Gene Therapy in Heart Failure

– SERCA2a as a Therapeutic Target –

Abstract

The treatment of heart failure (HF) may be entering a new era with clinical trials currently assessing the value of gene therapy as a novel therapeutic strategy. If these trials demonstrate efficacy then a new avenue of potential treatments could become available to the clinicians treating HF. In principle, gene therapy allows us to directly target the underlying molecular abnormalities seen in the failing myocyte. In this review we discuss the fundamentals of gene therapy and the challenges of delivering it to patients with HF. The molecular abnormalities underlying HF are discussed along with potential targets for gene therapy, focusing on SERCA2a. We discuss the laboratory and early clinical evidence for the benefit of SERCA2a gene therapy in HF. Finally, we discuss the ongoing clinical trials of SERCA2a gene therapy and possible future directions for this treatment. (Circ J 2014; 78: 2577–2587)

Heart failure (HF) is a major cause of morbidity and mortality in the developed world, affecting more than 23 million people.¹ Currently, the licensed pharmacological treatments for chronic HF are β-adrenergic blockers, angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor antagonists, aldosterone antagonists and recently, the selective sinus node inhibitor ivabradine, with diuretics for symptom control and fluid retention. The effectiveness of these interventions is limited by side effect profile, tolerability and efficacy. Implantable device treatments, including cardiac resynchronization therapy and left ventricular assist devices (LVADs), can be effective but are limited by cost, availability and complications including infection and anticoagulation. Gene therapy offers a potential paradigm shift in the way we approach the treatment of HF by allowing us to directly, and specifically, target the underlying molecular abnormalities.

The application of gene therapy to HF has been in development for more than 2 decades but has come to the forefront recently with the publication of the first 2 clinical trials using this novel approach, both demonstrating safety and a suggestion of efficacy.^2–4 Several other clinical trials of gene therapy in HF are now underway, in particular targeting the sarcoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a) gene, making this an opportune time to review gene therapy in HF and in particular SERCA2a gene therapy.

In this review we will discuss the fundamentals of gene therapy before describing the abnormalities of calcium (Ca²⁺) handling observed in HF, focusing on SERCA2a as a clinical target.

Gene Therapy Fundamentals

Definition

Gene therapy is a technology by which genes or small DNA or RNA molecules are delivered to human cells, tissues or organs to correct a genetic defect, or to provide new therapeutic functions for the ultimate purpose of preventing or treating diseases.⁵

Gene Selection

The first challenge in developing gene therapy as a treatment modality is the identification of a gene whose modification will lead to an improvement in the underlying pathophysiology that could translate into improved clinical outcomes. The expression of many hundreds of genes changes in the failing heart; some of these changes are beneficial while others confer negative effects such as adverse cardiac remodeling. These changes in gene expression arise when biomechanical stresses, such as pressure or volume overload, activate a series of signaling pathways, which eventually leads to the activation of transcription factors, co-regulators, and microRNAs in the cell nucleus.^6,7

Genes affected in HF can conceptually be placed in 1 of 3 groups. In the first group are the genes that code for proteins already known to play a role in HF; for example, proteins involved in Ca²⁺ handling and the β-adrenergic system. Some of these genes have been studied for more than 30 years and examples include downregulation of gene expression for SERCA2a, β-1 adrenoreceptors,^8,9 and AT-1 receptors.¹⁰ This group contains the most likely candidates for progressing to gene therapy as there is already an established link between HF and their coded proteins. However, whether normalizing the expression of a particular gene will affect clinical outcomes depends on the cause and effect relationship between gene expression and HF.

The second group comprises genes those whose expression is regulated in parallel to HF progression but that play no apparent causative role in the phenotype of HF. These genes may be useful as biomarkers, such as the upregulation of NPPB, which increases production of N-terminal pro-brain natriuretic peptide (NT-proBNP), but would not be good candidates for gene therapy.

The final group of genes whose expression is altered in HF comprises those that can be identified on microarray screening (discussed later). This technique can rapidly identify thousands of genes involved in the HF syndrome but the real challenge is determining which of them play a causative role in disease progression vs. those which mirror disease progression.

Once a valid gene has been selected as a target for gene therapy, other challenges include achieving transduction of a sufficient number of cells, limiting transduction to the target organ alone and achieving a durable effect of the treatment. Sufficient transduction of cardiomyocytes is partly dependent on vector selection and delivery method, which are discussed next (for a detailed review see Lyon et al¹¹).

Vector Selection

Gene Transfer Without a Vector (“Naked” DNA)

Most gene therapy research uses some form of vector to deliver the gene of interest. There are a number of barriers to using “naked” DNA, including the challenge of a negatively charged DNA strand crossing a negatively charged cell membrane and the rapid rate of breakdown of exposed DNA. Naked DNA has been used in a phase 1 clinical trial, which involved direct injection of a DNA plasmid encoding human stromal cell-derived factor-1 (SDF-1) in to the peri-infarct zones of patients with HF.⁴ Naked DNA has a transient effect, which though suitable in that study, which aimed to recruit stem cells over a short period of time to attempt myocardial regeneration, may not be ideal for the majority of HF gene therapy strategies where it is desirable to produce a sustained effect. To achieve a longer term effect, vectors are generally required.

Non-Viral Vectors

Non-viral vectors have the advantages of being low cost, non-pathogenic and non-immunogenic. In addition, there is no limit on the size of the transgene being delivered, which is a limitation of viral vectors. However, despite the use of delivery agents such as cationic lipids, the massive limitation of this approach is the insufficient transduction efficiency.

Viral Vectors

Viruses have a natural ability to deliver genes to cells and a variety of viral vectors have been used in laboratory studies, each species with its own advantages and disadvantages (Table 1). Although each virus transduces cells in a slightly different way (eg, they may bind to a different receptor on the cell membrane and some viruses will integrate the therapeutic gene in to the host genome while others do not) the overall principal is common (Figure 1). Viral vectors in general have the advantage of being able to deliver therapeutic genes with much greater transduction efficiency than non-viral means. In addition, viruses can be selected with greater cardiotropism to improve delivery to the target organ and limit the effect on other organs. A number of viral species have been used in gene therapy:

Table 1. Vectors Available to Deliver Genetic Material

Vectors	Advantages	Disadvantages
Non-viral	Low cost, non-pathogenic and non-immunogenic	Low transduction efficiency and lack of cardiotropism
Adenovirus	Easy to produce and all major cardiac cell types efficiently transduced	Immune and inflammatory reactions, short term gene expression and moderate transduction of all organs at high doses
Adeno-associated virus	Non-pathogenic, minimal immunogenicity, long term gene expression and cardiac tropism Efficient gene expression	Presence of neutralizing antibodies Short phase expression window not currently available
Lentivirus	Long-term gene expression	Inability to transduce non-dividing cells such as cardiac myocytes

Figure 1.

Adenoassociated virus (AAV)-mediated delivery of target gene. A modified AAV vector containing a therapeutic gene binds to the cardiomyocyte cell membrane (AAVs bind to cell surface heparan sulfate proteoglycan (HSPG) and are endocytosed via co-receptors such as integrin). Following endocytosis, the virus is released from the vesicle in the perinuclear region. The virus then releases the therapeutic gene in to the nucleus and the relevant protein is then produced. Adenoviral vectors behave in a similar manner, but to enter the cell they bind to coxsackie and adenovirus receptors.

Adenoviruses These are easy to produce and have a broad target cell tropism, with all major cardiac cell types being efficiently transduced. They are currently the most widely used vector in cardiovascular gene therapy in laboratory studies. Translation to clinical applications, however, has been significantly limited by moderate transduction efficiency (log-scales lower than adeno-associated viruses [AAVs]), short-term duration of gene expression, the immune and inflammatory reactions that adenoviruses provoke, and their tendency to infect all organs, especially the liver.

AAVs AAVs are the vector of choice in ongoing clinical trials in HF. They are derived from the parvovirus¹² and are not known to cause any human disease. There are 13 different AAV serotypes currently recognized (AAV1–AAV13), each with different tissue tropisms.¹³ AAV1, AAV6, AAV8 and AAV9 have been shown to transduce skeletal and cardiac muscle efficiently. Wild-type AAVs are able to integrate in to the host genome. Although this often occurs in a site-specific location on chromosome 19, it can occur at sites of DNA damage, making insertional mutagenesis, though rare, still a possibility. However, the recombinant AAVs used in clinical trials are not known to integrate in to the host genome, but instead persist in the cell nucleus in a concatemeric episomal form, making the risk of insertional mutagenesis theoretically very remote. The main limitations of AAVs are the production of high-titer vector stocks, and the existence of neutralizing antibodies in human populations. The prevalence of neutralizing antibodies has been reported to be in the order of 20–60%, but this depends on the serotype. The presence of neutralizing antibodies means a significant proportion of patients (~50%) are excluded from current clinical trials.

Lentiviruses These vectors integrate in to the host genome and as such may theoretically persist for the lifetime of the cell. Mouse studies using lentiviurses have demonstrated gene expression for the lifetime of the animal.^14,15 Persistence of beneficial effects up to 2 years has been demonstrated in a small number of patients with X-linked adrenoleukodystrophy.¹⁶ This year, 2 studies have reported safety and benefits of lentiviral-delivered gene therapy targeting hematopoietic stem cells.^17,18 An important consideration with viruses that integrate in to the host genome is the site of integration. If this does not occur in a predictable, site-specific location and instead the virus integrates at a random site, it could be oncogenic. This complication was seen over 10 years ago when retroviruses were used to treat children with severe combined immunodeficiency.¹⁹ It can be minimized with appropriate choice of vector and by loading the vector with self-inactivating promoter sequences that only allow expression of the therapeutic gene. The main limitation of retroviral vectors in cardiac disease is their inability to transduce non-dividing cells such as cardiomyocytes. However, lentiviral vectors, based on the human immunodeficiency virus type 1,²⁰ can transduce non-dividing cells. The experimental use of such lentiviruses in preclinical models is expanding in the cardiovascular system, but application for human cardiac gene therapy has not yet been studied.

Delivery Systems for Gene Therapy

The ideal delivery system would be a peripheral intravenous injection with a vector that selectively transduces only cardiomyocytes. In humans, this is problematic because the large blood volume causes a dilutional effect and viruses infect organs other than the heart, particularly the liver. Some of the AAV serotypes have strong cardiac tropism and conceivably this approach may be feasible in the future. In the meantime, as the heart is a relatively compartmentalized organ, multiple techniques, both percutaneous and surgical, can be used to deliver gene therapy (Table 2).

Table 2. Advantages and Disadvantages of Delivery Methods Available for Gene Therapy

Delivery method	Advantages	Disadvantages
Antegrade intracoronary infusion without coronary artery occlusion*	Homogeneous delivery to whole myocardium Tolerated in patients with heart failure	Limited in patients with significant coronary artery disease
Antegrade intracoronary infusion with coronary artery occlusion	Homogeneous delivery to whole myocardium Allows flow of vector to occur without dilution	Limited in patients with significant coronary artery disease
Closed loop recirculation Vector is infused into a coronary artery, removed from the circulation from the coronary sinus, oxygenated extracorporeally and redelivered down the coronary artery	Vector has a longer exposure time to myocardium and has been shown to improve transduction in an animal model21	Complicated system and requires anticoagulation
Retrograde infusion through coronary sinus	High transduction efficiency in large animal models.22–24 Still feasible in the presence of significant coronary artery disease	High coronary pressure can result in myocardial edema or hemorrhage
Direct myocardial injection*	Avoids first pass of liver, the effect of neutralizing antibodies and the inflammatory response	Limited vector delivery because of restricted area of injection. Myocardial injury from injection
Peripheral intravenous infusion	Simplest, most convenient delivery method	Dilutional effect and no vector with high enough cardiotropism to be clinically viable currently
Pericardial injection	Feasible and safe when guided by imaging modalities and could potentially allow a high concentration of vector to be in contact with a large area of myocardium for a prolonged period of time	Vectors in this space preferentially transduce the pericardial cells with transduction in the myocardial layers of only superficial extent. Penetration of deeper myocardium can be achieved with proteolytic agents but this causes cardiac toxicity

*Technique used in human trials.

From a clinical perspective, the most practical route, and the one adopted in the clinical trials of SERCA2a gene therapy, is a 10-min antegrade coronary artery infusion without coronary artery occlusion. The various adjuncts to antegrade intracoronary infusion, such as coronary artery occlusion or the closed loop recirculation method, have not been adopted in humans because of the added complexity of the procedures and the potential damage to the myocardium. One adjunct to intracoronary infusion, which is used in clinical trials, is the use of concomitant intravenous glyceryl trinitrate (GTN), because this was shown to significantly increase transduction in a large-animal model. Intravenous GTN increased SERCA2a mRNA by more than 2-fold compared with no GTN.²¹ The mechanism of action for this improvement in transduction is unknown but may relate to coronary vasodilatation, changes in vascular permeability or increasing myocardial perfusion through a reduction in left ventricular end-diastolic pressure. Of note, intracoronary GTN did not increase SERCA2a expression,²¹ suggesting the effect is not simply related to coronary vasodilatation.

The only other approach used in a clinical trial is direct intramyocardial injection of a gene therapy product using SDF-1 naked DNA.⁴ This was a suitable approach because the purpose was to deliver the gene to peri-infarct zones only. It was performed through a percutaneous approach but could also be performed surgically. A significant advantage of this approach is that bypassing the endothelium and avoiding blood means a high local concentration of vector can be achieved. Although this method may be applicable to targeting well-circumscribed areas such as infarct zones, the global nature of LV impairment means that this approach is not generally suitable for HF.

Abnormalities of Cellular Calcium in HF (A Target for Gene Therapy)

There are a wide range of pathological abnormalities recognized in failing myocytes, many of which could conceivably be targets for gene therapy. Table 3 demonstrates a number of processes that are deranged in HF. With so many processes affected, it demonstrates the first challenge of gene therapy in HF, which is to select the most appropriate gene to target. Abnormalities of Ca²⁺ handling have come to the forefront, and in particular SERCA2a has become the target of choice in clinical trials. We will go on to focus on the abnormalities of Ca²⁺ handling observed in HF and the pivotal role that SERCA2a plays in cardiomyocyte Ca²⁺ homeostasis.

Table 3. Summary of the Major Changes That Occur in the Failing Ventricular Cardiomyocyte

Morphological changes
Cardiomyocyte size and shape	Hypertrophy characteristics dependent on aetiology of HF 　· HF due to pressure overload: increase in width and length of cardiomyocytes 　· HF due to volume overload: predominantly increase in length of cardiomyocytes
Surface topology	Overall spatial disruption leading to delayed excitation-contraction coupling 　· Loss of Z grooves 　· Loss of t-tubule openings
Intercellular communication	Reduced electrical and functional coupling 　· Connexin 43 redistribution away from intercalated discs 　· Reduced connexin expression
Extracellular matrix	Fibroblasts driven by neurohormonal activation leads to: 　· Increased fibroblast number and metabolic activity 　· Increased production and deposition of extracellular matrix leading to spatial uncoupling of adjacent myocytes
Functional changes
Impaired β-adrenoceptor signaling	· β-AR expression and density reduced (predominantly β1-AR is reduced) 　· β1-AR: β2-AR reduced from around 4:1 in normal hearts to 1:1 　· Increased Gi:Gs ratio. Along with the increased importance of β2-AR, which couple to Gi proteins, this contributes to the β-AR desensitisation and a negatively inotropic effect of β-AR stimulation in chronic HF
Abnormalities of Ca2+ handling	· Down regulation and reduced activity of SERCA2a 　· Overall reduction in PLN but relative increase in active (unphosphorylated) fraction thus inhibitory to SERCA2a 　· Elevated PP1 activity resulting in dephosphorylation of PLN increasing its inhibition of SERCA2a 　· Impaired Ca2+ re-uptake with prolonged Ca2+ transients with a smaller peak amplitude. Higher resting Ca2+ concentration in cytoplasm but reduced Ca2+ stores in the SR leading impaired contraction and relaxation 　· Prolonged action potential duration 　· Hyperphosphorylation and increased oxidation of RyR2 causing Ca2+ leak and increased Ca2+ sparks 　· Increased importance of NCX for Ca2+ extrusion
Abnormalities of Na+ handling	Elevated intracellular [Na+]. Possible mechanisms: 　· Reduced Na+/K+ ATPase gene expression or activity 　· Increased Na+ influx via the NCX (NHE) 　· Increased Na+ influx via the late Na+ current Elevated [Na+] effects: 　· Increase in NCX reverse mode activity during systole leading to Ca2+ influx. Ca2+ cannot be cleared by SERCA2a (due to SERCA2a down regulation) so cleared by the forward mode of NCX during diastole which may lead to cellular triggered activity via after depolarisations 　· Disrupts Ca2+ uptake in to mitochondria, through mitochondrial NCX, leading to energetic inefficiency · Reduces the electrochemical gradient for clearance of H+ by the NHE resulting in impaired intracellular acid-base balance
Mitochondrial dysfunction	Reduced phosphocreatine:ATP ratio reflecting impaired mitochondrial function. Increased oxidative stress with increased ROS due to mitochondrial dysfunction. Effects of increased ROS: 　· Increased SR Ca2+ leak via RyR2 oxidation 　· Impaired Na+/K+ ATPase and SERCA2a activity 　· Predisposition to apoptosis 　· Self generation of further ROS
Increased apoptosis	Increased apoptosis due to a variety of factors including: 　· Increased mitochondrial ROS causes mitochondrial dysfunction and ultimately mitochondrial rupture, releasing several pro-apoptotic factors 　· Abnormal Ca2+ homeostasis leads to impaired protein production in the endoplasmic reticulum
Abnormal gene expression	Activation of fetal gene expression. Results in physiology more akin to foetal ventricular myocardium: 　· Switch from FFA to carbohydrate metabolism 　· Changes in t-tubule and SR physiology 　· Changes in sarcolemmal ion channel expression 　· Alteration of myofilament myosin heavy chain isoforms

β-AR, β-adrenergic receptor; Ca²⁺, calcium; FFA, free fatty acid; H⁺, hydrogen ion; HF, heart failure; Na⁺, sodium; Na⁺/K⁺ ATPase, sodium-potassium adenosine triphosphatase; NCX, Na⁺−Ca²⁺ exchanger; PLN, phospholamban; ROS, reactive oxygen species; RyR2, ryanodine receptor 2; SERCA2a, sarcoplasmic (endoplasmic) reticulum Ca²⁺ ATPase 2a; SR, sarcoplasmic reticulum; t-tubule, transverse tubule.

It is the regulatory role of Ca²⁺ in excitation-contraction coupling that is of particular importance in understanding its role in HF. Normal Ca²⁺ cycling (Figure 2) begins with the cardiac action potential depolarizing the surface membrane and triggering a small Ca²⁺ current into the cytoplasm through L-type Ca²⁺ channels. This triggers a much larger release of Ca²⁺ from the SR store through the ryanodine receptor (RyR). This calcium-induced calcium release triggers contraction through the binding of Ca²⁺ to the troponin C component of the cardiac myofilaments. During diastole, Ca²⁺ is taken back up in to the SR through the action of SERCA2a and some is extruded from the cell by the Na⁺–Ca²⁺ exchange (NCX). These changes in cytoplasmic Ca²⁺ concentration occur rapidly in normal myocytes. Over 25 years ago Gwathmey et al first reported that Ca²⁺ handling is abnormal in the myocardium of patients with endstage HF.²² In particular, during diastole cytoplasmic Ca²⁺ concentrations were slow to fall and myocytes from failing hearts were unable to restore low resting Ca²⁺ levels during diastole, reflected in impaired diastolic relaxation. These observations are a result of impaired Ca²⁺ reuptake into the SR during diastole. Impaired reuptake of Ca²⁺ contributes to the diastolic impairment observed in HF and there is also less Ca²⁺ available for systolic contraction, contributing to systolic impairment. Abnormalities of Ca²⁺ handling also contribute to the ventricular arrhythmias seen in HF: increased levels of Ca²⁺ in the cytoplasm are partially compensated for by increased expression of the NCX²³ as a mechanism to remove Ca²⁺. In addition, the RyRs have a higher opening probability in failing hearts²⁴ and both these factors increase Ca²⁺ leakage in diastole, which promotes the delayed afterdepolarizations and triggered activity that can ultimately result in ventricular arrhythmias.²⁵

Figure 2.

Schematic of a cardiomyocyte showing normal Ca²⁺ handling during excitation-contraction coupling. (1) Normal Ca²⁺ cycling begins with the cardiac action potential depolarizing the surface membrane and triggering a small Ca²⁺ current into the cytoplasm through L-type Ca²⁺ channels. (2) Triggering of a much larger influx of Ca²⁺ from the sarcoplasmic reticulum (SR) store through the ryanodine receptor (RyR). (3) Calcium-induced calcium release triggers contraction through the binding of Ca²⁺ to the troponin C component of the cardiac myofilaments. (4) During diastole, Ca²⁺ is taken back up in to the SR through the action of sarcoplasmic (endoplasmic) reticulum Ca²⁺ ATPase 2a (SERCA2a) and extruded from the cell by the Na⁺–Ca²⁺ exchange (NCX). (5) SERCA2a function is regulated by phospholamban (PLN).

Thus, the abnormalities of Ca²⁺ handling in HF can play a role in systolic dysfunction, diastolic dysfunction and arrhythmogenesis, making it an attractive system to target therapeutically. There are numerous molecules involved in Ca²⁺ handling, but altered SERCA2a appears to play a critical role. In both human failing myocardium and various animal models of HF, mRNA levels of SERCA2a are reduced^26–28 and the activity of SERCA2a is consistently shown to be reduced in failing myocardium.^29–31 Further support for the importance of SERCA2a activity in HF comes from evidence that a SERCA2a knock-out mouse develops measurable indices of systolic and diastolic dysfunction in vivo,³² that SERCA2a gene therapy improves important cardiac parameters in HF (see later) and that targeting the regulators of SERCA2a (ie, PLN and post-translational modifications such as small ubiquitin-related modifier 1 (SUMO1); see later) also have beneficial effects on cardiac function.

SERCA2a as a Target

Pharmacological agents have thus far been unable to target SERCA2a with sufficient efficacy to make conventional drug development viable, perhaps because of the lack of SERCA2a protein to target, but gene therapy can succeed where pharmacology has failed and restore levels of both protein and activity. Gene therapy can be used to either target SERCA2a directly or to manipulate the regulators of SERCA2a activity: PLN and post transcriptional modification by SUMO-1 (Figure 3). When PLN is unphosphorylated it inhibits SERCA2a function and it has been shown that increased levels of PLN in a rabbit model are detrimental and result in HF.³³ Also, the SERCA2a:PLN ratio is reduced in patients with advanced HF, with a relative increase in the unphosphorylated PLN fraction allowing PLN to inhibit SERCA2a. When PLN is phosphorylated by protein kinase A (PKA) or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) it forms a pentamer, relieving the inhibition of SERCA2a (Figure 3).³⁴ The activity of PKA and CaMKII in normal physiology is increased by β-adrenergic stimulation, which has the effect of relieving the inhibition of SERCA2a and resulting in increased Ca²⁺ reuptake in to the SR, allowing more rapid relaxation and more Ca²⁺ available for contraction.^35,36 PKA also has an indirect mechanism for increasing the phosphorylation of PLN by increasing the activity of inhibitor-1 (I-1), which in turn inhibits protein phosphatase 1 (PP1).

Figure 3.

Regulation of SERCA2a by phospholamban (PLN). When PLN is unphosphorylated it acts as an inhibitor of SERCA2a activity (Left). When PLN is phosphorylated it forms a pentamer and the inhibition of SERCA2a is relieved. Factors increasing the activity of SERCA2a are displayed. Ca²⁺, calcium; CaMkII, Ca²⁺/calmodulin-dependent kinase II; I-1, inhibitor 1; PKA, protein kinase A; PP1, protein phosphatase 1.

SUMOs are a group of proteins that alter the function of other proteins. This action is meditated through post-translational modification, described as SUMOylation. The levels of cytoplasmic SUMO1 are regulated in parallel to the levels and activity of SERCA2a and SUMOylation of SERCA2a increases its stability and activity. Laboratory studies have demonstrated that SUMOylation of SERCA2a is reduced in HF.³⁷

Therefore, strategies to increase the activity of SERCA2a include: (1) increased expression of SERCA2a ; (2) any strategy that reduces the quantity of inhibitory unphosphorylated PLN present (commonly by increasing the phosphorylation of PLN); and (3) increasing SUMO activity.

Laboratory Evidence for Targeting SERCA2a

There is laboratory evidence supporting each of the 3 different strategies of increasing SERCA2a activity:

Increased Expression of SERCA2a via Gene Delivery

Our research group and others have repeatedly demonstrated that SERCA2a gene transfer improves a range of cardiac physiological parameters in a variety of experimental models of HF. Some of these beneficial effects include increased contractility, normalization of LV size, reduction in both arrhythmias and arrhythmia susceptibility, as well as normalization of Ca²⁺ transients, cardiac microstructure and fetal gene expression (Table 4).

Table 4. Physiological Parameters Enhanced by SERCA2a Gene Therapy in Preclinical Studies

Physiological parameter	Model	Effect of SERCA2a gene therapy
Cellular-Ca2+ transient alteration	Neonatal rat cardiomyocytes (non-failing)	Increased amplitude of Ca2+ transients and faster relaxation kinetics38
	Rat cardiomyocytes treated with PMA (reduces endogenous SERCA2a expression)	Shortens Ca2+ transients39
	Isolated failing human cardiomyocytes	Normalized Ca2+ transients40
Contractility	Neonatal rat cardiomyocytes (non-failing)	Enhanced contraction measured by shortening of myocytes38
	Rabbit myocytes (non-failing)	Reduced time to peak contraction and 50% relaxation41
	Isolated failing human cardiomyocytes	Faster contraction velocity and enhanced relaxation velocity of myocytes40
	In vivo rat model of HF (aortic banding)	Improved rate of change of LV systolic pressure and rate of isovolumic relaxation42
	In vivo porcine model of HF (MR)	Increased rate of change of systolic LV pressure43
LV remodeling	In vivo rat model of HF (aortic banding)	Normalized LV volumes44
	In vivo porcine model of HF (MR)	Reduced LV size43
Arrhythmia	In vivo rat model og HF (post-infarction)	Reduced in vivo ventricular arrhythmias, reduced susceptibility to arrhythmias during programmed stimulation ex vivo, reduced Ca2+ leak45
	Guinea pig model of HF (aortic banding)	Reduced cardiac alternans and susceptibility to inducible ventricular arrhythmias46
Biomarkers	In vivo porcine model of HF (MR)	BNP stabilized in treatment group (increased in placebo)43
Cardiac microstructure	Rat model of HF (post-infarction)	Restoration of the sarcolemmal and t-tubule microarchitecture47 which is severely perturbed in chronic HF48
Fetal gene expression	Rat model of HF (aortic banding)	Partial normalization of the transcriptome49
	Rat model of HF (post-infarction)	Partial normalization of microRNA signature50

BNP, B-type natriuretic peptide; LV, left ventricular; MR, mitral regurgitation. Other abbreviations as in Table 3.

Reduction in Phospholamban-Mediated Inhibition of SERCA2a

Another approach to improve Ca²⁺ handling in HF involves relieving the PLN-mediated inhibition of SERCA2a to indirectly increase its activity. This can be achieved by directly targeting PLN or by targeting PP1 and I-1 to increase the phosphorylation of PLN.

Gene Therapy Targeting PLN AAV-mediated overexpression of a mutant form of PLN prevented deterioration in cardiomyopathic hamsters and post-myocardial infarction rats.^38,39 Large-animal studies have confirmed these findings in a sheep model of HF treated with an inhibitory phospholamban peptide, resulting in improved SERCA2a activity together with improved systolic and diastolic LV function.⁴⁰ Similar findings have been observed in human cardiomyocytes, whereby suppressing levels of PLN improved contraction and relaxation velocities similar to the benefit seen with gene transfer of SERCA2a.⁴¹

As an alternative to conventional gene therapy, RNA interference therapy was used for the first time in a rat model of HF to suppress PLN expression. Both adenoviral and AAV vectors were used to generate small hairpin RNA to silence PLN and this resulted in suppressed PLN protein expression. As a consequence, SERCA2a activity increased, resulting in improved systolic and diastolic cardiac function.⁴² These results should be treated cautiously, however, when translating this animal work of PLN inhibition in to a potential treatment for HF in humans. A naturally occurring mutation of PLN in the human population is not associated with improved cardiac function; conversely, it leads to a severe HF phenotype.⁴³

Targeting PP1 and I-1 The inhibitory effect of PLN on SERCA2a activity is maximized when PLN is unphosphorylated. PP1 dephosphorylates PLN, thereby increasing the inhibitory action of PLN on SERCA2a (Figure 3). PP1 in turn is inhibited by I-1. As such, PP1 indirectly inhibits SERCA2a activity whereas I-1 increases SERCA2a activity. HF is associated with elevated PP1 activity in humans. Murine models in which either I-1 is over expressed in a constitutively active form (I-1c) or PP1 is inhibited have shown improved cardiac function and protection from HF.^44–46

Increased SERCA2a Activity Through Enhanced Post-Translational Modification (SUMOylation)

Gene therapy with AAV9.SUMO1 vector to increase SUMO1 expression in an animal model of HF led to markedly improved cardiac function, comparable to SERCA2a gene delivery,³⁷ and was additive when dual AAV9.SERCA and AAV9.SUMO was applied to the failing heart in vivo.

Clinical Evidence for Targeting SERCA2a

There are only 2 clinical trials reporting the results of gene therapy in patients with HF and therefore clinical evidence for this new therapeutic approach is limited. One of the studies was a phase 1 study investigating the safety of SDF-1 DNA injected into the peri-infarct zone of patients with HF.⁴ The hypothesis under investigation was that SDF-1 attracts stem cells and enhanced cell-mediated repair mechanisms, which would support myocardial regeneration. That study demonstrated safety and some improvement in measures of angina.

The other reported study delivered AAV1-mediated SERCA2a gene therapy (AAV1.SERCA2a), commercially known as MYDICAR^®, to patients with advanced HF (CUPID [Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease] study).^2,47,48 This was the first ever clinical trial of gene therapy in patients with HF and remains the only clinical trial of SERCA2a gene therapy to have been reported. The CUPID trial (ClinicalTrials.gov identifier: NCT00454818) initially enrolled 12 patients in an open-label phase 1 study to investigate the safety of 4 doses of AAV1.SERCA2a. This was followed up with the phase 2 study in which 39 patients with advanced HF were enrolled and randomized to receive 1 of 3 doses of intracoronary AAV1.SERCA2a vs. placebo. AAV1.SERCA2a consists of an AAV1 capsid and the human SERCA2a cDNA flanked by inverted terminal repeats. Patients were enrolled with severe HF of ischemic and non-ischemic etiology, often on cardiac transplantation waiting lists, though this was not mandatory. Treatment success was determined by examining concordant trends in the following endpoints: patient’s symptoms (New York Heart Association functional class, Minnesota Living With Heart Failure Questionnaire), functional status (6-min walk test, and V̇O₂max), NT-proBNP levels, and echocardiographic measures.

Initial results at 1 year revealed that patients in the “high-dose” treatment group demonstrated improvement or stabilization of symptoms, functional class, NT-proBNP level, and LV end-systolic volume (LVESV).⁴⁹ There was also a significant increase in time-to-cardiovascular events and a decreased frequency of cardiovascular events per patient in all patients receiving AAV1.SERCA2a. Importantly, patients in the treatment arm showed no increase in adverse events, disease-related events, or arrhythmias, which correlates with our preclinical study demonstrating the antiarrhythmic effects of SERCA2a gene therapy in the context of chronic HF.⁵⁰ Recently, 3-year follow-up data have been reported,³ which show sustained benefit in the high-dose arm, with no safety concerns up to this time point, and evidence of effective and persistent transgene expression measured on PCR in 3 patients from the high-dose arm with tissue samples available.

Ongoing Clinical Trials of SERCA2a Gene Therapy for HF

The CUPID trial has paved the way for 3 other clinical trials of SERCA2a gene therapy in patients with HF. These trials are either underway or due to start in 2014 and are investigating complementary aspects of the potential benefit of SERCA2a gene therapy; clinical outcomes, gene and protein expression and the effects on LV remodeling.

Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease 2 (CUPID2) Trial (ClinicalTrials.gov identifier: NCT01643330)

This is the first gene therapy trial for HF to recruit patients from outside the USA. It is a phase 2, double-blind, placebo-controlled, international, multicenter, randomized study evaluating the safety and efficacy of intracoronary administration of AAV1.SERCA2a in subjects with HF. Recruitment has now completed.

Patients who had chronic HF and an ejection fraction ≤35% with NYHA 3 or 4 class symptoms despite optimal medical therapy were recruited. Patients were also required to have either been admitted with decompensated HF in the previous 6 months or a raised NT-proBNP (>1,200 ng/L in sinus rhythm or >1,600 ng/L in atrial fibrillation). Patients who met these criteria were pre-screened to determine if the neutralizing antibody to AAV1 was present. If the antibody was present they were excluded from the trial.

The 250 patients were recruited from over 50 sites across the world including our own center. They were randomized 1:1 to receive a one-off 10-min intracoronary infusion of placebo or the “high dose” of AAV1.SERCA2a (1×10¹³ DNAse resistant particles). GTN was co-administered intravenously during the investigational product infusion.

The primary endpoint of the trial is time-to-recurrent HF-related hospitalizations in the presence of terminal events (all-cause death, heart transplantation, LVAD implantation). This will be analyzed using the joint frailty model and results are expected in 2015–2016.

SERCA2a Gene Therapy in LVAD Patients (SERCA-LVAD)

This trial is approved to start at 2 centers in the UK in 2014. Patients with advanced ischemic or non-ischemic HF who have received a LVAD for chronic HF are to be recruited. Patients will receive an intracoronary infusion of either AAV1.SERCA2a or placebo. Myocardial tissue samples will be taken at the time of LVAD implantation, before gene therapy treatment, and follow-up samples will be taken either at the time of transplantation or via percutaneous biopsy. The unique aspect of this trial is that by taking tissue biopsies the investigators will be able to correlate changes in clinical outcome with the expression of SERCA2a and correction of the underlying calcium handling abnormalities of HF. Importantly, both neutralizing antibody positive and negative patients will be enrolled, to evaluate the effect of the antibody on gene expression efficacy, and whether the presence of neutralizing antibodies is a justifiable exclusion criterion.

AGENT-HF TRIAL (AAV1.SERCA2a GENe Therapy Trial in Heart Failure)

This trial is a phase 2 single-center, double-blind, randomized, placebo-controlled study at the Pitié-Salpêtrière Hospital Institute of Cardiology, Paris, France. The primary objective of this study is to investigate the effect of AAV1.SERCA2a on cardiac remodeling parameters in patients with severe HF. The primary outcome will be the effect of gene therapy on LVESV (measured with a 256-slice CT-scan) 6 months after treatment. Secondary endpoints will include changes in the ejection fraction, diastolic volumes, V̇O₂max, echocardiographic remodeling, BNP and biological safety profile. This trial has begun recruitment.

Potential Future for Gene Therapy in HF

SERCA2a is only 1 target for gene therapy that may benefit patients with HF and the results of ongoing studies will be available in the coming years. There is a host of other molecular abnormalities that could ultimately be targeted by gene therapy in the future. These include various other molecules modifying SERCA2a function such as PLN, SUMO-1 and I-1; components of the β-adrenergic system such as G-protein-coupled receptor kinase or adenyl cyclase; the Ca²⁺ -binding protein S100A1; or pathways of cell repair and regeneration. Furthermore, there have been major advances in the way we approach the relationship between genetics and disease in the modern era with the widespread use of microarrays. Rather than selecting a gene of interest on the basis of a link to an underlying molecular process, it is now common to analyze thousands of genes simultaneously and the challenge is then to determine which of the genes with changed expression in disease are physiologically important. Microarray profiles of human hypertrophic, dilated and failing hearts representing >10,000 genes have been reported;^51,52 621 genes were upregulated in dilated cardiomyopathy (DCM), with 37 being induced by more than 2.5-fold. 263 genes were downregulated in DCM. Regulated genes were represented across functional classes, with a tendency for more genes in the categories of protein synthesis and metabolism. In addition, there is a pro-apoptotic shift in the tumor necrosis factor-α pathway.⁵³ Such studies have led to the identification of a large number of genes that are regulated in response to HF, but teasing out which are important to the underlying pathology and which can then be intervened upon for therapeutic effect is sometime away.

In addition to increasing the number of targets for gene therapy, it is conceivable that future strategies may be to target more than 1 gene in an individual. In laboratory research, this approach demonstrated additive benefit in a HF model when SERCA2a and SUMO-1 were targeted together.³⁷ This approach would be analogous to current medical therapy in which multiple systems are targeted (eg, using ACEIs and β-blockers).

For gene therapy to be a viable treatment option in the future, there are a number of hurdles to be tackled. The first is continued evidence of safety from ongoing clinical trials and long term follow-up. There is also the significant limitation of the prevalence of neutralizing antibodies to AAV vectors, which excludes approximately 50% of patients from receiving this therapy. There are various strategies under consideration to overcome this barrier. Lastly, excluding gene therapy targets involved in cell regeneration, the majority of targets (such as SERCA2a) require surviving myocytes to derive benefit from therapy. It is conceivable that patients with large territories of infarcted myocardium may derive less or no benefit if they have only a small amount of surviving myocardium.

Conclusions

Gene therapy provides optimism that there may be a new armament of treatment options available to physicians treating patients with HF in the future. Laboratory studies have shown that SERCA2a gene therapy can improve contractility and reduce ventricular arrhythmias in animal models of HF in an energetically favorable manner. This may be the first positive inotrope that reduces the risk of arrhythmias. There are still a significant number of hurdles to overcome and the clinical trials are still at an early stage in the development pathway. Crucially, it also remains to be seen whether the failing human heart can be successfully transduced with therapeutic DNA in sufficient levels to affect the underlying failing myocardial substrate and initiate clinically meaningful reverse remodeling.

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