Viruses are highly infectious simple microorganisms. They have evolved with extraordinary skills to enter our body. It is expected that vectors based on viruses may lead to much more robust gene transfer than plasmid-based nonviral vectors . Several viral vectors have been explored for heart gene delivery. These include lentiviral, adenoviral and adeno-associated virus (AAV) viral vectors (Figure 1).
Lentiviral vectors for heart gene delivery
Lentiviral vectors are originally developed from HIV, a RNA virus . They can package an approximately 8 kb genome. In contrast to traditional retroviral vectors, lentiviral vectors mediate stable gene transfer in terminally differentiated nondividing cells.
Initial studies in cultured cardiomyocytes suggest that lentiviral vectors are extremely powerful. The transduction efficiency reached 70% in adult cardiomyocytes and 100% in neonatal cardimyocytes [22, 23]. Using a heterotopic heart transplantation model, Zhao et al.  observed robust transduction at 7 days after direct injection of a green fluorescence protein (GFP) lentiviral vector. In a separate study, Fleury et al.  showed persistent heart gene transfer up to 10 weeks after direct myocardial injection of the lentiviral GFP vector. Transduction efficiency reached a peak at 3–7 days post-injection but declined thereafter. Additional studies suggested that the drop of gene expression was likely a result of gene transfer-associated myocardial inflammation at the injection site .
More recently, Niwano et al.  tested whether lentivirus-mediated SERCA2a expression could protect against heart failure in a rat myocardial infarction model. At 21 days after hypothermic intracoronary injection (Figure 2), transduction efficiency reached approximately 40% in the heart but was essentially not detectable in the liver and spleen. Importantly, favorable myocardial remodeling was observed. Echocardiography and a cardiac catheter assay showed significant functional improvement. The survival rate was almost doubled in treated animals when challenged with myocardial infarction 6 months later.
Adenoviral vectors for heart gene delivery
Adenovirues are double-stranded DNA viruses. They carry an approximately 30-kb genome. Adenoviral vectors have been shown to effectively transduce several tissues such as the airway epithelium [25, 26]. The use of recombinant adenoviruses for heart gene delivery was initiated in early 1990s (Figure 1). Today, adenoviral vectors have been tested for cardiac gene therapy in rodents, large animals and in humans.
In 1992, Stratford-Perricaudet et al.  showed that intravenous injection of an adenoviral LacZ vector to neonatal mice resulted in widespread gene transfer in several tissues, including the heart and skeletal muscle (Figure 2). Sustained LacZ expression was detected for at least 12 months. However, similar injection protocol resulted in minimal LacZ expression at 3 weeks after gene transfer in adult mice . This result suggests that the mammalian heart is amenable to adenovirus transduction but there are important limitations. Several studies published in the subsequent 2 years confirmed the transient nature of adenoviral mediated heart gene transfer [28, 29]. Guzman et al.  showed that adenoviral vectors were significantly more efficient than plasmids after direct myocardial injection. Robust expression was detected at the injection site during the first week but it diminished to the background level by 30 days. Kass-Eisler et al.  obtained similar results with a different adenoviral vector. These results suggest that adenoviral vectors represent an efficient but unstable gene delivery vehicle for the heart.
To further extend the findings in rodents, French et al.  explored adenoviral gene transfer in the heart of domestic swine. Adenoviral vectors were injected at multiple locations in the ventricular wall. Similar to that reported in rodents, expression peaked at 7 days and declined thereafter. A clear dose response was also noted. Nevertheless, expression did not spread far from the injection site and, furthermore, a marked leukocytic infiltration was observed near transduced cardiomyocytes .
Heart transplantation has become a standard procedure. Ex vivo manipulation of the donor heart by viral gene transfer may further enhance therapeutic outcome. Gojo et al.  tested whether a hypothermically preserved heart graft could be transduced by adenovirus. The vector was perfused through the coronary artery of the donor heart at 4 °C before heterotopic transplantation. Interestingly, the heart infected with low-dose virus (1 × 109 plaque forming units, pfu) showed high transduction for 4 weeks. However, strong inflammation was observed around the transduced cardiomyocytes in the heart that was infected with high-dose adenovirus (≥ 1 × 1010 pfu). Furthermore, minimal expression was detected at 2 weeks after operation in the high-dose group .
Direct myocardial injections only lead to limited transduction at the injection site. To obtain broader myocardial transduction, Fromes et al.  developed an intrapericardial injection strategy (Figure 2). Collagenase and hyaluronidase were injected together with adenovirus to the pericardial sac in rats. This protocol significantly increased adenovirus diffusion into the myocardium. Up to 40% myocardium was efficiently transduced at 7 days post-injection. Nevertheless, expression declined thereafter and became undetectable by 28 days .
To achieve global myocardial gene transfer, Hajjar et al.  developed a catheter-based delivery method (Figure 2). Briefly, a catheter was inserted through the apex of the left ventricle until it reached just above the root of the aortic valve. The aorta and pulmonary artery were briefly blocked using a clamp while adenovirus was injected. This technique allowed efficient circulation of adenovirus through the coronary arteries. Almost homogeneous cardiac gene transfer was achieved with several different adenoviruses. Importantly, the application of this method to an adenovirus carrying the phospholamban gene resulted in significant change of left ventricular function . Although adenoviral transduction of the lung and liver was also noted, the study demonstrated for the first time that a gene transfer technique could be used to modulate overall heart function. Subsequently, a modified catheter-based technique was investigated in rabbits . To perfuse the coronary beds, Maurice et al.  rapidly injected 1.5 ml of adenovirus via a catheter placed in the chamber of the left ventricle. During injection, they transiently clamped the aorta. Diffuse multi-chamber expression was achieved, with peak expression at 6 days post-injection. Using this system, it was also shown that a therapeutic β-adrenergic receptor adenovirus significantly enhanced heart contractility and hemodynamic performance in rabbits .
The first successful demonstration of adenovirus-mediated gene therapy in a cardiomyopathy model was performed by Ikeda et al.  2002 . δ-sarcoglycan deficient hamster is a naturally occurring model of dilated cardiomyopathy. Interestingly, only limited gene transfer (approximately 5%) was achieved when a brief aortic and pulmonary artery occlusion was applied. However, a combination of core temperature reduction, partial cardioplegia and transient aorta/pulmonary artery blocking resulted in an impressive transduction efficiency of approximately 77% . Similar to previous adenovirus studies, inflammation was observed approximately 1 week later and transduction remained short-term. Nevertheless, left ventricular function was significantly improved at 3 weeks after the δ-sarcoglycan adenovirus was delivered.
To achieve persistent adenoviral gene transfer in the heart, Christensen et al.  explored embryonic and neonatal intracardiac delivery. Embryonic adenovirus injection appeared to have minimal impact on pregnancy. Adenovirus injection in 1-day-old mice resulted in stable myocardial transduction that lasted for several months. Importantly, there was no inflammatory reaction in the heart of neonatally injected mice. This technique has provided an excellent platform for studying gene function in murine cardiomyocytes through in vivo gene transfer.
Accumulatively, adenovirus-mediated heart gene transfer has been conducted in a relatively large number of human patients by many research groups [37-41]. A particular focus of these studies is to treat coronary artery disease with an adenovirus that expresses VEGF121. In most trials, 4 × 1010 particle units of adenoviral vectors were delivered to the heart. The procedure was well tolerated [37-41]. However, controversy exists regarding the therapeutic efficacy. Although some studies have demonstrated reduced myocardial ischemia during exercise, as well as increased new blood vessel formation , others have failed to show an improvement in exercise capacity and myocardial perfusion .