Therapeutic transcription factor decoy oligodeoxynucleotides for Marfan syndrome and heart failure

The inhibition of transcription factor activation can be translated into a novel therapeutic strategy for a wide range of disease models. Decoy oligodeoxynucleotides (ODNs) containing the consensus binding site of a target transcription factor (TF) subsequently prevent binding of the TF to the promoter regions of its target genes associated with certain disorders. Decoy ODNs were shown to be safe and effective, but until now the main delivery route consisted of local application. However, depending on the target organ, this approach is not always feasible. Therefore, the main aim of this study is the establishment of an adeno-associated virus (AAV)-based method of decoy ODNs delivery, which allows continous generation of the therapeutic nucleic acid compound as a short hairpin RNA (shRNA) in AAV-transduced cells. In Marfan syndrome, increased Transforming growth factor-β (TGF-β) bioavailability induces enhanced activity of activator protein-1 (AP-1), which in turn upregulates expression of matrix metalloproteinases (MMPs). These endopeptidases were proven to be involved in the elastin degradation and aortic fragility which causes aortic aneurysm formation and dissection. The effect of ”naked” hpAP-1 decoy ODNs application and AAV9SLR transduction was analysed using primary mgR/mgR SMCs as an in vitro model and AP-1 activation was induced by interleukin-1 β (IL-1β) treatment. Both treatments led to decreased mRNA level of AP-1 target genes MMP9 and monocyte chemotactic protein-1 (MCP-1), as well as decreased MMP activity and smooth muscle cells (SMCs) migration capacity. Moreover, IL-1β-induced reactive oxygen species (ROS) production was reduced. In vivo experiments were performed using mgR/mgR mice. By F.I.S.H. experiments, the expression of the hpAP-1 RNA decoy ODNs in endothelial cells and SMCs was demonstrated, 4 weeks after ex vivo tissue transduction and reimplantation into mgR/mgR mice. Furthermore, AP-1 target genes MMP9 and MCP-1 were downregulated and MMP protein level and activity was significantly reduced. Additionally, AAV9SLR transduction caused a remarkable decrease in the number of infiltrating macrophages and improved endothelial tight junction integrity. Importantly, AP-1 inhibition significantly decreased the level of elastin degradation, as shown by a reduction in the number of islands of damage. The second aim of this study was the preclinical validation of AAV9 vector express- ing hpNFAT decoy ODNs as a therapeutic approach in an experimental model of left ventricular hypertrophy and heart failure in mice (transverse aortic constriction, TAC). The effect of hpNFAT decoy ODNs and AAV9 transduction was first assessed in vitro by employing the cardiomyocyte cell line HL-1, as well as primary neonatal cardiomyocytes, which were treated with the pro-hypertrophic stimulus endothelin-1 (ET-1). The results revealed a significant downregulation of the fetal gene programme, represented by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) as well as decreased myosin heavy chain-β (β-MHC) protein expression. Moreover, hpNFAT decoy ODNs and AAV9 expressing hpNFAT RNA decoy ODNs reduced ET-1 induced protein synthesis rate. Two weeks prior to inducing myocardial hypertrophy through aortic stenosis by TAC surgery, AAV9 was injected systemically. The vector led to the intracellular synthesis of the active RNA decoy ODNs in cardiomyocytes and decreased expression of fetal gene programme. In addition, markers of myocardial hypertrophy heart weight/tibia length (HW/TL) and heart failure lung weight/tibia length (LW/TL) were significantly reduced in treated mice. At the same time, heart function was markedly improved and fibrosis markers were reduced by NFAT neutralization through AAV9 injection. In conclusion, AAV-mediated decoy ODN delivery to the target organ may be a powerful tool to regulate the expression of genes involved in the progression of certain diseases. The present study showed the therapeutic efficacy of this concept by investigating two cardiovascular disease models

Fibin regulates cardiomyocyte hypertrophy and causes protein-aggregate-associated cardiomyopathy in vivo

Despite the identification of numerous molecular pathways modulating cardiac hypertrophy its pathogenesis is not completely understood. In this study we define an unexpected role for Fibin (“fin bud initiation factor homolog”) in cardiomyocyte hypertrophy. Via gene expression profiling in hypertrophic murine hearts after transverse aortic constriction we found a significant induction of Fibin. Moreover, Fibin was upregulated in another mouse model of cardiac hypertrophy (calcineurin-transgenics) as well as in patients with dilated cardiomyopathy. Immunoflourescence microscopy revealed subcellular localization of Fibin at the sarcomeric z-disc. Overexpression of Fibin in neonatal rat ventricular cardiomyocytes revealed a strong anti-hypertrophic effect through inhibiting both, NFAT- and SRF-dependent signalling. In contrast, transgenic mice with cardiac-restricted overexpression of Fibin developed dilated cardiomyopathy, accompanied by induction of hypertrophy-associated genes. Moreover, Fibin overexpression accelerated the progression to heart failure in the presence of prohypertrophic stimuli such as pressure overload and calcineurin overexpression. Histological and ultrastructural analyses surprisingly showed large protein aggregates containing Fibin. On the molecular level, aggregate formation was accompanied by an induction of the unfolded protein response subsequent UPR-mediated apoptosis and autophagy. Taken together, we identified Fibin as a novel potent negative regulator of cardiomyocyte hypertrophy in vitro. Yet, heart-specific Fibin overexpression in vivo causes development of a protein-aggregate-associated cardiomyopathy. Because of close similarities to myofibrillar myopathies, Fibin represents a candidate gene for cardiomyopathy and Fibin transgenic mice may provide additional mechanistic insight into aggregate formation in these diseases

Translational Medicine: Towards Gene Therapy of Marfan Syndrome

Marfan syndrome (MFS) is one of the most common inherited disorders of connective tissue caused by mutations of the fibrillin-1 gene (FBN1). Vascular abnormalities, such as the enlargement of the aorta with the risk of life-threatening rupture are frequently observed. However, current treatment is limited and therapeutic options focus solely on symptomatic therapy. Gene therapy focuses on genetically modifying cells to produce a therapeutic effect and may be a promising treatment option for MFS. Here, we first provide an overview of the historical background and characterization of MFS. Subsequently, we summarise current gene therapy options and possible translational concepts for this inherited disorder that affects connective tissue

AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach

The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice

Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-gamma via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated. Rangrez et al. show that overexpression of the HECT domain E3 ubiquitin protein ligase 3 (HectD3) reduces cardiac hypertrophy while reducing macrophage infiltration in mice. This study provides a cardioprotective mechanism, where HectD3 targets SUMO2 and Stat1 to exert its anti-hypertrophic and anti-inflammatory effects

Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue

Gene therapeutic approaches to aortic diseases require efficient vectors and delivery systems for transduction of endothelial cells (ECs) and smooth muscle cells (SMCs). Here, we developed a novel strategy to efficiently deliver a previously described vascular-specific adeno-associated viral (AAV) vector to the abdominal aorta by application of alginate hydrogels. To efficiently transduce ECs and SMCs, we used AAV9 vectors with a modified capsid (AAV9SLR) encoding enhanced green fluorescent protein (EGFP), as wild-type AAV vectors do not transduce ECs and SMCs well. AAV9SLR vectors were embedded into a solution containing sodium alginate and polymerized into hydrogels. Gels were surgically implanted around the adventitia of the infrarenal abdominal aorta of adult mice. Three weeks after surgery, an almost complete transduction of both the endothelium and tunica media adjacent to the gel was demonstrated in tissue sections. Hydrogel-mediated delivery resulted in induction of neutralizing antibodies but did not cause inflammatory responses in serum or the aortic wall. To further determine the translational potential, aortic tissue from patients was embedded ex vivo into AAV9SLR-containing hydrogel, and efficient transduction could be confirmed. These findings demonstrate that alginate hydrogel harboring a vascular-targeting AAV9SLR vector allows efficient local transduction of the aortic wall

AP-1 Oligodeoxynucleotides Reduce Aortic Elastolysis in a Murine Model of Marfan Syndrome

Marfan syndrome is characterized by high expression of matrix metalloproteinases (MMPs) in aortic smooth muscle cells (AoSMCs) associated with medial elastolysis and aortic root aneurysm. We aimed to reduce aortic elastolysis through decrease of MMP expression with decoy oligodeoxynucleotides (dODNs) neutralizing the transcription factor activating factor-1 (AP-1). AP-1 abundance in nuclear extracts as well as MMP-2 and MMP-9 expression were significantly increased in isolated mAoSMC of mgR/mgR Marfan mice compared to wild-type cells. Exposure to AP-1 neutralizing dODNs resulted in a significant reduction of basal and interleukin-1β-stimulated MMP expression and activity in mAoSMCs. Moreover, increased migration and formation of superoxide radical anions was substantially decreased in mAoSMCs by AP-1 dODN treatment. Aortic grafts from donor Marfan mice were treated with AP-1- dODN ex vivo and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Pretreatment of aortic grafts with AP-1 dODN led to reduced elastolysis, macrophage infiltration, and MMP activity. Permeability of the endothelial monolayer was increased for dODN in mgR/mgR aortae with observed loss of tight junction proteins ZO-1 and occludin, enabling dODN to reach the tunica media. Targeting AP-1 activity offers a new potential strategy to treat the vascular phenotype associated with Marfan syndrome