Periodic Reporting for period 4 - NORVAS (Therapeutic and Biomarker Potential of long non-coding RNAs in Vascular Disease)
Período documentado: 2020-11-01 hasta 2021-04-30
Main aim of NORVAS is to identify non-coding RNA based therapies and biomarkers that enable us to combat the burden of vascular diseases in general, and abdominal aortic aneurysms and carotid artery stenosis and subsequent stroke in particular. Abdominal aortic aneurysms (AAAs) are defined as a permanent dilation of the abdominal aorta that predisposes to the fatal consequence of rupture. The diagnosis of a AAA is commonly an accidental finding, although there is an increasing number of screening programs targeting high-risk populations. A number of screens demonstrate that the disease prevalence is approximately 5% in men and 1 % in women over 60 years of age. Sixty percent of patients with AAAs die of other cardiovascular causes, such as stroke or myocardial infarction, suggesting a relationship between AAAs and atherosclerosis. Currently, the only available treatment option remains surgical repair or endovascular stenting. Besides being not feasible to treat the early stages of the disease, both interventional procedures do carry a potential operative risk, and thus appear only effective in preventing aortic rupture. With a mortality rate of 30%, stroke is the fourth leading cause of death and also the leading cause of adult disability in Western countries, with ischemic stroke accounting for approximately 85% of all cases. Vulnerable atherosclerotic plaques in the common carotid arteries are considered the most dominant initiator for ischemic forms of stroke (approx. 85% of all strokes). Some of the mechanisms proposed to contribute to unstable carotid atheroma development are identical to the ones being involved in AAA development and rupture, including apoptosis and insufficient proliferation of smooth muscle cells within the fibrous cap of advanced atherosclerotic carotid lesions. Again, the most common form of treatment in patients with an ischemic stroke is surgical removal of the affected carotid artery plaque. Thus, here all novel forms of treatments that can stabilise late stage and rupture prone carotid artery plaques are highly desirable. The key contribution of non-coding RNAs in regulating gene expression has recently received great attention. Using transcriptomic profiling technology on unique diseased human biobank material, we have identified several microRNAs (e.g. miR-21, miR-210) and long non-coding RNAs (H19, MIAT, NUDT6, SLFNL-AS1) as novel key regulators of smooth muscle cell survival in the vascular system. We performed studies in disease-relevant experimental in vivo models (rodents and LDLR-/- Yucatan mini-pigs) and developed novel in vitro models (aorta-on-a-chip) to functionally assess how inhibition of these non-coding RNAs influence aortic aneurysm progression and atherosclerotic plaque vulnerability. To enhance the translational feasibility of our findings, we will continue to utilise clinically established delivery tools, such as drug eluting stents and balloons, to locally administer our ncRNA modulators of interest to the vascular system. Furthermore, we screened longitudinal cohort studies that follow-up on individuals at different stages of aortic aneurysm disease, enabling us to investigate the prospective biomarker potential of ncRNAs in recognizing aneurysm growth patterns and patient’s acute and future risk of aortic rupture.
The main focus of our investigations in fighting the pathologies of aneurysm and carotid artery disease have thus shifted towards the development of local delivery tools and techniques for antisense oligonucleotides (ASOs), taking us away from systemic injections that carry the disadvantage of substantial side effects (Aims 1, 2 & 4 of proposal). For our NORVAS studies, we decided to utilise a novel atherosclerosis and cardiovascular disease-relevant LDLR-/- Yucatan mini-pig model, allowing us to test our therapeutic approach of local, DEB-assisted delivery of ASOs to the pig aorta (Aims 2 & 4). During the project were we able to show that blocking the long non-coding RNA H19 halts abdominal aortic aneurysm progression through suppression of the transcription factor HIF1a (Li DY et al, Circulation 2018). Other NORVAS-supported studies in our lab revealed that local miR-21 inhibition is feasible by using drug eluting stents to limit the occurrence of in-stent restenosis (Wang H et al, Arterioscler Thromb Vasc Biol 2016; planned Phase I clinical trial in 2022/2023), and inhibition of miR-210 enhances smooth muscle cell survival and plaque stabilization (Eken SM et al, Circ Res 2017). Also, we were able to show that triggering local uptake of miR-21 mimics through the support of ultrasound-mediated nanoparticle burst can also stabilize advanced atherosclerotic lesions through inhibition of the transcription factor REST in smooth muscle cells of fibrous caps (Jin H et al, Mol Ther 2018). Further, we have sufficiently tested our large animal (mini-pig) model in regards of feasibility to mimic human aneurysm and carotid artery disease. Here, we were able to show in a drug repurposing study that the tyrosine kinase inhibitor (TKI) lenvatinib, which is already being clinically used to treat patients with thyroid cancers, can limit experimental AAA growth and rupture in preclinical mouse and mini-pig models (Busch et al, JCI Insight 2021). For the very first time a drug eluting balloon (with lenvatinib) was used to limit AAA growth. In addition to the preclinical models described in the proposal and the mentioned published manuscripts, we have started to explore options of organs-on-chips models to mimic not only the vascular disease of interest, but also to use such a device as a drug testing platform. We have developed an aorta-on-a-chip using patient derived cells with aortic aneurysm disease that can be utilised to test novel treatment strategies under miniature patient conditions in a 3D cell system (Paloschi et al, Cardiovasc Res 2020). Finally, we have three manuscripts still under review that explore the prospective biomarker potential of microRNAs in patients with advancing AAAs (Aim 3 of proposal), as well as three additional long non-coding RNAs (MIAT and NUDT6/SLFNL-AS1) in carotid artery disease. These three missing publications will further highlight the great importance and functional relevance lncRNAs play in the disease context.
We believe that we have identified novel and disease-relevant roles and therapeutic benefit for several non-coding RNAs (long non-coding RNAs and microRNAs) in aortic aneurysm and carotid artery disease. We were able to test their potential as novel molecular treatments to limit the burden of these vascular diseases, for which currently very limited forms of treatment exist. Our novel disease models (preclinical large animal and in vitro aorta-on-a-chip) are useful tools for drug development in aneurysm disease and late stage atherosclerosis. Utilizing these advanced disease models enables us to test our novel ncRNA-based strategies in a feasible translational setting before entering Phase 1 clinical trials in humans. Furthermore has our work provided the very first suitable biomarker in aortic aneurysm disease to predict future expansion rates and the risk of rupture. This biomarker will also be derived from non-coding RNA transcripts, and will most likely be tissue specific. The NORVAS Starting Grant has definitely enabled us to reach a new level of research on RNA therapies and their utilisation to tackle the burden of vascular diseases.