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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Coll Cardiol. Author manuscript; available in PMC 2012 March 8.
Published in final edited form as:
PMCID: PMC3070185

Rescuing the Failing Heart by Targeted Gene Transfer


Congestive heart failure is a major cause of morbidity and mortality in the US. While progress in conventional treatments is making steady and incremental gains to reduce heart failure mortality, there is a critical need to explore new therapeutic approaches. Gene therapy was initially applied in the clinical setting for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as congestive heart failure. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Furthermore, the recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure.

Keywords: Gene therapy, heart failure, sarcoplasmic reticulum, calcium


Accumulating preclinical studies are providing an increasingly sound scientific basis for cautious human evaluation of additional gene therapy strategies. Furthermore, the anatomical compartmentalization of the heart and its accessibility by surgical and percutaneous approaches render the myocardium a highly amenable target system for gene therapy. Improvement in our understanding of the molecular mechanisms of heart failure along with the development of novel and safer vectors for gene delivery have led to the identification of novel targets that are difficult to manipulate pharmacologically but may be more amenable to gene therapy. In this review, we will highlight new strategies for the treatment of heart failure by gene transfer focusing on the vectors, targets, and delivery methods.


Non-Viral Vectors

Non-viral vectors can be loosely grouped as plasmid DNA, liposome-DNA complexes (lipoplexes) and polymer-DNA complexes (polyplexes) (1). Oligonucleotides and their analogues, either alone or in complexes, are also an example of non-viral vector mediated gene transfer. Although myocardial plasmid-mediated gene transfer is relatively inefficient (2) it has been the vector system upon which several therapeutic angiogenesis trials have been based. In this setting, transient secretion of angiogenic factors by a modest number of gene-modified cells is sufficient for the desired phenotypic effect.

More recently degradable polymer nanoparticles have been developed for gene delivery. Nanoparticle systems, polymers of hydroxy acids such as poly (lactic-co-glycolic) acid (PLGA), which is FDA approved, have the capacity to release DNA material over prolonged time frames. Nanoparticles are well suited for gene delivery applications, as they have a large nucleic acid cargo capacity and avoid the safety risks of standard adenoviral transfection (3). With the development of degradable polymer nanoparticles made of end-modified poly(β-amino ester) that are easy to synthesize, and that can self-assemble with DNA to form stable nanoparticles which degrade via hydrolytic cleavage of backbone ester groups there is now hope that these nanoparticles can be used in cardiac gene transfer.

Viral Vectors

The predominant use of viral vector systems in preclinical models of gene therapy is a reflection of the increased gene transfer efficiencies achievable with these systems. The most commonly used viral vector systems will be covered in the following discussion and in table 1, we list the three most commonly used vectors for cardiovascular gene transfer.

Table 1
Comparison of major viral vector systems

Adenoviral Vectors

Recombinant human adenoviral vectors are the most commonly used vectors in experimental gene therapy models. The strengths of this vector system include the relative ease of production, high functional vector titers achievable and broad target cell tropism, particularly within the cardiovascular system. All major cardiac cell types can be efficiently transduced by adenoviral vectors, both in vitro and in vivo. With regard to cardiomyocytes, efficient in vivo transduction has been demonstrated in gene therapy models from several mammalian species. (4,5). The adenovirus uses the coxsackie adenovirus receptor to integrate into the cell. Physical approaches to enhance adenoviral gene transfer in the myocardium include methods that rely on catheter-based or surgically-induced trans-coronary pressure gradients to increase gene delivery to the myocardium (6) or the application of ultrasound energy to disperse circulating vector as it traverses the myocardium (7).

Although adenoviral vectors are useful research tools, translation to clinical applications has challenging for a number of reasons. Adenoviral vectors have a widely appreciated capacity for evoking intense immune and inflammatory reactions which limits their expression. Additional constraints of these vectors include the promiscuity with which they infect all organs (especially the liver) even when delivered in a distant organ.

A number of approaches have been explored to increase the biosafety of adenoviral vectors. These include vectors with increasingly reduced amounts of parental virus genes such that current generation “gutted” adenoviral vectors are devoid of almost all of these. This development results in reduced production of toxic viral proteins and may extend transgene expression over time.

Adeno-Associated Virus (AAV) Vectors

Recombinant adeno-associated virus (rAAV) vectors are derived from the dependant parvovirus (8). This vector system has a number of clinically favorable attributes such as lack of parental agent pathogenicity and vector-related cytotoxicity, minimal immunogenecity, and the capacity for stable long-term transgene expression through genomic integration and/or stable episome maintenance. Major limitations of rAAV vector systems include the production of high-titer vector stocks, a limited packaging capacity and the existence of neutralizing antibodies in human populations.

Adeno-associated viruses (AAV) have many characteristics that make them particularly well suited as vectors for cardiac gene transfer. First, AAV effectively transfects slowly- or non-dividing cells making them ideal vectors for gene transfer to cardiomyocytes. Second, AAV is not known to cause human disease, suggesting that the risks to patients, health care workers, and the public at large are low. AAV vectors contain no viral genes, further increasing their safety and decreases immune responses thereby allowing it to persist and express transgenes in cells long-term (>4 years in certain trials (9)). A total of 12 different AAV serotypes are known, including AAV1 through AAV12, each with different tissue tropisms.(10). AAV1 and AAV6 have been shown to transduce skeletal and cardiac muscle efficiently while AAV9 have very high tropisms to the heart. Dimeric, or self-complementary molecules (scAAV) spontaneously re-anneal, alleviating the requirement for host-cell DNA synthesis. Samulski and colleagues engineered such vectors and found that these scAAV resulted in rapid and higher levels of transgene expression than a conventional single-stranded vector(11). These novel scAAV vectors represent a biochemical intermediate in rAAV transduction and should provide new insights into the biology of vector transduction. However, the size of the cassette sizes have to be half of a normal size single stranded vector (ie 2.3 kb) which further limits the number of genes that can be used with scAAV.

Limiting expression of the transgene to the heart is undoubtedly beneficial for clinical applications. One way of achieving expression exclusively in the heart is the use of a cardiomyocyte specific promoter. However, while this is an attractive approach, it would be more desirable to achieve cardiomyocyte specific expression by targeting the vector itself to the heart. Vector targeting will likely allow the use of lower vector doses when compared to non-targeted AAV. Infusing lower vector doses are not only preferable from a production point of view but, maybe more importantly safer in patients. It has been previously shown that chimeric viruses, i.e. virions composed of capsid proteins of more than one serotype, show distinct transduction profiles (12). Based on these results, viruses that are composed of hybrid capsid proteins, i.e. capsid proteins encoded by amino acid segments of several serotypes, should result in distinct tropism. To identify hybrid viruses that can selectively transduce cardiomyocytes in vivo, a combinatorial approach has been used whereby the generation of a library of diverse AAV variants, obtained by DNA shuffling, with an enrichment of cardiotropic AAV variants by directed evolution are combined (13).

Retroviral & Lentiviral Vectors

Retroviral vectors based on Moloney Murine leukaemia virus (MoMLV) have been used widely in preclinical models. More recently this vector has been used in the successful French trial of gene therapy for X-linked severe combined immunodeficiency (X-SCID) (14). The main limitations of retroviral vectors are an inability to tranduce non-dividing cells such as cardiac mycoytes. As a result, many investigators have sought the overcome this block by inducing the in vivo proliferation in target cells or by employing an ex vivo strategy (2).

Lentiviral vectors, based on the human immunodeficiency virus type 1 (HIV-1) (15), transduce target cells by genomic integration. In contrast to retroviral vectors, these vectors are capable of transducing mitotically quiescent cells, a property that broadens the range of target cells, particularly within the cardiovascular system. Vector modifications addressing biosafety concerns associated with contemporary HIV-1-derived lentiviral vectors include, the deletion of all accessory protein genes from the packaging system, separation of packaging elements into multiple plasmids, and the use of a chimeric 5’ long terminal repeat (LTR) and a self-inactivating 3’ LTR in the vector plasmid (16). The experimental use of this relatively new vector system in preclinical models is rapidly expanding and its potential application for human gene therapy has not yet been explored. The strengths of this system include the ability to confer long-term stable transgene expression. The major limitations relate to biosafety. Recently, three out of 10 children from the French X-SCID trial developed premalignant clonal T-cell proliferation that was directly attributable to dysregulation of a gene at the proviral integration site (17). The risk of insertional mutagenesis with integrating vector use had previously been considered minimal, a view now under revision.

Design of the Expression Cassette

The basic components of an expression cassette include promoter/enhancer elements, the gene(s) of interest and an appropriate mRNA stabilizing polyadenylation signal. Other frequently employed cis-acting elements include internal ribosome entry site (IRES) sequences to allow expression of two or more genes without the need for an additional promoter, and introns and post-transcriptional regulatory elements to improve transgene expression.

Tissue specific promoters can be used to restrict transgene expression to the desired target cell population and avoid unintended cells such as antigen presenting cells. For example, cardiomyocyte-specific promoters such as alpha-myosin heavy chain have been employed to restrict gene expression to the myocardium. Similarly, the smooth muscle-specific promoter SM22α has been demonstrated to restrict gene expression to cells of this type.

In a number of instances, it is desirable to have precise regulation of a therapeutic gene expression in vivo. Natural and synthetic enhancer-promoters can be utilized to drive gene transcription in a temporal, spatial or environmental signal-inducible manner in response to heat shock, hypoxia, radiation, chemotherapy, or epigenetic agents. Hypoxia, intravascular shear stress and left ventricular strain have all been used in models of this type of regulation. In the setting of heart failure regulatable system would be important if the gene of interest needs to be turned on or off for a short period of time. This would include genes driving angiogenesis or stem cell recruitment or expansion.

Targeted Nucleases

Meganucleases and zinc finger proteins can be engineered to induce double-strand breaks at specific DNA sequences (18) (19). These breaks are repaired by homologous recombination or by non-homologous end joining, which results in insertions or deletions of a few base pairs. They can then be used to restore the normal reading frame of a gene with a specific mutation. Similarly engineered zinc finger protein transcription factors to specifically activate or repress virtually any gene. Meganucleases and zinc finger proteins have been used to target dystrophin mutations and VEGF expression by gene transfer (20). With the ever expanding genetic mutations in dilated and hypertrophic cardiomyopathies (21), these nucleases can be used in the future to repair DNA in the affected organs by gene therapy.

Methods of vector delivery

In addition to the major advances in engineering vehicles for myocardial gene therapy, novel delivery methods have been developed to specifically target the heart. Regardless of the delivery methods used chemical approaches which include the use of vasodilatory and permeabilizing agents have been used to facilitate transfer of vector form the vascular lumen to the myocardium (22). In fact a number of agents that increase the permeability of the vascular bed have been used in preclinical trials including nitroglycerin, nitroprusside, serotonin, bradykinin, histamine, substance P, and VEGF (Vascular endothelial growth factor) (23). Clinically and in the setting of heart failure, these agents must be used in caution as not to decrease systemic blood pressure. The multiple methods of gene delivery are illustrated in Figures 1 and and22.

Figure 1Figure 1Figure 1
Antegrade injections of Vectors through Coronary Vessels
Figure 2Figure 2
Retrograde injections of Vectors through Coronary Vessels

Antegrade injection

Antegrade injection in the coronary circulation is the most straightforward way of delivering vectors to the myocardium. It has the potential to homogenously deliver the vector to the whole myocardium. There are multiple techniques of antegrade coronary injection:

a. Antegrade injection while blocking the coronary artery (Figure 1a)

This involves inflating a balloon to block the coronary flow while injecting downstream of the balloon. This method allows flow of the viral vector to occur without dilution however blocking antegrade flow may not be well tolerated even for a short period of time

b. Prolonged Antegrade injection without blocking the coronary arteries (Figure 1b)

This simple yet effective technique works best with AAV vectors. There is no obstruction of flow and can be an optimal method for patients with heart failure which may not tolerate coronary artery blockade. Even though this method of antegrade injection does not infect all cardiomyocytes (~60% of myocytes), it follows the normal pattern of flow in the coronaries and has been shown to improve ventricular function in a large animal model of heart failure (24). Of note, this is the method that was used clinically in patients with severe heart failure receiving AAV1.SERCA2a.

c. Circulatory Perfusion Technique (Figure 1c)

The V-Focus delivery system allows for a minimally invasive, percutaneous procedure to establish isolated regional perfusion of the myocardium. It enables a closed circuit to be percutaneously established between the coronary arteries and the coronary sinus. Through percutaneous catheters, it selectively isolates and circulates the gene transfer agent through the coronary circulation. Adequate oxygenation of the myocardium is maintained in part by circulating the perfusate through an extracorporeal membrane oxygenation system (ECMO). Once a closed circuit is established, the vector is directly injected into the circuit and circulates through the myocardium for approximately 10 minutes. This technique has allowed widespread transduction of SERCA2a in an ovine model of heart failure with a substantial increase in contractility both with adenoviral and AAV gene transfer (25,26).

Retrograde injection

a. Retrograde delivery through the coronary sinus (Figure 2a)

Using the coronary vein, which is disease free in most cases, for the route to deliver the therapeutic material is attractive especially in the clinical setting where the patient has a diseased coronary artery (2729). The increase of the efficacy has been achieved by pressure-regulated retro-perfusion using a specialized device in this approach. Retrograde infusion of vector via the coronary veins constitutes a novel catheter based technique for myocardial gene delivery and in studies pioneered by Boekstegers et al it was shown to be superior to antegrade coronary injections (27). However other studies showed that antegrade coronary injections was superior to retrograde injections (30). However, this method can be somewhat problematic in patients who may not tolerate coronary occlusion.

b. Transcatheter Device (Figure 2b)

A composite catheter system (TransAccess) incorporates a phased-array ultrasound tip for guidance and a sheathed, extendable nitinol needle for transvascular myocardial access is advanced from the coronary sinus retrograde. A microinfusion (IntraLume) catheter is advanced through the needle, deep into remote myocardium, and the autologous cell-hydrogel suspension is injected into normal heart. This allows the delivery of vectors directly into specific areas of the myocardium accessible through the coronary sinus (31).

Direct injection

Direct injection of the vector into the myocardium can be achieved surgically or percutaneously. It overcomes numerous potential drawbacks that can be encountered with the intravascular route including the first-pass effect of the liver and spleen, the effect of neutralizing antibodies, the T-cell response and the impermeability of the endothelial barrier. Surgical gene transfer can be performed through a subxiphisternal or transthoracic approach and can be an attractive approach when the myocardium is easily accessible as during cardiothoracic surgeries (Figure 3a). Catheter-based needle endomyocardial injection can be performed with several types of catheters and under different guidance modalities including fluoroscopy, electromagnetism and 3D mapping systems (Figure 3b). Both the surgical and the percutaneous approaches have limited vector delivery secondary to the restricted area of injection and the leakage at the site of the myocardial injection.

Figure 3Figure 3Figure 3
Ventricular injections of Vectors

Pericardial delivery (Figure 3c)

The pericardial sac is a closed space in close proximity to the myocardium and is accessible to both surgical and percutaneous delivery. Vectors in this space preferentially transduce the pericardial cells with minimal myocardial expression. A percutaneous pericardial approach allows a safe and effective pericardial access appropriate for the delivery of therapeutic agents.

The optimal method of delivery depends largely on the proportion of target tissue to be genetically modified. Focal transduction might be desired to salvage an ischemic area whereas diffuse gene expression is more likely to reverse global myocardial dysfunction.

Surgical Delivery

Surgical delivery in animal models usually involves an open chest procedure where the heart is arrested in hypothermia or the aorta & pulmonary arteries are cross clamped in rodent models (6) (32) (33) or the animals are placed on bypass in large animal models. On bypass, the viral vector can be delivered both in antegrade or retrograde fashion (34). More recently, Bridges et al has have developed a complete surgical isolation of the heart in situ with retrograde administration of vectors which results in very high myocyte transduction efficiency (35,36). However it is not clear whether patients with advanced heart failure could tolerate such a procedure for gene transfer.


The last twenty years witnessed significant evolution in our understanding of the pathophysiology of heart failure in its molecular and cellular dimensions which broadened the scope of interventions available for gene therapy. We will discuss in this part some of the most important systems targeted to restore the function of failing cardiomyocytes.

For targets to be validated, it is important that they rescue function in animal models when heart failure has been already established, that the rescue is not associated with arrhythmogenesis and that a gene-dose effect is established, ie with increasing expression of the gene of interest there is a concomitant improvement in function.

In Figure 4, the various targets in excitation contraction coupling are presented

Figure 4
Targets within Cardiac Myocytes

1. Targeting the β-adrenergic system

The β-adrenergic system is affected by multiple alterations including β-ARs downregulation, upregulation of β-ARs kinase and increased Gi function. These alterations lead to desensitization of the β-ARs and decreased signaling through their pathway. Several gene-based experiments tested the hypothesis that genetic manipulation of the myocardial β-AR system can enhance cardiac function.

a. Overexpression of β-AR

Overexpression of β-AR was initially tested as a simple way to overcome β-AR downregulation. Transgenic mice overexpressing the human β1-ARs suffered from severe cardiomyopathy (37). This finding reinforces the hypothesis that β-AR downregulation is a protective mechanism in the failing heart. In contrast, mice with cardiac overexpression of β2-AR demonstrated increased basal myocardial adenylyl cyclase activity with increased left ventricular function (38). Both direct and intracoronary myocardial delivery of Adenovirus containing the human β2-AR transgene resulted in enhanced cardiac performance in rabbits (39) (40).

b. Inhibition of G protein-coupled receptor kinases (GRKs)

The interaction between activated β-ARs and G proteins is regulated by kinases that modulate the receptor activity by phosphorylation of its carboxyl terminus. Agonist-dependent desensitization is mediated by a family of GRKs which phosphorylate the agonist-occupied receptors resulting in functional uncoupling. GRK2 is the most expressed GRK in the heart. It has been implicated in the pathogenesis of dysfunctional cardiac β-AR signaling accounting for a deleterious activity in the failing heart (41). Studies in mice in which HF was induced by a myocardial infarction, showed that selective GRK2 ablation 10 days postinfarction resulted in increased survival, halted ventricular remodeling and enhanced cardiac contractile performance (42). A peptide termed βARKct capable of inhibiting GRK2 mediated β-AR desensitization has been evaluated in vivo in animals. Using intracoronary adenovirus-mediated βARKct transgene delivery to rabbits 3 weeks after induced myocardial infarction demonstrated a marked reversal of ventricular dysfunction (43).

c. Activation of cardiac AC expression

Although detrimental outcomes were demonstrated with multiple elements of the β-adrenergic system used to improve the expression of cAMP, activation of AC type VI (AC VI) seems to have a unique favorable profile. Overexpression of AC VI in transgenic mice resulted in improved cardiac function in response to adrenergic stimulation along with increased cAMP production in isolated cardiac myocytes. Importantly, AC VI had a neutral effect on basal heart function and was not associated with any structural heart abnormalities (44). In a pacing model of HF in pigs, intracoronary delivery of adenovirus encoding AC VI resulted in improved LV function and remodeling, associated with increased cAMP generating capacity (23). The favorable effects of AC VI in preclinical studies are encouraging and this approach is currently under investigation for initiation of clinical trials in patients with HF (45).

2. Targeting Ca2+ cycling proteins

Ca2+ plays a crucial role in contraction and relaxation phases of the cardiac cycle (Figure 1). Ryanodine receptors (RyR) are proteins forming a link between the T tubules in the cardiomyocytes and the sarcoplasmic reticulum (SR). In a process known as “Ca2+ induced Ca2+ release”, depolarization activates voltage-operated L-type Ca2+ channels of the T tubule to allow Ca2+ entry into the cardiomyocyte. Ca2+ reaches the RyR protein modifying its molecular configuration which in turn opens the Ca2+ release channel of the SR releasing Ca2+ into the cytosol. This process greatly increases the concentration of Ca2+ in the cytosol allowing it to interact with troponin C triggering the contraction process. Relaxation occurs when Ca2+ detaches from troponin C and is either taken up by the SR via the action of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) or extruded from the cardiac cell by the sarcolemnal Na+/Ca2+ exchanger (NCX). Phospholamban (PLN) is a protein implicated in regulation of SERCA2a activity. In its unphosphorylated form PLN can inhibit SERCA2a. In contrast, phosphorylation of PLN relieves its inhibitory effect and results in increased SERCA2a activity with improved Ca2+ handling. Protein phosphatase 1 (PP1) is a type of serine/threonine phosphatase which has a major role in dephosphorylation of PLN in the heart (4648). In particular, stimulation of the β-adrenergic axis induces phosphorylation of a phosphatase inhibitor Protein-1 (I-1) resulting in PP1 inhibition and enhancement of myocardial contractile function (4648). HF is characterized by multiple defects in Ca2+-handling proteins with impaired SR Ca2+ loading and release. Reversal of those defects by gene therapy techniques has shown very promising results. We will review the main aspects of those novel therapies in this section.

a. Overexpression of SERCA2a

More than twenty years ago, Gwathmey et al first reported that calcium cycling is abnormal in human heart failure (49) and was found to be partially due to decreased SERCA2a activity regardless of the etiology of the heart failure (5052) (53). Improvement in cardiac contractility after gene transfer of SERCA2a has been demonstrated in a large number of experimental models of heart failure (54) (55). More importantly, long-term overexpression of SERCA2a by intracoronary delivery of AAV carrying SERCA2a has been associated with preserved systolic function and improved ventricular remodeling in a swine volume-overload model of HF (24). Beyond their effects on enhancing contractility, SERCA2a gene transfer has been shown to restore the energetics state of the heart both in terms of energy supply and utilization, decrease ventricular arrhythmias, and enhance coronary flow through activation of eNOS in endothelial cells (56) (57) (58) (59).

b. PLN inhibition

Another approach to improve Ca2+ handling involves inhibition of PLN. Decreasing PLN in human cardiac myocytes showed an improvement in contraction and relaxation velocities similar to the benefit seen with gene transfer of SERCA2a (60). Silencing of PLN expression in a sheep HF model resulted in improved SERCA activity along with improved systolic and diastolic LV function (26). In addition to the above conventional gene therapy strategies, RNAi therapy was used for the first time in a model of cardiac disease, specifically in rats with HF, in an attempt to suppress phospholamban expression. A rAAV-RNAi vector generated stable cardiac production of a regulatory RNA sequence, which in turn suppressed phospholamban expression. SERCA2a protein was subsequently increased accompanied by restoration of systolic and diastolic cardiac function (61).

c. Active I-1 and Inhibition of PP1

HF is associated with elevated PP1 activity in humans resulting in dephosphorylation of PLN. Overexpression of PP1 or ablation of I-1 in murine hearts has been associated with decreased β-AR-mediated contractile responses, depressed cardiac function and premature death consistent with HF (4648). Expression of a constitutively active I-1 in transgenic mice led to PP1 inhibition with increased phosphorylation of PLN and improved cardiac contractility. A recent study on transgenic mice expressing active I-1 confirmed the relationship between phosphorylation of PLN and SERCA2a activity. I-1 expression ameliorated ischemia/reperfusion-induced injury by reducing the infarct size and improving contractile recovery in addition to decreasing biomarkers of apoptosis and ER stress response (4648).

d. S100A1

S100 is a multigenic family of Ca2+-modulated proteins implicated in intracellular and extracellular regulatory activities. S100A1 is the most abundant S100 protein isoform in the heart. It promotes cardiac contractile and relaxation function through enhancing the activity of both RYRs and SERCA2a (62). In a rat model of HF, AAV6-mediated long term expression of S100A1 resulted in a sustained in vivo reversal of LV dysfunction and remodeling (63,64).

3. Targeting cell death

Apoptosis is a process of programmed cell death that is involved in normal organ development. In models of acute and subacute ischemia/reperfusion, overexpression of the anti-apoptotic protein Bcl-2 or akt reduces the rate of cardiomyocyte apoptosis and improved heart function [49]. Despite the early encouraging results in animal studies, anti-apoptotic strategies face multiple challenges before being considered for human trials. There remain some uncertainties about the reliability of the techniques currently used to estimate the rate of apoptotic cell death [53]. In addition, loss of the normal cellular regulation role provided by apoptosis can generate a serious risk of malignant cellular proliferation and autoimmune injuries. Finally, while ischemia/reperfusion injury appears to be amenable to intervention, it is less clear if other forms of cardiac injury such as hypertrophy and HF can benefit from anti-apoptotic strategies.

4. Homing of stem cells

It has been well established that damaged and inflamed tissues send out signals to attract stem cells to the injured tissue, and many of these signals have been identified, including stromal derived factor 1 (SDF-1). There has been growing evidence that SDF-1:CXCR4 (its receptor) axis plays an important role in regulating myocardial repair following ischemic injury. SDF-1 has been shown to be increased following myocardial ischemia and a number of studies have shown that enhancing SDF1 levels around the infarct improves myocardial remodeling following infarction. Currently the strategy of delivering SDF1 naked DNA by percutaneous measures to the peri-infarct area is being exploited clinically (65).

Translating Pre-Clinical Results to Designing Clinical Trials

Once a target is validated in cells, rodents, and large animal models, the appropriate vector has been identified and a route of delivery is chosen, a number of challenges remain to starting clinical trials. These include: evaluation of the immune response, clinical parameters to be examined and evaluating the risk of arrhythmias.

Immune Response

One of the challenges with viral gene transfer is the pre-existence of neutralizing antibodies. In different studies, it has been shown that about a significant proportion of adults are seropositive for AAV2 (up to ~80%)% AAV1 (~50%), AAV5 (~40%) and AAV6 (~30%) (66) (67). The presence of pre-existing antibodies can have considerable implications for cardiovascular gene therapy because it has been shown in several studies that these AAV specific antibodies are neutralizing (66) (67) and can severely compromise the utility of this approach. These results highlight the necessity to determine neutralizing antibody titers against the specific vector used in the patient population.

Another issue that can occur is that cells may transiently express AAV capsid protein on their cell surface. T-cell response could occur in any organ but highest concern for liver or the heart (site of injection). This response is dose dependent and in clinical trials to treat hemophilia or liportein lipase deficiency, higher doses (>1013 viral genomes) were associated with activation of capsid specific T cells and elimination of the transgene (68) (69). In the clinical trials, to evaluate potential development of a T-cell response, it is important to use an ELISPOT assay to detect anti-AAV capsid T cell responses (IFN-γ release when patient’s peripheral blood monocytes are exposed to capsid peptide).

Evaluation of Clinical Parameters Post Gene Transfer

It is critical that the assessment of the efficacy of gene therapy trials be robust enough to detect biological signals. In pre-clinical trials and large animal studies, investigators have relied mainly on hemodynamic measurements coupled with non-invasive techniques such as echocardiography and MRI (Magnetic resonance Imaging). MRIs, which provide the most detailed structural measurements of the heart cannot be routinely used in advanced heart failure patients since a majority of them would have ICDs (Implantable cardiac defibrillators) or biventricular pacemakers. Clinically, invasive monitoring is obviously difficult to perform, however there are a number of clinical measures that can be followed.


Arrhythmias have been clearly shown to be a consequence of pharmacological inotropic therapy. In the context of gene therapy there may be a number of reasons for arrhythmias to occur. The overexpression or knock-down of specific genes such as channels or ionic transporters may induce changes in electrical remodeling that can predispose the cells to arrhythmias. In addition, inhomogeneous expression within the myocardium can induce areas of heterogeneously contracting cardiomyocytes that can also lead to arrhythmias. It is therefore essential that in pre-clinical studies, detection of arrhythmias be thoroughly studied following gene transfer using holter monitors or implantable continuous monitoring. Clinically, it would be important to protect the patient receiving the gene therapy product with ICDs.

Clinical trials

Despite the initial hope that gene therapy had at its inception, the failures of the clinical trials coupled with the serious adverse events brought the field close to collapse ten years ago. The well publicized complications included the death a patient with a mild form of the monogenic disorder ornithine transcarbamylase deficiency which was caused by multi-organ failure following the injection of a recombinant adenovirus (7072), and the development of new T cell lymphomas in three out of the ten children with severe combined immunodeficiency (SCID) after treatment with retroviruses (14,7375). However, the field of gene therapy learned valuable lessons from these failures and has rebounded recently with rigorous regulatory barriers along with a new wave of novel vectors. There is now a growing number of gene therapy trials for various diseases, most notably inherited blindness (whereby gene transfer by AAV vectors partially restored vision in a pediatric patient with Leber's Congenital Amaurocis, a major cause of congenital blindness (76)), cancer, infectious diseases, monogenic diseases, and cardiovascular diseases.

Specifically, in heart failure, there are currently a number of trials ongoing or in the planning stages targeting various pathways for rescuing the failing myocardium (Table 1). The targets that have been taken forward towards clinical trials include SERCA2a, adenylyl cyclase type 6, and SDF-1.

The first clinical trial of gene therapy in patients with HF was launched in the United States in 2007 (77,78). CUPID (Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) is a multicenter trial designed to evaluate the safety profile and the biological effects of gene transfer of the SERCA2a cDNA by delivering a recombinant AAV1 (AAV1.SERCA2a) in patients with advanced HF. Participants in this trial were administered a single intracoronary infusion of AAV1.SERCA2a in an open-label approach (77,78). Cohorts 1–4 of 3 patients each received sequentially a single escalating dose of AAV1.SERCA2a. Twelve month follow-up of these patients showed an acceptable safety profile (77,78). Improvement was detected in several patients, reflected by symptomatic (5 patients), functional (4 patients), biomarker (2 patients) and LV function/remodeling (6 patients) parameters. Although this was a phase 1 study involving a small number of patients, early results found that AAV1.SERCA2a treatment conferred quantitative biological benefit. In the phase 2 trial, 39 patients with advanced HF were randomized to receive intracoronary adeno-associated virus 1 (AAV1) mediated SERCA2a gene delivery (in one of 3 doses (low dose − 6 × 1011 DRP, middle dose − 3 × 1012 DRP and high dose − 1 × 1013 DRP) versus placebo. Patient’s symptoms (NYHA class, Minnesota Living With Heart Failure Questionnaire [MLWHFQ]), functional status (6 minute walk test [6MWT] and VO2 max), NT-proBNP levels and echocardiographic measures were evaluated over 6 months. Treatment success was determined by examining concordant trends in the above endpoints for group- and patient-based comparisons, as well as clinical outcomes. The AAV1.SERCA2a high-dose group met the pre-specified criteria for success at the group and individual patient levels. AAV1.SERCA2a treated patients, versus placebo, demonstrated improvement or stabilization in NYHA class, MLWHFQ, 6MWT, VO2 max, NT-proBNP levels, and LV end-systolic volumes. Significant increases in time to adjudicated CV events, and a decreased frequency of CV events per patient were observed in all patients receiving AAV1.SERCA2a. No increases in adverse events, disease-related events, laboratory abnormalities or arrhythmias were observed in AAV1.SERCA2a treated patients compared to placebo.

Two other clinical trials targeting SERCA2a are currently enrolling patients. The first trial is in patients with advanced heart failure having received left ventricular assist devices at least one month prior to treatment and who will receive either AAV6.SERCA2a or saline. This trial is being conducted in the United Kingdom. A second Phase 2 monocenter double blind randomized placebo-controled, parallel study will be held in the Institut of Cardiology Pitié-Salpêtrière, Paris, France with the primary objective to investigate the impact of AAV6-CMV-SERCA2a on cardiac remodeling parameters in patients with severe heart failure.

In a separate clinical study adenovirus-5 encoding human adenylyl cyclase type 6 (is being delivered through intracoronary injection to patients with congestive heart failure. Intracoronary delivery of Ad5.hAC6 or PBS in 3:1 randomization with dose escalation, The patients will be randomized in a dose dependent fashion starting at 3.2 × 109 viral particles to 3.2 × 10^12 viral particles in 6 dose groups using a 3:1 randomization fashion with PBS (buffered saline being used for control). The trial is currently enrolling patients.

An additional trial is examining the effects of injecting SDF-1 directly into the myocardium of patients with ischemic heart disease. An open label dose escalation study to evaluate the safety of a single escalating dose of SDF-1 administered by endomyocardial injection to cohorts of adults with ischemic heart failure is currently enrolling patients. SDF-1 naked DNA will be injected directly into the myocardium at multiple sites through a percutaneous, left ventricular approach.


An increase in our knowledge of molecular mechanisms of heart failure along with improved gene therapy technology has led to substantial efforts in pre-clinical testing of anumber of targets and more recetly in the successful completion of a phase 2 trial in gene tehrapy for heart failure. Now that the safety of AAV vectors has been established for the treatment of heart failure along with the efficiacy of SERCA2a in the treatment of heart failure, the field is now open for testing novel targets that are pharmacologically difficult to modulate with more advanced AAV based vector systems.

Table 2
Major Clinical Trials in Gene Therapy for Hear Failure


This work is supported by Leducq Foundation (RJH), by NIH R01 HL093183, HL088434, HL071763, HL080498, HL083156, and P20HL100396 (RJH). DL was supported in part by an award from the German Research Foundation (DFG).


Adeno-associated vector
Adenylyl Cyclase
Beta Adrenergic Receptor
DNAse resistant Particles
Protein Phosphatase Inhibitor-1
Left Ventricular Assist Device
Neutralizing Antibodies
Sarcoplasmic Reticulum Ca2+ ATPase
Sarcoplasmic Reticulum


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