Gene Therapy For Heart Failure Research Articles

Cardiac-targeting magnetic lipoplex delivery of SH-IGF1R plasmid attenuate norepinephrine-induced cardiac hypertrophy in murine heart.

Recent studies have demonstrated a number of molecular mechanisms contributing to the initiation of cardiac hypertrophy response to pressure overload. IGF1R (insulin-like growth factor-1 receptor), an important oncogene, is overexpressed in hypertrophic heart and mediates the hypertrophic pathology process. In this study, we applied with liposomal magnetofection that potentiated gene transfection by applying an external magnetic field to enhance its transfection efficiency. Liposomal magnetofection provided high efficiency in transgene expression in vivo. In vivo, IGF1R-specific-shRNA (small-hairpin RNA) by magnetofection inhibited IGF1R protein expression by 72.2±6.8, 80.7±9.6 and 84.5±5.6%, at 24, 48 and 72 h, respectively, after pGFPshIGF1R injection, indicating that liposomal magnetofection is a promising method that allows the targeting of gene therapy for heart failure. Furthermore, we found that the treated animals (liposomal magnetofection with shIGF1R) showed reduced septal and posterior wall thickness, reduced HW:BWs (heart weight-to-body weights) compared with controls. Moreover, we also found that liposomal magnetofection-based shIGF1R transfection decreased the expression level of p-ERK (phosphorylated extracellular-signal-regulated kinase)1/2, p-AKT1 (phosphorylated protein kinase B1) compared with untreated hearts. These results suggested that liposomal magnetofection-mediated IGF1R-specific-shRNA may be a promising method, and suppression the IGF1R expression inhibited norepinephrine-induced cardiac hypertrophic process via inhibiting PI3K (phosphoinositide 3-kinase)/AKT pathway.

An Innovative Needleless Liquid Jet Injection Delivery Method for Heart Failure Gene Therapy: Preliminary Study Featuring AAV9 Demonstrates Greater Cardiac Specificity Versus Standard Approaches

An Innovative Needleless Liquid Jet Injection Delivery Method for Heart Failure Gene Therapy: Preliminary Study Featuring AAV9 Demonstrates Greater Cardiac Specificity Versus Standard Approaches

Clinical Trials of Gene Therapy for Heart Failure: Quantitation of the Cardiac Calcium Pump by Immunochemistry

We have used quantitative immunoblot, enzyme-linked immunosorbant assay (ELISA), and capillary western to measure absolute cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a isoform) content in order to determine specific activity. Celladon, Inc., is developing a gene therapy treatment for heart failure (HF) where SERCA2a is the target protein, and they need an accurate measure of SERCA2a to calculate specific activity of enzyme produced by the gene therapy construct. Patients with HF produce too little SERCA2a or SERCA2a that has too low of activity to maintain normal calcium cycling that is responsible for cardiac contraction. SERCA2a produced by MYDICAR, an adeno- associated viral (AAV) vector, must have comparable activity to endogenous SERCA2a to be useful as a gene therapy product. Immunoblot showed linear SERCA2a detection. We conclude that immunoblot measures absolute SERCA2a content, useful in measuring specific activity. This work was funded by Celladon and by NIH grants to DDT (R01 GM27906, P30 AR0507220, T32 AR007612).

Gene therapy in heart failure. SERCA2a as a therapeutic target.

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.

SERCA2a gene therapy in heart failure: an anti-arrhythmic positive inotrope.

Therapeutic options that directly enhance cardiomyocyte contractility in chronic heart failure (HF) therapy are currently limited and do not improve prognosis. In fact, most positive inotropic agents, such as β-adrenoreceptor agonists and PDE inhibitors, which have been assessed in HF patients, cause increased mortality as a result of arrhythmia and sudden cardiac death. Cardiac sarcoplasmic reticulum Ca(2)(+) -ATPase2a (SERCA2a) is a key protein involved in sequestration of Ca(2)(+) into the sarcoplasmic reticulum (SR) during diastole. There is a reduction of SERCA2a protein level and function in HF, which has been successfully targeted via viral transfection of the SERCA2a gene into cardiac tissue in vivo. This has enhanced cardiac contractility and reduced mortality in several preclinical models of HF. Theoretical concerns have been raised regarding the possibility of arrhythmogenic adverse effects of SERCA2a gene therapy due to enhanced SR Ca(2)(+) load and induction of SR Ca(2)(+) leak as a result. Contrary to these concerns, SERCA2a gene therapy in a wide variety of preclinical models, including acute ischaemia/reperfusion, chronic pressure overload and chronic myocardial infarction, has resulted in a reduction in ventricular arrhythmias. The potential mechanisms for this unexpected beneficial effect, as well as mechanisms of enhancement of cardiac contractile function, are reviewed in this article.

Heart Failure Gene Therapy

Gene therapy, aimed at the correction of key pathologies being out of reach for conventional drugs, bears the potential to alter the treatment of cardiovascular diseases radically and thereby of heart failure. Heart failure gene therapy refers to a therapeutic system of targeted drug delivery to the heart that uses formulations of DNA and RNA, whose products determine the therapeutic classification through their biological actions. Among resident cardiac cells, cardiomyocytes have been the therapeutic target of numerous attempts to regenerate systolic and diastolic performance, to reverse remodeling and restore electric stability and metabolism. Although the concept to intervene directly within the genetic and molecular foundation of cardiac cells is simple and elegant, the path to clinical reality has been arduous because of the challenge on delivery technologies and vectors, expression regulation, and complex mechanisms of action of therapeutic gene products. Nonetheless, since the first demonstration of in vivo gene transfer into myocardium, there have been a series of advancements that have driven the evolution of heart failure gene therapy from an experimental tool to the threshold of becoming a viable clinical option. The objective of this review is to discuss the current state of the art in the field and point out inevitable innovations on which the future evolution of heart failure gene therapy into an effective and safe clinical treatment relies.

AAV-mediated gene therapy for heart failure: enhancing contractility and calcium handling

Heart failure is a progressive, debilitating disease that is characterized by inadequate contractility of the heart. With an aging population, the incidence and economic burden of managing heart failure are anticipated to increase substantially. Drugs for heart failure only slow its progression and offer no cure. However, results of recent clinical trials using recombinant adeno-associated virus (AAV) gene delivery offer the promise, for the first time, that heart failure can be reversed. The strategy is to improve contractility of cardiac muscle cells by enhancing their ability to store calcium through increased expression of the sarco(endo)plasmic reticulum Ca2+-ATPase pump (SERCA2a). Preclinical trials have also identified other proteins involved in calcium cycling in cardiac muscle that are promising targets for gene therapy in heart failure, including the following: protein phosphatase 1, adenylyl cyclase 6, G-protein-coupled receptor kinase 2, phospholamban, SUMO1, and S100A1. These preclinical and clinical trials represent a “quiet revolution” that may end up being one of the most significant and remarkable breakthroughs in modern medical practice. Of course, a number of uncertainties remain, including the long-term utility and wisdom of improving the contractile performance of “sick” muscle cells. In this regard, gene therapy may turn out to be a way of buying additional time for actual cardiac regeneration to occur using cardiac stem cells or induced pluripotent stem cells.

Sarcoendoplasmic Reticulum Calcium Transport ATPase 2a

The development of cardiac-specific adeno-associated viral vectors capable of long-term transgenic expression has opened new avenues in the therapeutic approach to heart failure. Failing cardiomyocytes demonstrate altered calcium-handling secondary to depressed expression and activity of myocardial sarcoendoplasmic reticulum calcium ATPase 2a (SERCA2a). This observation has led to a large body of research investigating the therapeutic utility of SERCA2a gene therapy in heart failure.

Gene therapy for heart failure: where do we stand?

Advances in understanding of the molecular basis of myocardial dysfunction, together with the development of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Multiple components of cardiac contractility, including the Beta-adrenergic system, the calcium channel cycling pathway, and cytokine mediated cell proliferation, have been identified as appropriate targets for gene therapy. The development of efficient and safe vectors such as adeno-associated viruses and polymer nanoparticles has provided an opportunity for clinical application for gene therapy. The recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) has the potential to open a new era for gene therapy in the treatment of heart failure.

Potential of gene therapy as a treatment for heart failure

Advances in understanding the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, make gene-based therapy a promising treatment option for heart conditions. Cardiovascular gene therapy has benefitted from recent advancements in vector technology, design, and delivery modalities. There is a critical need to explore new therapeutic approaches in heart failure, and gene therapy has emerged as a viable alternative. Advances in understanding of the molecular basis of myocardial dysfunction, together with the development of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. The recent successful and safe completion of a phase 2 trial targeting the cardiac sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump (SERCA2a) has the potential to open a new era for gene therapy for heart failure.

Molecular targets in heart failure gene therapy: current controversies and translational perspectives

Use of gene therapy for heart failure is gaining momentum as a result of the recent successful completion of phase II of the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, which showed clinical safety and efficacy of an adeno-associated viral vector expressing sarco-endoplasmic reticulum calcium ATPase (SERCA2a). Resorting to gene therapy allows the manipulation of molecular targets not presently amenable to pharmacologic modulation. This short review focuses on the molecular targets of heart failure gene therapy that have demonstrated translational potential. At present, most of these targets are related to calcium handling in the cardiomyocyte. They include SERCA2a, phospholamban, S100A1, ryanodine receptor, and the inhibitor of the protein phosphatase 1. Other targets related to cAMP signaling are reviewed, such as adenylyl cyclase. MicroRNAs are emerging as novel therapeutic targets and convenient vectors for gene therapy, particularly in heart disease. We propose a discussion of recent advances and controversies in key molecular targets of heart failure gene therapy.

Gene delivery technologies for cardiac applications

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality in the Western society. Advances in understanding the molecular pathology of these diseases, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed these conditions within the reach of gene-based therapy. One of the cornerstones of limiting the effectiveness of gene therapy is the establishment of clinically relevant methods of genetic transfer. Recently there have been advances in direct and transvascular gene delivery methods with the use of new technologies. Current research efforts in IHD are focused primarily on the stimulation of angiogenesis, modify the coronary vascular environment and improve endothelial function with localized gene-eluting catheters and stents. In contrast to standard IHD treatments, gene therapy in HF primarily targets inhibition of apoptosis, reduction in adverse remodeling and increase in contractility through global cardiomyocyte transduction for maximal efficacy. This article will review a variety of gene-transfer strategies in models of coronary artery disease and HF and discuss the relative success of these strategies in improving the efficiency of vector-mediated cardiac gene delivery.

Phospholamban mutants compete with wild type for SERCA binding in living cells

We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca2+ cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLBM) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLBM in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLBM and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLBM for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.

Targeting GRK2 by gene therapy for heart failure: benefits above β-blockade

Heart failure (HF) is a common pathological end point for several cardiac diseases. Despite reasonable achievements in pharmacological, electrophysiological and surgical treatments, prognosis for chronic HF remains poor. Modern therapies are generally symptom oriented and do not currently address specific intracellular molecular signaling abnormalities. Therefore, new and innovative therapeutic approaches are warranted and, ideally, these could at least complement established therapeutic options if not replace them. Gene therapy has potential to serve in this regard in HF as vectors can be directed toward diseased myocytes and directly target intracellular signaling abnormalities. Within this review, we will dissect the adrenergic system contributing to HF development and progression with special emphasis on G-protein-coupled receptor kinase 2 (GRK2). The levels and activity of GRK2 are increased in HF and we and others have demonstrated that this kinase is a major molecular culprit in HF. We will cover the evidence supporting gene therapy directed against myocardial as well as adrenal GRK2 to improve the function and structure of the failing heart and how these strategies may offer complementary and synergistic effects with the existing HF mainstay therapy of β-adrenergic receptor antagonism.

Model‐specific selection of molecular targets for heart failure gene therapy

Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of β-adrenergic receptor signaling, Ca(2+) handling proteins and angiogenesis in the most common extrinsic models of HF.

Genetic treatment of heart rhythm disorders—where do we stand?

C d a i p p Groundbreaking discoveries on genetic loci that underlie arrhythmia syndromes, which commenced in the early 1990s, have immensely increased our understanding of these syndromes. These discoveries also rigorously changed daily clinical practice for cardiologists who were faced with the enquiries of presymptomatic family members of symptomatic individuals who were known with an arrhythmia syndrome. Now it has become possible to screen the genome for the risk of arrhythmias or even sudden death and, understandingly, this took a flight. Still, the holy grail of genetic discoveries in disease is in its therapeutic applicability and that promise is indeed coming our way. For example, in Duchenne muscular dystrophy, the increasing knowledge of the diseased locus has recently led to therapeutic gene therapy in a relatively small number of patients in whom the diseased exon was selectively bypassed. For rrhythmia syndromes, we are still in a preclinical phase, ut efforts to find curative options by means of genetic herapy are growing. Currently, genetic therapy for atrial brillation and bradycardia/conduction disease seems to dominate the field, but ventricular arrhythmias in ischemic conditions are also thoroughly investigated (for a recent review, see, eg, Boink and Rosen). The first in-human studies into the use of gene therapy in heart failure are even underway. In this issue of HeartRhythm, Soucek and colleagues from Heidelberg, Germany, presnt to us their preclinical work on genetic therapy for atrial brillation. Delay of atrial repolarization is considered one pproach to genetic therapy for atrial fibrillation, as coninuing propagation or reentry of fibrillation waves can be mpeded by prolonging the atrial effective refractory period ERP). The main currents responsible for the atrial ERP are outward potassium currents: IKr, IKur, IKs, and IK1. In their tudy, Soucek et al focused on suppressing the IKr current y means of introducing an engineered gene that encodes an Kr-suppressing (dominant-negative) protein into the atria of pigs with the use of an adenovirus vector. Their aim was to

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLBM) can compete with wild-type PLB (PLBW) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLBW, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in KCa, measured from the Ca2+-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLBW, while the potency of each of the LOF PLBM was about one third that of PLBW. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLBW and LOF PLBM, inhibitory potency was reduced relative to that of PLBW alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLBW was decreased by both (unlabeled) LOF PLBM. These results show that LOF PLBM can compete both physically and functionally with PLBW, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLBM targeted for gene therapy of heart failure in humans.

Structural Dynamics of SERCA and Phospholamban by TR-FRET and TIRF

We used fluorescence to investigate the structure and dynamics of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Time-resolved fluorescence energy transfer (TR-FRET) was used to study the oligomeric interaction between PLB and SERCA. Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. We used polarized TIRF to study the structure of PLB in the presence and absence of SERCA in a supported lipid bilayer. Polarized TIRF data shows that the cytoplasmic helix of PLB, labeled with bifunctional rhodamine, is close to be parallel to the membrane surface. SERCA binding lifts the cytoplasmic helix away from the membrane surface. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Gene therapy during cardiac surgery: role of surgical technique to minimize collateral organ gene expression

Effective gene therapy for heart failure has not yet been achieved clinically. The aim of this study is to quantitatively assess the cardiac isolation efficiency of the molecular cardiac surgery with recirculating delivery (MCARD™) and to evaluate its efficacy as a means to limit collateral organ gene expression. 10(14) genome copies (GC) of recombinant adeno-associated viral vector 6 encoding green fluorescent protein under control of the cytomegalovirus promoter was delivered to the nine arrested sheep hearts. Blood samples were assessed using real-time quantitative polymerase chain reaction (RT QPCR). Collateral organ gene expression was assessed at four-weeks using immunohistochemical staining. The blood vector GC concentration in the cardiac circuit during complete isolation trended from 9.59±0.73 to 9.05±0.65 (log GC/cm(3)), and no GC were detectable in the systemic circuit (P<0.001). The washing procedure performed prior to relinquishing the cardiac circuit decreased the systemic blood vector GC concentration >800-fold (P<0.001), consistent with >99% isolation efficiency. Conversely, incomplete isolation resulted in equalization of vector GC concentration in the circuits, leading to robust collateral organ gene expression. MCARD™ is an efficient, clinically translatable myocardial delivery platform for cardiac specific gene therapy. The cardiac surgical techniques utilized are critically important to limit collateral organ gene expression.