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Therapeutic transcription factor decoy oligodeoxynucleotides for Marfan syndrome and heart failure

Remes, Anca

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Abstract

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.

Item Type: Dissertation
Supervisor: Hecker, Prof. Dr. Markus
Date of thesis defense: 8 February 2018
Date Deposited: 09 Apr 2019 13:48
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Medizinische Fakultät Heidelberg > Institut fuer Physiologie und Pathophysiologie
Subjects: 570 Life sciences

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  • Therapeutic transcription factor decoy oligodeoxynucleotides for Marfan syndrome and heart failure. (deposited 09 Apr 2019 13:48) [Currently Displayed]
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