Kleopatra Rapti

Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models.

SUMO-1 Gene Transfer Improves Cardiac Function in a Large-Animal Model of Heart Failure

Cardiac gene delivery of small ubiquitin-related modifier 1 (SUMO-1) improved cardiac function and stabilized left ventricular volumes in a swine model of ischemic heart failure. Cardiac Gene Therapy to the Rescue Heart failure (HF) is one of the top reasons for hospitalization among the elderly and remains a leading cause of death in the Western world. Gene therapy has been proposed as a way to coerce the heart into being healthy by targeting cardiac-specific pathways. Replacing the gene sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2a) in patients has made it to phase 2b/3 trials, with early signs pointing to an improvement in HF-related events. To boost the effects of SERCA2a, Tilemann et al. designed a large-animal study that also tests the delivery of small ubiquitin-related modifier 1 (SUMO-1)—an important regulator of SERCA2a. The authors compared the efficacy of SUMO-1 gene transfer to SERCA2a gene transfer alone and to the combined delivery of both genes in a pig model of HF. In addition to being safe, administering SUMO-1 directly to the heart of these animals showed improved cardiac contractility and prevented left ventricular dilatation (two major aspects of HF). According to the authors, the functional improvements in this model of heart failure are most likely the result of improved SR Ca2+ ATPase activity afforded by increased SUMO-1 protein levels. Delivery of both SUMO-1 and SERCA2a suggested additional beneficial effects, but more mechanistic studies will be needed to understand this potential synergy. With the precedent set by the SERCA2a clinical trials, moving SUMO-1 gene therapy from pigs to humans seems likely in the short-term. Recently, the impact of small ubiquitin-related modifier 1 (SUMO-1) on the regulation and preservation of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2a) function was discovered. The amount of myocardial SUMO-1 is decreased in failing hearts, and its knockdown results in severe heart failure (HF) in mice. In a previous study, we showed that SUMO-1 gene transfer substantially improved cardiac function in a murine model of pressure overload–induced HF. Toward clinical translation, we evaluated in this study the effects of SUMO-1 gene transfer in a swine model of ischemic HF. One month after balloon occlusion of the proximal left anterior descending artery followed by reperfusion, the animals were randomized to receive either SUMO-1 at two doses, SERCA2a, or both by adeno-associated vector type 1 (AAV1) gene transfer via antegrade coronary infusion. Control animals received saline infusions. After gene delivery, there was a significant increase in the maximum rate of pressure rise [dP/dt(max)] that was most pronounced in the group that received both SUMO-1 and SERCA2a. The left ventricular ejection fraction (LVEF) improved after high-dose SUMO-1 with or without SERCA2a gene delivery, whereas there was a decline in LVEF in the animals receiving saline. Furthermore, the dilatation of LV volumes was prevented in the treatment groups. SUMO-1 gene transfer therefore improved cardiac function and stabilized LV volumes in a large-animal model of HF. These results support the critical role of SUMO-1 in SERCA2a function and underline the therapeutic potential of SUMO-1 for HF patients.

Delivery of gelfoam-enabled cells and vectors into the pericardial space using a percutaneous approach in a porcine model

Intrapericardial drug delivery is a promising procedure, with the ability to localize therapeutics with the heart. Gelfoam particles are nontoxic, inexpensive, nonimmunogenic and biodegradable compounds that can be used to deliver therapeutic agents. We developed a new percutaneous approach method for intrapericardial injection, puncturing the pericardial sac safely under fluoroscopy and intravascular ultrasound (IVUS) guidance. In a porcine model of myocardial infarction (MI), we deployed gelfoam particles carrying either (a) autologous mesenchymal stem cells (MSCs) or (b) an adenovirus encoding enhanced green fluorescent protein (eGFP) 48 h post-MI. The presence of MSCs and viral infection at the infarct zone was confirmed by immunoflourescence and PCR. Puncture was performed successfully in 16 animals. Using IVUS, we successfully determined the size of the pericardial space before the puncture, and safely accessed that space in setting of pericardial effusion and also adhesions induced by the MI. Intrapericardial injection of gelfoam was safe and reliable. Presence of the MSCs and eGFP expression from adenovirus in the myocardium were confirmed after delivery. Our novel percutaneous approach to deliver (stem-) cells or adenovirus was safe and efficient in this pre-clinical model. IVUS-guided delivery is a minimally invasive procedure that seems to be a promising new strategy to deliver therapeutic agents locally to the heart.

Targeted Gene Therapy for the Treatment of Heart Failure

Chronic heart failure is one of the leading causes of morbidity and mortality in Western countries and is a major financial burden to the health care system. Pharmacologic treatment and implanting devices are the predominant therapeutic approaches. They improve survival and have offered significant improvement in patient quality of life, but they fall short of producing an authentic remedy. Cardiac gene therapy, the introduction of genetic material to the heart, offers great promise in filling this void. In-depth knowledge of the underlying mechanisms of heart failure is, obviously, a prerequisite to achieve this aim. Extensive research in the past decades, supported by numerous methodological breakthroughs, such as transgenic animal model development, has led to a better understanding of the cardiovascular diseases and, inadvertently, to the identification of several candidate genes. Of the genes that can be targeted for gene transfer, calcium cycling proteins are prominent, as abnormalities in calcium handling are key determinants of heart failure. A major impediment, however, has been the development of a safe, yet efficient, delivery system. Nonviral vectors have been used extensively in clinical trials, but they fail to produce significant gene expression. Viral vectors, especially adenoviral, on the other hand, can produce high levels of expression, at the expense of safety. Adeno-associated viral vectors have emerged in recent years as promising myocardial gene delivery vehicles. They can sustain gene expression at a therapeutic level and maintain it over extended periods of time, even for years, and, most important, without a safety risk.

Cardiac I-1c Overexpression With Reengineered AAV Improves Cardiac Function in Swine Ischemic Heart Failure

Cardiac gene therapy has emerged as a promising option to treat advanced heart failure (HF). Advances in molecular biology and gene targeting approaches are offering further novel options for genetic manipulation of the cardiovascular system. The aim of this study was to improve cardiac function in chronic HF by overexpressing constitutively active inhibitor-1 (I-1c) using a novel cardiotropic vector generated by capsid reengineering of adeno-associated virus (BNP116). One month after a large anterior myocardial infarction, 20 Yorkshire pigs randomly received intracoronary injection of either high-dose BNP116.I-1c (1.0 × 10(13) vector genomes (vg), n = 7), low-dose BNP116.I-1c (3.0 × 10(12) vg, n = 7), or saline (n = 6). Compared to baseline, mean left ventricular ejection fraction increased by 5.7% in the high-dose group, and by 5.2% in the low-dose group, whereas it decreased by 7% in the saline group. Additionally, preload-recruitable stroke work obtained from pressure-volume analysis demonstrated significantly higher cardiac performance in the high-dose group. Likewise, other hemodynamic parameters, including stroke volume and contractility index indicated improved cardiac function after the I-1c gene transfer. Furthermore, BNP116 showed a favorable gene expression pattern for targeting the heart. In summary, I-1c overexpression using BNP116 improves cardiac function in a clinically relevant model of ischemic HF.

Stimulating Myocardial Regeneration with Periostin Peptide in Large Mammals Improves Function Post-Myocardial Infarction but Increases Myocardial Fibrosis

Aims Mammalian myocardium has a finite but limited capacity to regenerate. Experimentally stimulating proliferation of cardiomyocytes with extracellular regeneration factors like periostin enhances cardiac repair in rodents. The aim of this study was to develop a safe method for delivering regeneration factors to the heart and to test the functional and structural effects of periostin peptide treatment in a large animal model of myocardial infarction (MI). Methods and Results We developed a controlled release system to deliver recombinant periostin peptide into the pericardial space. A single application of this method was performed two days after experimental MI in swine. Animals were randomly assigned to receive either saline or periostin peptide. Experimental groups were compared at baseline, day 2, 1 month and 3 months. Treatment with periostin peptide increased the EF from 31% to 41% and decreased by 22% the infarct size within 12 weeks. Periostin peptide-treated animals had newly formed myocardium strips within the infarct scar, leading to locally improved myocardial function. In addition the capillary density was increased in animals receiving periostin. However, periostin peptide treatment increased myocardial fibrosis in the remote region at one week and 12 weeks post-treatment. Conclusion Our study shows that myocardial regeneration through targeted peptides is possible. However, in the case of periostin the effects on cardiac fibrosis may limit its clinical application as a viable therapeutic strategy.

Aortic Implantation of Mesenchymal Stem Cells after Aneurysm Injury in a Porcine Model

BACKGROUND Cell-based therapies are being evaluated in the setting of degenerative pathophysiologic conditions. The search for the ideal method of delivery and improvement in cell engraftment continue to pose a challenge. This study explores the feasibility of introducing mesenchymal stem cells (MSC) following aortic injury in a porcine model. METHODS Bone marrow-derived MSC were obtained from eight pigs, characterized for the MSC markers CD13 and CD 29, labeled with green fluorescent protein (GFP), and collected for autologous injection in a porcine model of abdominal aortic aneurysm (AAA). The pigs were euthanized (1-7 d) after the procedure to assess the histologic characteristics and presence of MSC in the aortic tissue. Negative controls included noninjured aorta. Tracking of the MSC was conducted by the identification of the GFP-labeled cells using immunofluorescence. RESULTS AAA sections stained with hematoxylin and eosin showed disorganization of the aortic tissue; collagen-muscle-elastin stain demonstrated fragmentation of elastin fibers. The presence of the implanted MSC in the aortic wall was evidenced by fluorescent microscopy showing GFP labeled cells. Engraftment of MSC up to 7 d after introduction was observed. CONCLUSION Autologous implantation of bone marrow-derived MSC following aortic injury in a porcine model may be successfully accomplished. The long-term impact and therapeutic value of such cell-based therapy will require further investigation.

Altered Spatiotemporal Dynamics of the Mitochondrial Membrane Potential in the Hypertrophied Heart

Chronically elevated levels of oxidative stress resulting from increased production and/or impaired scavenging of reactive oxygen species are a hallmark of mitochondrial dysfunction in left ventricular hypertrophy. Recently, oscillations of the mitochondrial membrane potential (DeltaPsi(m)) were mechanistically linked to changes in cellular excitability under conditions of acute oxidative stress produced by laser-induced photooxidation of cardiac myocytes in vitro. Here, we investigate the spatiotemporal dynamics of DeltaPsi(m) within the intact heart during ischemia-reperfusion injury. We hypothesize that altered metabolic properties in left ventricular hypertrophy modulate DeltaPsi(m) spatiotemporal properties and arrhythmia propensity.

Active Inhibitor-1 Maintains Protein Hyper-Phosphorylation in Aging Hearts and Halts Remodeling in Failing Hearts

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

Effectiveness of gene delivery systems for pluripotent and differentiated cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.