Enhancing the utility of adeno-associated virus gene transfer through inducible tissue-specific expression.

The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule.

Gene transfer in skeletal and cardiac muscle using recombinant adeno-associated virus.

Adeno-associated virus (AAV) is a DNA virus with a small (∼4.7 kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group.

Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping.

Although restoration of dystrophin expression via exon skipping in both cardiac and skeletal muscle has been successfully demonstrated in the mdx mouse, restoration of cardiac dystrophin expression in large animal models of Duchenne muscular dystrophy (DMD) has proven to be a challenge. In large animals, investigators have focused on using intravenous injection of antisense oligonucleotides (AO) to mediate exon skipping. In this study, we sought to optimize restoration of cardiac dystrophin expression in the golden retriever muscular dystrophy (GRMD) model using percutaneous transendocardial delivery of recombinant AAV6 (rAAV6) to deliver a modified U7 small nuclear RNA (snRNA) carrying antisense sequence to target the exon splicing enhancers of exons 6 and 8 and correct the disrupted reading frame.

Adeno-associated virus vector delivery to the heart.

Cardiac gene transfer may serve as a novel therapeutic approach in the treatment of heart disease. For it to reach its full potential, methods for highly efficient cardiac gene transfer must be available to investigators so that informative preclinical data can be collected and evaluated. We have recently optimized AAV-mediated cardiac gene transfer protocols in both the mouse and rat.

Myostatin is elevated in congenital heart disease and after mechanical unloading.

Background: Myostatin is a negative regulator of skeletal muscle mass whose activity is upregulated in adult heart failure (HF); however, its role in congenital heart disease (CHD) is unknown.

Methods: We studied myostatin and IGF-1 expression via Western blot in cardiac tissue at varying degrees of myocardial dysfunction and after biventricular support in CHD by collecting myocardial biopsies from four patient cohorts: A) adult subjects with no known cardiopulmonary disease (left ventricle, LV), (Adult Normal), (n = 5); B) pediatric subjects undergoing congenital cardiac surgery with normal RV size and function (right ventricular outflow tract, RVOT), (n = 3); C) pediatric subjects with worsening but hemodynamically stable LV failure [LV and right ventricle (LV, RV,)] with biopsy collected at the time of orthotopic heart transplant (OHT), (n = 7); and D) pediatric subjects with decompensated bi-ventricular failure on BiVAD support with biopsy collected at OHT (LV, RV, BiVAD), (n = 3).

Results: The duration of HF was longest in OHT patients compared to BIVAD.

Long-term systemic myostatin inhibition via liver-targeted gene transfer in golden retriever muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease affecting 1 in 3,500 newborn boys for which there is no effective treatment or cure. One novel strategy that has therapeutic potential for DMD is inhibition of myostatin, a negative regulator of skeletal muscle mass that may also promote fibrosis. Therefore, our goal in this study was to evaluate systemic myostatin inhibition in the golden retriever model of DMD (GRMD).

Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6-9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart.

Status of therapeutic gene transfer to treat cardiovascular disease in dogs and cats.

Gene therapy is a procedure resulting in the transfer of a gene(s) into an individual's cells to treat a disease, which is designed to produce a protein or functional RNA (the gene product). Although most current gene therapy clinical trials focus on cancer and inherited diseases, multiple studies have evaluated the efficacy of gene therapy to abrogate various forms of heart disease. Indeed, human clinical trials are currently underway.

Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques.

Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs).

Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB.

Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 in the canine.

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV) or plasmid DNA. While single-stranded AAV vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented by using self-complementary vectors.

Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of Duchenne muscular dystrophy.

Modulation of transforming growth factor-β (TGF-β) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-β family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver.

Status of therapeutic gene transfer to treat canine dilated cardiomyopathy in dogs.

Therapeutic gene transfer holds promise as a way to treat dilated cardiomyopathy from any underlying cause because the approach attempts to address metabolic disturbances that occur at the molecular level of the failing heart. Calcium-handling abnormalities and increased rates of apoptosis are abnormalities that occur in many types of heart disease, and gene therapies that target these metabolic defects have proven to be beneficial in numerous rodent models of heart disease. The authors are currently evaluating this approach to treat canine idiopathic dilated cardiomyopathy.

Myostatin is upregulated following stress in an Erk-dependent manner and negatively regulates cardiomyocyte growth in culture and in a mouse model.

Myostatin is well established as a negative regulator of skeletal muscle growth, but its role in the heart is controversial. Our goal in this study was to characterize myostatin regulation following cardiomyocyte stress and to examine the role of myostatin in the regulation of cardiomyocyte size. Neonatal cardiomyocytes were cultured and stressed with phenylephrine.

Myostatin activation in patients with advanced heart failure and after mechanical unloading.

Aims: Myostatin inhibits myoblast differentiation/proliferation and may play a role in heart failure (HF) and reverse remodelling after left ventricular assist device (LVAD) support. This study sought to characterize myostatin expression and activation in advanced HF before and after LVAD support.

Methods And Results: Left ventricular tissue pairs were collected at LVAD implantation (core) and at cardiac transplantation/LVAD explantation in patients with advanced ischaemic (ICM-ischaemic cardiomyopathy) and non-ischaemic (DCM-dilated cardiomyopathy) HF.

Systemic myostatin inhibition via liver-targeted gene transfer in normal and dystrophic mice.

Background: Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies.

Methodology/principal Findings: Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice.

Molecular analysis of vector genome structures after liver transduction by conventional and self-complementary adeno-associated viral serotype vectors in murine and nonhuman primate models.

Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs.

Gene therapy in large animal models of human cardiovascular genetic disease.

Several naturally occurring animal models for human genetic heart diseases offer an excellent opportunity to evaluate potential novel therapies, including gene therapy. Some of these diseases--especially those that result in a structural defect during development (e.g.

Sarcomeric-alpha-actinin defective in vinculin-binding causes Z-line expansion and nemaline-like body formation in cultured chick myotubes.

The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-alpha-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope.

Percutaneous Transendocardial Delivery of Self-complementary Adeno-associated Virus 6 Achieves Global Cardiac Gene Transfer in Canines.

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors.

Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 achieves global cardiac gene transfer in canines.

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors.

Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat.

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9.

Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance.

Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSCs) into the acutely ischemic myocardium.

Systemic administration of L-arginine benefits mdx skeletal muscle function.

A major consequence of muscular dystrophy is that increased membrane fragility leads to high calcium influx and results in muscle degeneration and myonecrosis. Prior reports have demonstrated that increased nitric oxide production via L-arginine treatment of normal and mdx mice resulted in increased expression of utrophin and increased activation of muscle satellite cells, which could ameliorate the dystrophic pathology. We delivered L-arginine to normal and mdx mice, and examined muscles for any functional changes associated with its administration.