheless, systemic infusion of targeted vectors may play an important role in the future, especially if congenital disorders like cardiomyopathy associated with muscular dystrophy are being targeted.

Table 2 shows an overview of some successfully applied transfer techniques for viral vectors.


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  		Table 2 Approaches for cardiac gene transfer (selected)

 

   6. Clinical Prospects
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
Acknowledgments
 References
 
The tragic death of Jesse Gelsinger after infusion of an adenoviral vector into the hepatic artery in a phase I trial in 1999 showed the limitations of translating basic gene therapy research into clinical applications [107]. While this patient suffered from a massive inflammatory response resulting in disseminated intravascular coagulation and multiorgan failure, another patient receiving a similar dose of the adenoviral vector did not demonstrate such adverse events. The mechanisms for this severe reaction have never been completely understood, but a previous immunization due to a viral infection has been hypothesised as a potential cause. Although recent years have shown significant improvements in efficiency and safety of vectors and application systems, many hurdles still have to be overcome. The immune system, partly responsible for the transient nature of adenoviral gene transfer [56–58], is still limiting its efficacy. Even AAV-2 vectors that appear to be well tolerated in laboratory animals, caused transient elevation of transaminases in a human hepatic gene therapy approach, most probably due to activation of memory T-cells against epitopes on the AAV-vector surface [108]. In contrast to laboratory animals, most patients have had contact with AAV-2 in their childhood and may have developed memory T-cells as a result. Therefore, future strategies for sustained gene transfer in patients using AAV-vectors require either immune suppression for the early period, when the vector capsid is present in the cell [109], or modification of the capsid in order to prevent activation of T-cells. The latter may be either achieved by choosing different serotypes or modification of the serotype 2 capsid itself. Interestingly, deletion of heparin binding reduced uptake in human dendritic cells and activation of capsid-specific T-cells [110]. However, further studies are required to prove applicability of this approach in humans. Finally, transient gene expression due to immune response may be advantageous in certain situation, such as stimulation of cardiac angiogenesis in ischemic heart disease.

Another area of concern, especially with AAV-mediated gene transfer, is the potential for germline transmission [111,112]. Although transferred DNA was transiently detectable in semen of patients upon AAV-2-mediated gene transfer, animal studies suggested that AAV-2 does not transduce spermatogonia [113–115]. Nevertheless, patients have been asked to bank sperms prior to enrolment in clinical trials and use barrier contraception until semen were tested negative for vector sequences [116]. Although the risk for germ line transmission is apparently low, these safety precautions seem to be justified. Especially modified vectors or serotype vectors need to be analyzed carefully for their potential of spermatogonia transduction prior to their use in clinical studies.

Presently, even cardiac specific promoter systems cannot guarantee exclusive gene expression in the heart, if the viral vector has contact with other tissues. Further developments in this area may improve efficacy and safety of cardiac gene therapy. More work is also needed to improve regulatory systems for clinical applications. Since low level gene expression mediated by AAV-2 vectors has been detected in muscular biopsies of patients for up to 3.7 years [116], a regulatory system is required to shut off gene expression in case of potential side effects by the therapeutic gene product. So far, no inducible system has been developed that has been tested in clinical trials.

Finally, more preclinical cardiovascular studies in large animal models are required, as done extensively for factor IX gene transfer prior to clinical trials [108,116]. Thus safety and efficiency of both vector and gene product can be evaluated in a system that resembles a clinical situation. Upon successful completion of such preclinical studies, careful design of clinical trials is of importance. By selecting appropriate patients and defining realistic and relevant clinical endpoints, applicability of clinical gene therapy could be evaluated. Technical and financial hurdles for GMP-grade vector production will necessitate finding industrial partners for these final steps.

In summary, significant advances in cardiac gene delivery have been achieved in the last years. However, gene therapy targeting the heart still remains a challenging aim. While non-viral vectors show remarkable organ specificity and low toxicity, they are severely limited by low transfer efficiency and transient expression. Improved delivery systems for non-viral vectors may overcome these limitations. In contrast, viral vectors have advanced to potent gene therapy vectors for the heart. Adenoviral and AAV vectors allow efficient cardiac gene transfer. In addition, AAV can lead to prolonged transgene expression. Combination of recent developments in transductional and transcriptional targeting together with novel application systems could be the basis for future gene therapy trials in patients with cardiac diseases.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
References
 
Raffi Bekeredjian is funded by the BioFuture grant of the Bundesministerium für Bildung und Forschung, Germany. Oliver J. Müller is supported by a Grant of the Deutsche Forschungsgemeinschaft (Mu1654/3-1) and Bundesministerium für Bildung und Forschung (BMBF 01GU0527).


   Notes
 
Time for primary review 16 days


   References
Top
 Abstract
 1. Introduction
 2. Delivery barriers of...
 3. Advances in vector...
 4. Regulatory sequences for...
 5. Advances in delivery...
 6. Clinical Prospects
 Acknowledgments
 References
 

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J. H. Boyd, B. Kan, H. Roberts, Y. Wang, and K. R. Walley
S100A8 and S100A9 Mediate Endotoxin-Induced Cardiomyocyte Dysfunction via the Receptor for Advanced Glycation End Products
Circ. Res., May 23, 2008; 102(10): 1239 - 1246.
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