REVIEW
European Heart Journal (2017) 0, 1–20 doi:10.1093/eurheartj/ehx165
Basic Science
Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives Wolfgang Poller1,2*, Stefanie Dimmeler3,4, Stephane Heymans5, Tanja Zeller6,7, Jan Haas8,9, Mahir Karakas6,7, David-Manuel Leistner1,2, Philipp Jakob1,2, Shinichi Nakagawa10,11, Stefan Blankenberg6,7, Stefan Engelhardt12,13, Thomas Thum14, Christian Weber15,13, Benjamin Meder8,9, Roger Hajjar16, and Ulf Landmesser1,2,17 1
Department of Cardiology, CBF, CC11, Charite Universit€atsmedizin Berlin, Campus Benjamin Franklin, Charite Centrum 11 (Cardiovascular Medicine), Hindenburgdamm 20, 12200 10 Berlin, Germany; 2German Center for Cardiovascular Research (DZHK), Site Berlin, Berlin, Germany; 3Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Johann Wolfgang Goethe Universit€at, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany; 4DZHK, Site Rhein-Main, Frankfurt, Germany; 5Center for Heart Failure Research, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands; 6Clinic for General and Interventional Cardiology, University Heart Center Hamburg, Martinistrasse 52, 20246 Hamburg, Germany; 7DZHK, Site Hamburg/Kiel/Lu¨beck, Hamburg, Germany; 8Institute for Cardiomyopathies Heidelberg (ICH), Universit€atsklinikum Heidelberg, Im Neuenheimer Feld 669, 69120 Heidelberg, Germany; 9DZHK, Site Heidelberg/Mannheim, Heidelberg, Germany; 10RNA Biology Laboratory, RIKEN Advanced Research Institute, Wako, Saitama 351-0198, Japan; 11RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan; 12Institute for Pharmacology and Toxikology, Technische Universit€at Mu¨nchen, Biedersteiner Strasse 29, 80802 Mu¨nchen, Germany; 13DZHK, Site Munich, Munich, Germany; 14Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany; 15Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-Universit€at, Pettenkoferstrasse 8a/9, 80336 Munich, Germany; 16Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; and 17Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany Received 10 November 2016; revised 14 January 2017; editorial decision 15 March 2017; accepted 15 March 2017
Recent research has demonstrated that the non-coding genome plays a key role in genetic programming and gene regulation during development as well as in health and cardiovascular disease. About 99% of the human genome do not encode proteins, but are transcriptionally active representing a broad spectrum of non-coding RNAs (ncRNAs) with important regulatory and structural functions. Non-coding RNAs have been identified as critical novel regulators of cardiovascular risk factors and cell functions and are thus important candidates to improve diagnostics and prognosis assessment. Beyond this, ncRNAs are rapidly emgerging as fundamentally novel therapeutics. On a first level, ncRNAs provide novel therapeutic targets some of which are entering assessment in clinical trials. On a second level, new therapeutic tools were developed from endogenous ncRNAs serving as blueprints. Particularly advanced is the development of RNA interference (RNAi) drugs which use recently discovered pathways of endogenous short interfering RNAs and are becoming versatile tools for efficient silencing of protein expression. Pioneering clinical studies include RNAi drugs targeting liver synthesis of PCSK9 resulting in highly significant lowering of LDL cholesterol or targeting liver transthyretin (TTR) synthesis for treatment of cardiac TTR amyloidosis. Further novel drugs mimicking actions of endogenous ncRNAs may arise from exploitation of molecular interactions not accessible to conventional pharmacology. We provide an update on recent developments and perspectives for diagnostic and therapeutic use of ncRNAs in cardiovascular diseases, including atherosclerosis/coronary disease, post-myocardial infarction remodelling, and heart failure.
................................................................................................................................................................................................... Keywords
Cardiovascular diseases coding genome
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Long non-coding RNAs
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microRNAs
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Short interfering RNAs
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Human non-
Introduction Key role of non-coding RNAs in health and disease About 99% of the human genome do not encode proteins, but are transcriptionally highly active and give rise to a broad spectrum of
.. .. .. .. .. ..
ncRNAs with regulatory and structural functions. The observation of a steeply increasing fraction of non-coding RNAs (ncRNAs), which is in contrast to the modest increase in the number of protein-coding genes during evolution from simple organisms to humans (Figure 1),
* Corresponding author. Tel: +49 030 450 513765, Fax: +49 030 450 513947, Email: [email protected] C The Author 2017. For permissions, please email: [email protected]. Published on behalf of the European Society of Cardiology. All rights reserved. V
2 suggests an overwhelming role of the ncRNAs1 in humans. ncRNAs such as microRNAs (miRs), small interference RNAs (siRNAs) and long non-coding RNAs (lncRNAs) are novel regulators of cardiovascular risk factors and cell functions and thus candidates to improve diagnostics and prognosis assessment. Beyond this, however, ncRNAs have now fundamentally expanded our spectrum of therapeutic options. With regard to disease pathogenesis, it has become evident from the Encyclopedia of DNA Elements (ENCODE) project2 and other studies3–10 that limiting analysis to protein-coding regions of the human genome is inadequate, since many non-coding variants are associated with important human diseases. Inclusion of the noncoding genomic elements in pathogenetic studies appears mandatory and comprehensive transcriptome mapping includes small and large ncRNAs in addition to the protein-coding genes. In the heart, miRs regulate post-transcriptional gene expression and have been shown to control cardiovascular development, inflammation, hypertrophy, fibrosis, and regeneration (see recent review11–13). Of note are also miRs that regulate cardiac contractility (miRs 25 and 22), regeneration (miR-302-367 and miR99/100 family), inflammation (miRs 155 and 221/222), and fibrosis (miRs 21, 208b, and 125b), as well as vascular functions.14–28
Non-coding RNAs as diagnostic and therapeutic tools Beyond their application as diagnostic and prognostic biomarkers (Section ‘Non-coding RNAs as biomarkers’), ncRNA can also be the targets (e.g. miRs, lncRNAs) or tools (e.g. siRNAs)29–32 of novel therapeutic strategies (Figure 2). Thus, RNA interference (RNAi)mediating siRNAs (‘RNAi triggers’) are highly versatile ‘general-purpose’ tools e.g. for the silencing of protein-encoding genes via targeting of their mRNA. In 2006, the Nobel prize of physiology or medicine was awarded to Dr Fire and Dr Mello ‘for their discovery of RNA interference—gene silencing by double-stranded RNA’32,35 which is now rapidly explored as a novel therapeutic principle for cardiovascular disease. Several studies demonstrated therapeutic potential of organ-targeted RNAi based on viral vectors36,37 or synthetic RNAs,38,39 and therapeutic strategies based on the modulation of miRs.40–45 An increasing spectrum of endogenous ncRNAs is employed for the development of novel therapeutic ncRNA tools optimized for specific therapeutic requirements.46–51 These investigations are only the beginning of therapeutic exploration, since 10 000 small ncRNAs arise from the human genome forming diverse RNA structures ranging from miRs to circular RNAs (circRNAs).52,53 These small ncRNAs are only part of a broader spectrum of ncRNAs including 16 000 lncRNAs up to many thousand basepairs in size.54–59 For lncRNAs, classification is still in its infancy60,61 and biological functions mostly unknown.62–66 However, recent studies strongly suggest that they constitute fundamentally new therapeutic targets67–70 with unusual RNA structures71,72 to be addressed using new drug types. Their importance for human health and disease is highlighted by observation that both small and large ncRNAs rapidly evolved during primate evolution and are often primate-specific and cannot be studied in common animal models of human disease.66,73 Overall, only a small part of the non-coding human genome has been investigated and explored with regard to possible therapeutic implications for cardiovascular medicine.
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.. .. Search for non-coding RNAs of clinical .. interest .. .. Because of the complexity of the non-coding genome, one may adopt .. .. a pragmatic heuristic approach directed to the identification of non-.. coding therapeutic targets in cardiovascular diseases (Figure 3). .. .. Insights from genome-wide phylogenetic studies, comprehensive .. sequencing work, and animal models of human cardiovascular dis.. .. eases constitute an indispensable first step towards target identifica.. tion. Importantly, the functions of the same ncRNA may differ .. .. between species, and most animal models differ significantly from the .. respective human disorder. Some ncRNAs do not even exist in ... humans73 or have only distant orthologues, so that their possible .. .. pathogenic relevance in humans cannot be investigated in animals. .. Therapeutic proof-of-concept studies of ncRNAs deregulated in animal .. .. models are therefore not necessarily predictive for effects in humans. .. It is therefore necessary to conduct screening studies for ncRNA .. .. of clinical interest also directly in humans, in particular in well-defined .. patient cohorts suffering from a carefully phenotyped cardiovascular .. .. disease of interest. Different principles build the basis of a successful .. screening project. First, relevant ncRNAs may be identified by .. .. genome-wide association studies (GWAS) indicating genomic loci .. conferring diseases risk via inherited mutations.74–83 A second ap.. .. proach is comprehensive transcriptome mapping in diseased organs, .. which may reveal genomically encoded defects, but also transcrip.. .. tome shifts developing during pathogenesis but not encoded at the .. 84 .. DNA level. Both approaches have already identified ncRNAs .. involved in cardiovascular disease pathogenesis. State-of-the-art ana.. .. lytical technologies enable both GWAS studies and comprehensive .. transcriptional mapping at high efficacy. However, some logistical and .. .. developmental aspects need to be considered that relate to funda.. mental limitations of these search strategies. Genome-wide associ.. .. ation studies commonly addresses early processes in disease .. pathogenesis by analysing the genome at the DNA level of inherited .. .. mutations acting from childhood. Late processes during disease de.. velopment, in contrast, are rather revealed by transcriptome map.. .. ping of already injured organs.85 In addition, the selection of properly .. phenotyped patients, sufficient cohort sizes, and proper control .. .. groups often constitutes a leading problem in such screening pro.. jects. In a third approach, high-throughput functional screening for .. .. relevant ncRNAs in cell types of pathogenic relevance .. (cardiomyocytes,86 vascular cells,87,88 cardiac fibroblasts,88–95 hep.. .. atocytes96,97) has been successfully used to identify new ncRNA with .. diagnostic and therapeutic potential. Disease-associated ncRNAs .. .. identified by any of these approaches may yield biomarkers to assess .. the risk of disease progression, thus facilitating treatment decisions, .. .. or to predict response-to-therapy to optimally allocate limited re.. sources. In addition, these ncRNAs may serve to develop and evalu.. .. ate novel therapeutic strategies beyond optimal current therapies. .. In the following, we discuss diagnostic and therapeutic perspec.. .. tives of ncRNAs in cardiovascular diseases. Three clinical goals are .. .. running as guiding threads throughout the article. First, the identifica.. tion of novel cardiovascular pathomechanisms and high-priority tar.. .. get diseases (Section ‘Non-coding RNAs as potential .. therapeutic targets in cardiovascular disease’). Second, the .. . development of clinically useful RNA-based therapeutics and
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Translational perspectives of the non-coding genome
identification of patients most likely to benefit (Section ‘Clinical translation of RNA therapeutics’). Third, the employment of RNA diagnostics to improve prognosis assessment and individualized clinical patient management (Section ‘Non-coding RNAs as biomarkers’). These issues are highlighted again in a Summarizing Illustration: Delineation of current high priority diseases, patients most likely to benefit, status of technical feasibility of ncRNA therapies, and the current value of RNA diagnostics for prognosis assessment and patient management.
Non-coding RNAs as potential therapeutic targets in cardiovascular disease Here, we review the identification of non-coding RNAs defining novel pathomechanisms, and important cardiovascular diseases significantly influenced by these novel processes.
MicroRNAs as therapeutic targets Post-myocardial infarction adverse cardiac remodelling and dysfunction MicroRNAs control important processes that contribute post-infarction injury and subsequent remodelling responses. For example, miRs can promote or inhibit cardiomyocyte cell death, regulate postischaemic neovascularization and control cardiac fibrosis (for an overview of miRs that control post-infarction repair see98). Cardiomyocyte cell death is induced by members of the miR-15 family, which are upregulated by myocardial ischaemia.99 Inhibition of miR-15 family members by short, locked nucleic acid (LNA)-based anti-miRs, which target the seed sequence of most miR-15 family members, reduced infarct size after ischaemia–reperfusion injury by derepressing the anti-apoptotic protein Bcl-2, the mitochondrial protecting factor ADP-ribosylation factor-like protein 2, and the deacetylase SIRT1 in mice.98,99 The feasibility of using miR-15 inhibitors has been documented also in pig models.99 Myocardial infarction additionally induces the expression of the pro-apoptotic miR-34 family members. Inhibition of miR-34 family members by different types of anti-miRs reduced infarct size, and augmented the recovery of heart function after acute myocardial infarction (AMI) by increasing the expression of cardioprotective SIRT1 and PNUTS.23,100 Inhibition of miR-15 or miR-34 family members also improved neovascularization after ischaemia.23,101 Members of the miR-1792 cluster, specifically miR-92a, control neovascularization and cardiomyo-cyte cell death after ischaemia.102,103 Pharmacological inhibition or genetic deletion of miR-92a increased capillary density and improved heart function after AMI in mice.102,103 Furthermore, catheter-based delivery of anti-miR-92a or encapsulated antagomiRs reduced infarct size in a large animal cardiac ischaemia/reperfusion pig model.104 Targets of miR-92a include integrin a5, KLF2 and SIRT1.102,104,105 miR-26a is increased after AMI and inhibits angiogenesis.106 Administration of a LNA-based anti-miR against miR-26a induced robust angiogenesis, reduced myocardial infarct size, and improved heart function.106 Mitochondrial function is controlled by miR-140, which reduces mitochondrial fission,107 thereby impairing cardiomyocyte survival. Pharmacological inhibition reduced infarct size after AMI in mice.107
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On the contrary, there are cardioprotective miRs108–111 including miR-210 which protects cardiomyo-cytes and vasculature. Intramyocardial injection of minicircle vectors carrying the miR-210 precursor reduced cell death and improved cardiac function and angiogenesis after AMI.108 Cardiac hypertrophy and heart failure One of the first reports about a miR involved in cardiac hypertrophy stem from the Olson laboratory, here miR-208 has been shown to be involved in hypertrophic signaling.112 Inhibition of miR-133 with an anti-miR-133 oligonucleotide generated cardiac hypertrophy in vivo showing a role of miR-133 in cardiac hypertrophy as well.113 Other miRs that are regulated during pathological stress include the miR-212/ 132 family which becomes activated during heart failure (HF) in humans114 and animal models.40 This miR family both regulates cardiac autophagy and hypertrophy in cardiomyocytes at least in part by modulation of transcription factor forkhead box O3. Inhibition of miR21 prevented HF development in mice.40 MicroRNAs also regulate intracellular calcium homeostasis, a process that is strongly deregulated during HF, thereby directly affecting cardiac contractility.86,92 For few specific cardiomyopathies, the therapeutic potential of ncRNAs has been investigated. Thus, recent studies identified novel miR therapeutic targets in coxsackievirus-B3 (CVB3) myocarditis. MicroRNAs 155, 146b, and 21 are upregulated in acute CVB3-myocarditis.115,116 Inhibition of miR-155114,117,118, miRs 21 and 146b119 by systemically delivered anti-miRs attenuates cardiac inflammation and myocardial damage in CVB3 or autoimmune myocarditis in mice. Atherosclerosis Atherosclerosis underlying coronary artery disease (CAD), stroke, and peripheral vascular disease is a chronic inflammatory reaction of the arterial wall characterized by maladaptive responses of endothelial cells at sites with disturbed flow conditions and of immune cells, in particular macrophages accumulating lipids without resolving inflammation in a context of dyslipidaemia. A plethora of miRs and relevant targets has emerged in mouse models of atherosclerosis to intricately orchestrate crucial mechanisms, thus forming a basis for potential therapeutic strategies using anti-miRs or miR mimics.120–122 An atheroprotective role in endothelial regeneration has been identified for the miR-126 strand pair. Depletion of miR-126-5p at predilection sites with altered flow under hyperlipidaemic stress limits the endothelial proliferative reserve and promotes lesion formation through derepression of DLK-1123 which can be restored by miR-126-5p mimics. Furthermore, delivery of miR-126-3p by microparticles from apoptotic to recipient endothelial cells amplifies CXCL12 expression by repressing RGS16 to recruit proangiogenic cells supporting endothelial recovery,124 a mechanism possibly impaired in CAD or diabetes.120 Similarly, transfer of KLF2-induced miR-143/miR-145 by endothelial microvesicles to smooth muscle cell (SMCs) attenuates atherosclerosis by generating a contractile phenotype, as with miR-145 overexpression.26,125 miR-181b targeting importin-a3 is upregulated by laminar flow and limits endothelial inflammation and atherosclerosis by inhibiting NF-jB activation.126 Conversely, miR-92a is upregulated by disturbed flow and modified lipoproteins to promote NF-jB-mediated endothelial inflammation and atherosclerosis by targeting KLF2/4, which is reversed by anti-miRs.127 Likewise, the pre-ribosomal RNA-
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A
B
Figure 1 Evolution of the non-coding human genome. (A) Whereas the number of protein-coding genes remains surprisingly similar from simple to complex species, it is the non-coding part of the genome that increases dramatically with morphologic complexity, reaching 99% of the entire genome in humans. Multiple evidence suggests important roles of ncRNAs transcribed from the non-coding genome in human health and disease. (B): Whereas a vast number of ncRNAs with grossly different structures (e.g. lncRNAs, miRs, circRNAs) have been identified, however, in the majority of these molecules their functions are essentially unknown (details in Supplementary material online, Figure S1).
derived miR-712 is upregulated by altered flow to elicit endothelial inflammation and atherosclerosis by targeting TIMP3.128 Macrophage subtypes show differential profiles of miR expression. Notably, miR-155 increases during atherogenesis or upon M1-type macrophage polarization, and promotes inflammatory activation and advanced atherosclerosis by targeting BCL6, a transcriptional regulator counteracting NF-jB, and by reducing cholesterol efflux.129,130 Released from competing targets in M1-macrophages, miR-342-5p suppresses Akt1 to cause inflammatory activation and atherogenesis by upregulating miR-155 and forming a functional tandem for antimiR strategies.131 The cholesterol efflux from macrophages to HDL is mediated by the ATP-binding cassette transporters ABCA1 and ABCG1. The ABCA1 30 UTR encompasses a wealth of binding sites for miRs including miR-33a/b, miR-19b, miR-144 and miR-148a.132 Beyond miR-33, several miRs targeting ABCA1/G1 can inhibit cholesterol efflux and have been implicated in atherogenesis.120,121,132 In contrast,
.. short-term antagonism of miR-33 in rodents and non-human pri.. .. mates significantly elevates plasma HDL, but overall the efficacy of .. 120–122,132 .. miR-33 silencing in atherogenesis remains controversial. .. Table 1 and Figure 4 provide short overviews of current miR.. targeting cardiovascular therapies. ... .. .. Long non-coding RNAs as potential .. .. therapeutic targets .. .. .. Long non-coding RNAs research is challenging due to the fact that .. most lncRNAs, in contrast to miRs, are not conserved among spe.. .. cies. Even when selecting the few being conserved, epigenetic effects .. and the processes of DNA and RNA assembly they influence may dif.. .. fer among species, making translation from rodents to humans chal.. lenging. Encouraging is the fact that lncRNAs have distinct functions .. .. in cardiovascular diseases (reviewed170–176) and therefore may open .. new therapeutic opportunities.177
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Figure 2 Endogenous non-coding RNAs as blueprints for RNA therapeutics. Non-coding RNAs may be addressed as therapeutic targets, but an increasing spectrum of endogenous ncRNAs are also employed as blueprints for the development of novel therapeutic tools. The spectrum of possible therapeutic targets has thus expanded beyond proteins, but also the therapeutic ‘toolbox’. One current topic is therapeutic RNA interference triggers (siRNAs) originally developed from endogenous siRNAs as blueprints, and made clinically applicable based on sophisticated chemical modifications and coupling to carriers/ligands for tissue targeting33,34 (details in Supplementary material online, Figure S2).
Supplementary material online, Figure S1 depicts unsolved issues in translational lncRNA research. First, development of a more advanced classification54,56,60,178–183 of the vast number of lncRNAs detected in humans—based on structure, subcellular localization, intracellular processing, functional modules—is needed to facilitate rational development of lncRNA-targeting treatments. Second, a peculiar aspect of lncRNAs to be addressed is their often extensive and specific post-translational processing with differential subcellular localization of the products, yielding complex RNA processing systems with different functions of the products69,184 which may be differentially addressable by drugs.185
Cardiac hypertrophy and heart failure Next to miRs, lncRNAs also play an important role in cardiac failure. The lncRNA Cardiac hypertrophy associated transcript (CHAST) is increased during cardiac hypertrophy in mice and humans and its inhibition both prevented and attenuated cardiac remodelling and HF.186 In the heart, suppression of Myosin heavy-chain-associated RNA transcript (MHRT) accelerated progression of hypertrophy to failure,187 whereas overexpression of CHAST induced cardiomyocyte
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hypertrophy. Gapmer-mediated CHAST silencing both prevented and attenuated pressure overload-induced pathological cardiac remodelling without evident early toxicological side effects.186 Knockdown of lncRNA Cardiac mesoderm enhancer associated ncRNA (CARMEN) inhibits cardiac specification and differentiation in cardiac precursor cells. This occurs independently of miR-143 and -145 expressions despite the fact that those two miRs located proximal to the enhancer sequences. CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2).188
Atherosclerosis Beyond miRs, lncRNAs may regulate atherosclerosis but their role and molecular mechanisms remain to be elucidated. As a strong genetic risk locus, the chromosome 9p21 (Chr9p21) region encodes the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL). Its transcript expression correlates with the severity of atherosclerosis, and ANRIL regulates target genes in trans through Alu motifs in their promoters, leading to increased cell proliferation and adhesion but decreased apoptosis, relevant to atherosclerosis.124 Conversely, lincRNA-p21 down-regulated in atherosclerotic plaques has been
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Search strategies to identify ncRNAs with clinical therapeutic potential PRECLINICAL genomewide transcriptome mapping studies encompassing mRNAs, miRs, lncRNAs, and other ncRNAs using
using RNA seq or other comprehensive methods
CLINICAL ncRNA expression profiling in cardiovascular disease patients first hints to biological functions and possible pathogenic roles of ncRNA in humans
EXPERIMENTAL ncRNA manipulation in animal model of disease LIMITATION: requires presence of the human transcript of interest in the animal
BIOLOGICAL FUNCTION ASSIGMENT
to human ncRNAs and processing products
CLINICAL TRANSLATION STRATEGIES
Traditional therapeutic proof-of-principle studies in animals LIMITATION: requires presence of human ncRNA in model species
Figure 3 Search strategies for non-coding RNAs of clinical interest. Genome-wide genomic and transcriptomic mapping have revealed a huge number of novel non-coding transcripts and provided structural information regarding their gene loci, but for the vast majority there is as yet no functional assignment. Functional characterization is complicated by their often extensive processing. Prospecting of this plethora of ncRNA for possible clinical significance may be critically facilitated by state-of-the-art transcriptional mapping of cardiovascular patients, in order to obtain first functional hints to guide subsequent in-depth experimental investigations including therapeutic proof-of-principle studies.
found to control neointima formation, repressing SMC proliferation and inducing apoptosis by enhancing p53 activity.125 Intense investigations into the function on other miRs and lncRNAs yet to be identified are currently underway. The potential of targeting lncRNAs was first described in models of angiogenesis or cell growth. Silencing of Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) reduced capillary growth not only in a mouse model of hind limb ischaemia (detrimental),68 but also in a rat model of diabetic retinopathy (beneficial).189 MALAT1-derived mascRNA is involved in innate immunity and viral myocarditis,190 but appears to be dispensable in pressure overload-induced HF in mice.191 Next, inhibition of lincRNA-p21 aggravated neointimal hyperplasia in a carotid artery injury model in ApoE knockout mice.192 Smooth muscle and endothelial cell-enriched migration/differentiation associated RNA (SENCR) is a vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.87 All of the above was obtained in rodent models, however, with only correlative human data on cardiac expression186,187 or blood levels.193 Since lncRNAs are poorly conserved between species, translation of animal findings to patients will be challenging. Whether the use of more relevant human cell lines, like inducible pluripotent stem cells may help to overcome these limitations of animal studies, remains to be determined. Identification of human-specific lncRNAs, either as human orthologues from rodent-discovered lnRNAs,194 or being expressed during embryonic stem cell differentiation into cardiomyocytes,195 will be important for further advance of lncRNA-based treatments in humans.
.. .. Clinical translation of RNA .. .. therapeutics .. .. .. Several novel pathomechanisms influenced by ncRNAs, discussed .. above, cannot be therapeutically addressed using conventional .. .. pharmacology. ncRNA therapeutics offer new options to influence .. these hitherto inaccessible disease processes. .. .. .. .. .. .. .. Versatility of therapeutic non-coding .. .. RNA structures .. ... Small interference RNAs and related structures mediating RNAi and .. thus target gene silencing are the currently most advanced and fre.. quently used types of ncRNA for therapeutic purposes, and part of a .. .. continuously expanding spectrum of therapeutic ncRNA tools de.. veloped from endogenous ncRNA as blueprints (Figure 2). .. .. Fundamentally different from DNA, RNAs are carrying information .. not only in their linear sequences of nucleotides (primary structure), .. .. but local nucleotide pairing creates secondary structures e.g. Hairpins, .. and interactions among distantly located sequences create tertiary .. .. structures.196 In fact, this structural versatility needs to be considered .. for RNAs as therapeutic tools as well as targets. The plethora of .. .. RNA types, sequences, and structures created by evolution is a treas.. ure trove of potential therapeutic tools and targets (Figure 2).
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Translational perspectives of the non-coding genome
Figure 4 Cardiovascular RNA drugs address multiple organ systems. RNA drugs do not need to address heart (A) or vasculature (B) directly. Primarily liver-targeted RNA drugs (C) are the currently most successful development in the cardiovascular field. Another group of strategies addresses the immune system (D), in particular monocytes-macrophages. Carrier denotes a synthetic nanoparticle and/or receptor ligand employed to deliver an RNA drug to its tissue target.29,31,33,34,155–160 ‘Carrier’ is bound to and serves to stabilize the RNA drug within the circulation, and to endow it with at least partial selectivity for the target cells, in order to minimize side effects. ‘AAV9’ denotes a cardiac-targeting recombinant adenoassociated viral vector containing a genome from which the therapeutic RNA sequence is continuously transcribed (for transcript types see Figure 5). apo(a), apolipoprotein (a); CCR2, chemokine C-C motif receptor 2; CHAST, Cardiac hypertrophy associated transcript; PCSK9, proprotein convertase subtilisin/kexin type 9; PLB, phospholamban.
Classification of non-coding RNA therapeutics Figure 5 provides an overview of currently available ncRNA therapeutics and their key properties: (i) sufficient stability or continuous in vivo production of therapeutic ncRNA, or the ability to redose if needed, (ii) high specificity of therapeutic ncRNA for the molecular target, (iii) proper targeting to the correct cells type or tissue by use of appropriate vectors plus transcriptional targeting, (iv) side effects induced by the therapeutic ncRNA itself or its delivery system need to be minimized, and (v) regulatability for certain applications.197 Overall, RNA therapeutics may be chemically synthesized nucleic acids delivered using chemical delivery systems,33,34,198–200 or produced in vivo by recombinant viral vectors targeting different tissues.201–209 Viral vectors have the capacity to act as long-term productive ‘RNA drug factories’, whereas chemical synthesis allows introduction of RNA modifications that cannot be generated biologically (details in Supplementary material online, Figure S3). Table 1
.. .. summarizes studies employing ncRNAs in clinical trials, preclinical .. animal models, or other experimental studies in vivo. .. .. .. .. .. .. Challenges in translation .. Clinical translation of these fascinating and far-reaching options faces .. .. challenges ranging from ncRNA drug design and delivery (Box 1) to .. regulatory issues (Box 2). Whereas animal studies are most valuable .. .. and indispensable for proof of principle, most of these studies use .. young otherwise healthy animals and thus rarely reflect the clinical .. .. reality. Taking HF as an example, few if any animal models mimic the .. human HF reality, a chronic systemic condition lasting years to dec.. .. ades. Models of chronic HF in animals are rare and only recently a .. model emerged which combines diabetes, obesity, hypertension, and .. .. leads to kidney dysfunction.210,211 On the solid foundation of animal .. studies, few pioneering clinical trials were successful in demonstrating .. . the technical and clinical feasibility of ncRNA therapeutic approaches.
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Table 1
Translational studies addressing the therapeutic potential of non-coding RNAs
Investigation
Strategy
Technology
Clinical Trials LDL-C reduction
Therapeutic RNAi targeting PCSK9 reduced LDL-C Synthetic siRNA via nanoparticle carrier
Reference
.................................................................................................................................................................................................................... 133
in non-human primates LDL-C reduction
RNAi drug targeting PCSK9 reduced LDL-C in vol- Synthetic siRNA via nanoparticle carrier unteers (intravenous siRNA administration in lipid
134
LDL-C reduction
RNAi drug targeting PCSK9 reduced LDL-C in vol- synthetic siRNA conjugated to GalNAC unteers (subcutaneous siRNA administration con-
LDL-C reduction
ASO targeting PCSK9 reduced LDL-C in non-human LNA-ASO via nanoparticle carrier primates
136
Lp(a) reduction
ASO targeting apolipoprotein(a) reduced Lp(a) in a 20 -MOE-ASO via nanoparticle carrier randomised, double-blind, placebo-controlled
137–139
Transthyretin suppression
Clinical RNAi therapy for transthyretin amyloidosis Synthetic siRNA via nanoparticle carrier inhibited hepatic synthesis of mutant TTR protein
140
Antiviral
RNAi-based antiviral treatment in non-human
Synthetic siRNA via nanoparticle carrier
141
LNA-ASO intracoronary injection (swine)
104,142
nanoparticles) 135
jugated to GalNAC)
clinical study
primates Experimental Research Vascular-targeted anti-miR
miR-92a inhibition of ischaemia/reperfusion injury, and improvement of re-endothelialization following vascular injury
Cardiac-targeted RNAi
Cardiotropic silencing of Ca2+ cycle regulator phos- Cardiac-targeted AAV9-vector-derived pholamban for the treatment of severe heart failure
Monocyte-targeted RNAi
Nanoparticle-encapsulated synthetic siRNA for silencing of monocytic CCR2
36
shRNA (rat) Non-viral, lipid nanoparticle-mediated siRNA 38 delivery targeting particularly monocytes (mouse)
RNAi imaging in vivo
PHD2-shRNA followed by a hypoxia response
Cardiac RNAi
element-containing promoter RNAi induced alloimmune tolerance in heart trans- siRNA (rat)
Imaging of RNAi in space and time (mouse) 143 144
plantation model TLR adaptor silencing Vasculature-targeted RNAi RNAi visualization
RNAi to silence chymase increased plaque stability Lentivirus-based RNAi Visualizing lipid-formulated siRNA release and target Development of strategies to improve the
Improvement of stem cells by RNAi
RNAi to enhance survival and function of transplanted cells
Enhancement of the efficacy of cardiovascu- 147 lar stem cell therapies
Allele-specific RNAi
Allele-specific RNAi for human induced pluripo-
Rescue of autosomal dominant-negative
knockdown
tency stem cell cardiomyocytes
145 146
delivery of candidate RNAi drugs
148
disorders
Safety issues Scientific and Regulatory Policy Committee:
149
Biotherapeutics Scientific and Regulatory Policy Committee:
150
Antisense Oligonucleotides Emerging technologies Gene silencing
siRNA vs miRNA for gene silencing
Comparison of therapeutic siRNAs and
144,148,151,152
miRNAs RNA engineering RNAi visualization
High-throughput cellular RNA device engineering Visualizing lipid-formulated siRNA release and target To facilitate development of rational stratknockdown
RNAi-based functional profiling
RNAi-based functional profiling of loci from genome-wide association studies
ASO, antisense oligonucleotide; LNA, locked nucleic acid; RNAi, RNA interference.
153 146
egies to improve the cytosolic delivery of candidate drugs RNAi to analyse 133 candidate genes in 56 loci identified by GWAS
154
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Translational perspectives of the non-coding genome
Box 1 Goals and challenges of non-coding RNA therapeutics research
• Required Knowledge
• Required Properties of RNA Drug
Posttranscriptional processing and intracellular kinetics (lncRNAs) Expression patterns in different cell types and organs Cellular function(s) may grossly differ between cell types (pleiotropism)
Biological function(s) derived from germline or tissue-specific animal models
• Patient Selection High unmet clinical need
High specificity of RNA drug - RNA target interaction High stability of RNA drug in the circulation and within target cells
• RNA Drug Delivery System Selective targeting to diseased organ or specific cell type (to avoid off-target effects at organ level)
Clinical phenotype allows for rapid and significant improvement
Clinical Aspects
Subcellular localization (miRs, lncRNAs)
Technological Aspects
Rationale for ncRNAs as Therapeutic Targets
Key characteristics of ncRNA-based drugs, clinical goals, and challenges to overcome in translation from preclinical research to clinical applications.
Patient population straightforward to define and identify Endpoints well defined and easily measurable with detectable effects in
European Heart Journal (2017) 0, 1–20 doi:10.1093/eurheartj/ehx165
Basic Science
Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives Wolfgang Poller1,2*, Stefanie Dimmeler3,4, Stephane Heymans5, Tanja Zeller6,7, Jan Haas8,9, Mahir Karakas6,7, David-Manuel Leistner1,2, Philipp Jakob1,2, Shinichi Nakagawa10,11, Stefan Blankenberg6,7, Stefan Engelhardt12,13, Thomas Thum14, Christian Weber15,13, Benjamin Meder8,9, Roger Hajjar16, and Ulf Landmesser1,2,17 1
Department of Cardiology, CBF, CC11, Charite Universit€atsmedizin Berlin, Campus Benjamin Franklin, Charite Centrum 11 (Cardiovascular Medicine), Hindenburgdamm 20, 12200 10 Berlin, Germany; 2German Center for Cardiovascular Research (DZHK), Site Berlin, Berlin, Germany; 3Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Johann Wolfgang Goethe Universit€at, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany; 4DZHK, Site Rhein-Main, Frankfurt, Germany; 5Center for Heart Failure Research, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, Netherlands; 6Clinic for General and Interventional Cardiology, University Heart Center Hamburg, Martinistrasse 52, 20246 Hamburg, Germany; 7DZHK, Site Hamburg/Kiel/Lu¨beck, Hamburg, Germany; 8Institute for Cardiomyopathies Heidelberg (ICH), Universit€atsklinikum Heidelberg, Im Neuenheimer Feld 669, 69120 Heidelberg, Germany; 9DZHK, Site Heidelberg/Mannheim, Heidelberg, Germany; 10RNA Biology Laboratory, RIKEN Advanced Research Institute, Wako, Saitama 351-0198, Japan; 11RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan; 12Institute for Pharmacology and Toxikology, Technische Universit€at Mu¨nchen, Biedersteiner Strasse 29, 80802 Mu¨nchen, Germany; 13DZHK, Site Munich, Munich, Germany; 14Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany; 15Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximilians-Universit€at, Pettenkoferstrasse 8a/9, 80336 Munich, Germany; 16Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; and 17Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany Received 10 November 2016; revised 14 January 2017; editorial decision 15 March 2017; accepted 15 March 2017
Recent research has demonstrated that the non-coding genome plays a key role in genetic programming and gene regulation during development as well as in health and cardiovascular disease. About 99% of the human genome do not encode proteins, but are transcriptionally active representing a broad spectrum of non-coding RNAs (ncRNAs) with important regulatory and structural functions. Non-coding RNAs have been identified as critical novel regulators of cardiovascular risk factors and cell functions and are thus important candidates to improve diagnostics and prognosis assessment. Beyond this, ncRNAs are rapidly emgerging as fundamentally novel therapeutics. On a first level, ncRNAs provide novel therapeutic targets some of which are entering assessment in clinical trials. On a second level, new therapeutic tools were developed from endogenous ncRNAs serving as blueprints. Particularly advanced is the development of RNA interference (RNAi) drugs which use recently discovered pathways of endogenous short interfering RNAs and are becoming versatile tools for efficient silencing of protein expression. Pioneering clinical studies include RNAi drugs targeting liver synthesis of PCSK9 resulting in highly significant lowering of LDL cholesterol or targeting liver transthyretin (TTR) synthesis for treatment of cardiac TTR amyloidosis. Further novel drugs mimicking actions of endogenous ncRNAs may arise from exploitation of molecular interactions not accessible to conventional pharmacology. We provide an update on recent developments and perspectives for diagnostic and therapeutic use of ncRNAs in cardiovascular diseases, including atherosclerosis/coronary disease, post-myocardial infarction remodelling, and heart failure.
................................................................................................................................................................................................... Keywords
Cardiovascular diseases coding genome
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Long non-coding RNAs
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microRNAs
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Short interfering RNAs
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Human non-
Introduction Key role of non-coding RNAs in health and disease About 99% of the human genome do not encode proteins, but are transcriptionally highly active and give rise to a broad spectrum of
.. .. .. .. .. ..
ncRNAs with regulatory and structural functions. The observation of a steeply increasing fraction of non-coding RNAs (ncRNAs), which is in contrast to the modest increase in the number of protein-coding genes during evolution from simple organisms to humans (Figure 1),
* Corresponding author. Tel: +49 030 450 513765, Fax: +49 030 450 513947, Email: [email protected] C The Author 2017. For permissions, please email: [email protected]. Published on behalf of the European Society of Cardiology. All rights reserved. V
2 suggests an overwhelming role of the ncRNAs1 in humans. ncRNAs such as microRNAs (miRs), small interference RNAs (siRNAs) and long non-coding RNAs (lncRNAs) are novel regulators of cardiovascular risk factors and cell functions and thus candidates to improve diagnostics and prognosis assessment. Beyond this, however, ncRNAs have now fundamentally expanded our spectrum of therapeutic options. With regard to disease pathogenesis, it has become evident from the Encyclopedia of DNA Elements (ENCODE) project2 and other studies3–10 that limiting analysis to protein-coding regions of the human genome is inadequate, since many non-coding variants are associated with important human diseases. Inclusion of the noncoding genomic elements in pathogenetic studies appears mandatory and comprehensive transcriptome mapping includes small and large ncRNAs in addition to the protein-coding genes. In the heart, miRs regulate post-transcriptional gene expression and have been shown to control cardiovascular development, inflammation, hypertrophy, fibrosis, and regeneration (see recent review11–13). Of note are also miRs that regulate cardiac contractility (miRs 25 and 22), regeneration (miR-302-367 and miR99/100 family), inflammation (miRs 155 and 221/222), and fibrosis (miRs 21, 208b, and 125b), as well as vascular functions.14–28
Non-coding RNAs as diagnostic and therapeutic tools Beyond their application as diagnostic and prognostic biomarkers (Section ‘Non-coding RNAs as biomarkers’), ncRNA can also be the targets (e.g. miRs, lncRNAs) or tools (e.g. siRNAs)29–32 of novel therapeutic strategies (Figure 2). Thus, RNA interference (RNAi)mediating siRNAs (‘RNAi triggers’) are highly versatile ‘general-purpose’ tools e.g. for the silencing of protein-encoding genes via targeting of their mRNA. In 2006, the Nobel prize of physiology or medicine was awarded to Dr Fire and Dr Mello ‘for their discovery of RNA interference—gene silencing by double-stranded RNA’32,35 which is now rapidly explored as a novel therapeutic principle for cardiovascular disease. Several studies demonstrated therapeutic potential of organ-targeted RNAi based on viral vectors36,37 or synthetic RNAs,38,39 and therapeutic strategies based on the modulation of miRs.40–45 An increasing spectrum of endogenous ncRNAs is employed for the development of novel therapeutic ncRNA tools optimized for specific therapeutic requirements.46–51 These investigations are only the beginning of therapeutic exploration, since 10 000 small ncRNAs arise from the human genome forming diverse RNA structures ranging from miRs to circular RNAs (circRNAs).52,53 These small ncRNAs are only part of a broader spectrum of ncRNAs including 16 000 lncRNAs up to many thousand basepairs in size.54–59 For lncRNAs, classification is still in its infancy60,61 and biological functions mostly unknown.62–66 However, recent studies strongly suggest that they constitute fundamentally new therapeutic targets67–70 with unusual RNA structures71,72 to be addressed using new drug types. Their importance for human health and disease is highlighted by observation that both small and large ncRNAs rapidly evolved during primate evolution and are often primate-specific and cannot be studied in common animal models of human disease.66,73 Overall, only a small part of the non-coding human genome has been investigated and explored with regard to possible therapeutic implications for cardiovascular medicine.
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.. .. Search for non-coding RNAs of clinical .. interest .. .. Because of the complexity of the non-coding genome, one may adopt .. .. a pragmatic heuristic approach directed to the identification of non-.. coding therapeutic targets in cardiovascular diseases (Figure 3). .. .. Insights from genome-wide phylogenetic studies, comprehensive .. sequencing work, and animal models of human cardiovascular dis.. .. eases constitute an indispensable first step towards target identifica.. tion. Importantly, the functions of the same ncRNA may differ .. .. between species, and most animal models differ significantly from the .. respective human disorder. Some ncRNAs do not even exist in ... humans73 or have only distant orthologues, so that their possible .. .. pathogenic relevance in humans cannot be investigated in animals. .. Therapeutic proof-of-concept studies of ncRNAs deregulated in animal .. .. models are therefore not necessarily predictive for effects in humans. .. It is therefore necessary to conduct screening studies for ncRNA .. .. of clinical interest also directly in humans, in particular in well-defined .. patient cohorts suffering from a carefully phenotyped cardiovascular .. .. disease of interest. Different principles build the basis of a successful .. screening project. First, relevant ncRNAs may be identified by .. .. genome-wide association studies (GWAS) indicating genomic loci .. conferring diseases risk via inherited mutations.74–83 A second ap.. .. proach is comprehensive transcriptome mapping in diseased organs, .. which may reveal genomically encoded defects, but also transcrip.. .. tome shifts developing during pathogenesis but not encoded at the .. 84 .. DNA level. Both approaches have already identified ncRNAs .. involved in cardiovascular disease pathogenesis. State-of-the-art ana.. .. lytical technologies enable both GWAS studies and comprehensive .. transcriptional mapping at high efficacy. However, some logistical and .. .. developmental aspects need to be considered that relate to funda.. mental limitations of these search strategies. Genome-wide associ.. .. ation studies commonly addresses early processes in disease .. pathogenesis by analysing the genome at the DNA level of inherited .. .. mutations acting from childhood. Late processes during disease de.. velopment, in contrast, are rather revealed by transcriptome map.. .. ping of already injured organs.85 In addition, the selection of properly .. phenotyped patients, sufficient cohort sizes, and proper control .. .. groups often constitutes a leading problem in such screening pro.. jects. In a third approach, high-throughput functional screening for .. .. relevant ncRNAs in cell types of pathogenic relevance .. (cardiomyocytes,86 vascular cells,87,88 cardiac fibroblasts,88–95 hep.. .. atocytes96,97) has been successfully used to identify new ncRNA with .. diagnostic and therapeutic potential. Disease-associated ncRNAs .. .. identified by any of these approaches may yield biomarkers to assess .. the risk of disease progression, thus facilitating treatment decisions, .. .. or to predict response-to-therapy to optimally allocate limited re.. sources. In addition, these ncRNAs may serve to develop and evalu.. .. ate novel therapeutic strategies beyond optimal current therapies. .. In the following, we discuss diagnostic and therapeutic perspec.. .. tives of ncRNAs in cardiovascular diseases. Three clinical goals are .. .. running as guiding threads throughout the article. First, the identifica.. tion of novel cardiovascular pathomechanisms and high-priority tar.. .. get diseases (Section ‘Non-coding RNAs as potential .. therapeutic targets in cardiovascular disease’). Second, the .. . development of clinically useful RNA-based therapeutics and
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identification of patients most likely to benefit (Section ‘Clinical translation of RNA therapeutics’). Third, the employment of RNA diagnostics to improve prognosis assessment and individualized clinical patient management (Section ‘Non-coding RNAs as biomarkers’). These issues are highlighted again in a Summarizing Illustration: Delineation of current high priority diseases, patients most likely to benefit, status of technical feasibility of ncRNA therapies, and the current value of RNA diagnostics for prognosis assessment and patient management.
Non-coding RNAs as potential therapeutic targets in cardiovascular disease Here, we review the identification of non-coding RNAs defining novel pathomechanisms, and important cardiovascular diseases significantly influenced by these novel processes.
MicroRNAs as therapeutic targets Post-myocardial infarction adverse cardiac remodelling and dysfunction MicroRNAs control important processes that contribute post-infarction injury and subsequent remodelling responses. For example, miRs can promote or inhibit cardiomyocyte cell death, regulate postischaemic neovascularization and control cardiac fibrosis (for an overview of miRs that control post-infarction repair see98). Cardiomyocyte cell death is induced by members of the miR-15 family, which are upregulated by myocardial ischaemia.99 Inhibition of miR-15 family members by short, locked nucleic acid (LNA)-based anti-miRs, which target the seed sequence of most miR-15 family members, reduced infarct size after ischaemia–reperfusion injury by derepressing the anti-apoptotic protein Bcl-2, the mitochondrial protecting factor ADP-ribosylation factor-like protein 2, and the deacetylase SIRT1 in mice.98,99 The feasibility of using miR-15 inhibitors has been documented also in pig models.99 Myocardial infarction additionally induces the expression of the pro-apoptotic miR-34 family members. Inhibition of miR-34 family members by different types of anti-miRs reduced infarct size, and augmented the recovery of heart function after acute myocardial infarction (AMI) by increasing the expression of cardioprotective SIRT1 and PNUTS.23,100 Inhibition of miR-15 or miR-34 family members also improved neovascularization after ischaemia.23,101 Members of the miR-1792 cluster, specifically miR-92a, control neovascularization and cardiomyo-cyte cell death after ischaemia.102,103 Pharmacological inhibition or genetic deletion of miR-92a increased capillary density and improved heart function after AMI in mice.102,103 Furthermore, catheter-based delivery of anti-miR-92a or encapsulated antagomiRs reduced infarct size in a large animal cardiac ischaemia/reperfusion pig model.104 Targets of miR-92a include integrin a5, KLF2 and SIRT1.102,104,105 miR-26a is increased after AMI and inhibits angiogenesis.106 Administration of a LNA-based anti-miR against miR-26a induced robust angiogenesis, reduced myocardial infarct size, and improved heart function.106 Mitochondrial function is controlled by miR-140, which reduces mitochondrial fission,107 thereby impairing cardiomyocyte survival. Pharmacological inhibition reduced infarct size after AMI in mice.107
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On the contrary, there are cardioprotective miRs108–111 including miR-210 which protects cardiomyo-cytes and vasculature. Intramyocardial injection of minicircle vectors carrying the miR-210 precursor reduced cell death and improved cardiac function and angiogenesis after AMI.108 Cardiac hypertrophy and heart failure One of the first reports about a miR involved in cardiac hypertrophy stem from the Olson laboratory, here miR-208 has been shown to be involved in hypertrophic signaling.112 Inhibition of miR-133 with an anti-miR-133 oligonucleotide generated cardiac hypertrophy in vivo showing a role of miR-133 in cardiac hypertrophy as well.113 Other miRs that are regulated during pathological stress include the miR-212/ 132 family which becomes activated during heart failure (HF) in humans114 and animal models.40 This miR family both regulates cardiac autophagy and hypertrophy in cardiomyocytes at least in part by modulation of transcription factor forkhead box O3. Inhibition of miR21 prevented HF development in mice.40 MicroRNAs also regulate intracellular calcium homeostasis, a process that is strongly deregulated during HF, thereby directly affecting cardiac contractility.86,92 For few specific cardiomyopathies, the therapeutic potential of ncRNAs has been investigated. Thus, recent studies identified novel miR therapeutic targets in coxsackievirus-B3 (CVB3) myocarditis. MicroRNAs 155, 146b, and 21 are upregulated in acute CVB3-myocarditis.115,116 Inhibition of miR-155114,117,118, miRs 21 and 146b119 by systemically delivered anti-miRs attenuates cardiac inflammation and myocardial damage in CVB3 or autoimmune myocarditis in mice. Atherosclerosis Atherosclerosis underlying coronary artery disease (CAD), stroke, and peripheral vascular disease is a chronic inflammatory reaction of the arterial wall characterized by maladaptive responses of endothelial cells at sites with disturbed flow conditions and of immune cells, in particular macrophages accumulating lipids without resolving inflammation in a context of dyslipidaemia. A plethora of miRs and relevant targets has emerged in mouse models of atherosclerosis to intricately orchestrate crucial mechanisms, thus forming a basis for potential therapeutic strategies using anti-miRs or miR mimics.120–122 An atheroprotective role in endothelial regeneration has been identified for the miR-126 strand pair. Depletion of miR-126-5p at predilection sites with altered flow under hyperlipidaemic stress limits the endothelial proliferative reserve and promotes lesion formation through derepression of DLK-1123 which can be restored by miR-126-5p mimics. Furthermore, delivery of miR-126-3p by microparticles from apoptotic to recipient endothelial cells amplifies CXCL12 expression by repressing RGS16 to recruit proangiogenic cells supporting endothelial recovery,124 a mechanism possibly impaired in CAD or diabetes.120 Similarly, transfer of KLF2-induced miR-143/miR-145 by endothelial microvesicles to smooth muscle cell (SMCs) attenuates atherosclerosis by generating a contractile phenotype, as with miR-145 overexpression.26,125 miR-181b targeting importin-a3 is upregulated by laminar flow and limits endothelial inflammation and atherosclerosis by inhibiting NF-jB activation.126 Conversely, miR-92a is upregulated by disturbed flow and modified lipoproteins to promote NF-jB-mediated endothelial inflammation and atherosclerosis by targeting KLF2/4, which is reversed by anti-miRs.127 Likewise, the pre-ribosomal RNA-
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A
B
Figure 1 Evolution of the non-coding human genome. (A) Whereas the number of protein-coding genes remains surprisingly similar from simple to complex species, it is the non-coding part of the genome that increases dramatically with morphologic complexity, reaching 99% of the entire genome in humans. Multiple evidence suggests important roles of ncRNAs transcribed from the non-coding genome in human health and disease. (B): Whereas a vast number of ncRNAs with grossly different structures (e.g. lncRNAs, miRs, circRNAs) have been identified, however, in the majority of these molecules their functions are essentially unknown (details in Supplementary material online, Figure S1).
derived miR-712 is upregulated by altered flow to elicit endothelial inflammation and atherosclerosis by targeting TIMP3.128 Macrophage subtypes show differential profiles of miR expression. Notably, miR-155 increases during atherogenesis or upon M1-type macrophage polarization, and promotes inflammatory activation and advanced atherosclerosis by targeting BCL6, a transcriptional regulator counteracting NF-jB, and by reducing cholesterol efflux.129,130 Released from competing targets in M1-macrophages, miR-342-5p suppresses Akt1 to cause inflammatory activation and atherogenesis by upregulating miR-155 and forming a functional tandem for antimiR strategies.131 The cholesterol efflux from macrophages to HDL is mediated by the ATP-binding cassette transporters ABCA1 and ABCG1. The ABCA1 30 UTR encompasses a wealth of binding sites for miRs including miR-33a/b, miR-19b, miR-144 and miR-148a.132 Beyond miR-33, several miRs targeting ABCA1/G1 can inhibit cholesterol efflux and have been implicated in atherogenesis.120,121,132 In contrast,
.. short-term antagonism of miR-33 in rodents and non-human pri.. .. mates significantly elevates plasma HDL, but overall the efficacy of .. 120–122,132 .. miR-33 silencing in atherogenesis remains controversial. .. Table 1 and Figure 4 provide short overviews of current miR.. targeting cardiovascular therapies. ... .. .. Long non-coding RNAs as potential .. .. therapeutic targets .. .. .. Long non-coding RNAs research is challenging due to the fact that .. most lncRNAs, in contrast to miRs, are not conserved among spe.. .. cies. Even when selecting the few being conserved, epigenetic effects .. and the processes of DNA and RNA assembly they influence may dif.. .. fer among species, making translation from rodents to humans chal.. lenging. Encouraging is the fact that lncRNAs have distinct functions .. .. in cardiovascular diseases (reviewed170–176) and therefore may open .. new therapeutic opportunities.177
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Figure 2 Endogenous non-coding RNAs as blueprints for RNA therapeutics. Non-coding RNAs may be addressed as therapeutic targets, but an increasing spectrum of endogenous ncRNAs are also employed as blueprints for the development of novel therapeutic tools. The spectrum of possible therapeutic targets has thus expanded beyond proteins, but also the therapeutic ‘toolbox’. One current topic is therapeutic RNA interference triggers (siRNAs) originally developed from endogenous siRNAs as blueprints, and made clinically applicable based on sophisticated chemical modifications and coupling to carriers/ligands for tissue targeting33,34 (details in Supplementary material online, Figure S2).
Supplementary material online, Figure S1 depicts unsolved issues in translational lncRNA research. First, development of a more advanced classification54,56,60,178–183 of the vast number of lncRNAs detected in humans—based on structure, subcellular localization, intracellular processing, functional modules—is needed to facilitate rational development of lncRNA-targeting treatments. Second, a peculiar aspect of lncRNAs to be addressed is their often extensive and specific post-translational processing with differential subcellular localization of the products, yielding complex RNA processing systems with different functions of the products69,184 which may be differentially addressable by drugs.185
Cardiac hypertrophy and heart failure Next to miRs, lncRNAs also play an important role in cardiac failure. The lncRNA Cardiac hypertrophy associated transcript (CHAST) is increased during cardiac hypertrophy in mice and humans and its inhibition both prevented and attenuated cardiac remodelling and HF.186 In the heart, suppression of Myosin heavy-chain-associated RNA transcript (MHRT) accelerated progression of hypertrophy to failure,187 whereas overexpression of CHAST induced cardiomyocyte
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hypertrophy. Gapmer-mediated CHAST silencing both prevented and attenuated pressure overload-induced pathological cardiac remodelling without evident early toxicological side effects.186 Knockdown of lncRNA Cardiac mesoderm enhancer associated ncRNA (CARMEN) inhibits cardiac specification and differentiation in cardiac precursor cells. This occurs independently of miR-143 and -145 expressions despite the fact that those two miRs located proximal to the enhancer sequences. CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2).188
Atherosclerosis Beyond miRs, lncRNAs may regulate atherosclerosis but their role and molecular mechanisms remain to be elucidated. As a strong genetic risk locus, the chromosome 9p21 (Chr9p21) region encodes the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL). Its transcript expression correlates with the severity of atherosclerosis, and ANRIL regulates target genes in trans through Alu motifs in their promoters, leading to increased cell proliferation and adhesion but decreased apoptosis, relevant to atherosclerosis.124 Conversely, lincRNA-p21 down-regulated in atherosclerotic plaques has been
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Search strategies to identify ncRNAs with clinical therapeutic potential PRECLINICAL genomewide transcriptome mapping studies encompassing mRNAs, miRs, lncRNAs, and other ncRNAs using
using RNA seq or other comprehensive methods
CLINICAL ncRNA expression profiling in cardiovascular disease patients first hints to biological functions and possible pathogenic roles of ncRNA in humans
EXPERIMENTAL ncRNA manipulation in animal model of disease LIMITATION: requires presence of the human transcript of interest in the animal
BIOLOGICAL FUNCTION ASSIGMENT
to human ncRNAs and processing products
CLINICAL TRANSLATION STRATEGIES
Traditional therapeutic proof-of-principle studies in animals LIMITATION: requires presence of human ncRNA in model species
Figure 3 Search strategies for non-coding RNAs of clinical interest. Genome-wide genomic and transcriptomic mapping have revealed a huge number of novel non-coding transcripts and provided structural information regarding their gene loci, but for the vast majority there is as yet no functional assignment. Functional characterization is complicated by their often extensive processing. Prospecting of this plethora of ncRNA for possible clinical significance may be critically facilitated by state-of-the-art transcriptional mapping of cardiovascular patients, in order to obtain first functional hints to guide subsequent in-depth experimental investigations including therapeutic proof-of-principle studies.
found to control neointima formation, repressing SMC proliferation and inducing apoptosis by enhancing p53 activity.125 Intense investigations into the function on other miRs and lncRNAs yet to be identified are currently underway. The potential of targeting lncRNAs was first described in models of angiogenesis or cell growth. Silencing of Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) reduced capillary growth not only in a mouse model of hind limb ischaemia (detrimental),68 but also in a rat model of diabetic retinopathy (beneficial).189 MALAT1-derived mascRNA is involved in innate immunity and viral myocarditis,190 but appears to be dispensable in pressure overload-induced HF in mice.191 Next, inhibition of lincRNA-p21 aggravated neointimal hyperplasia in a carotid artery injury model in ApoE knockout mice.192 Smooth muscle and endothelial cell-enriched migration/differentiation associated RNA (SENCR) is a vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.87 All of the above was obtained in rodent models, however, with only correlative human data on cardiac expression186,187 or blood levels.193 Since lncRNAs are poorly conserved between species, translation of animal findings to patients will be challenging. Whether the use of more relevant human cell lines, like inducible pluripotent stem cells may help to overcome these limitations of animal studies, remains to be determined. Identification of human-specific lncRNAs, either as human orthologues from rodent-discovered lnRNAs,194 or being expressed during embryonic stem cell differentiation into cardiomyocytes,195 will be important for further advance of lncRNA-based treatments in humans.
.. .. Clinical translation of RNA .. .. therapeutics .. .. .. Several novel pathomechanisms influenced by ncRNAs, discussed .. above, cannot be therapeutically addressed using conventional .. .. pharmacology. ncRNA therapeutics offer new options to influence .. these hitherto inaccessible disease processes. .. .. .. .. .. .. .. Versatility of therapeutic non-coding .. .. RNA structures .. ... Small interference RNAs and related structures mediating RNAi and .. thus target gene silencing are the currently most advanced and fre.. quently used types of ncRNA for therapeutic purposes, and part of a .. .. continuously expanding spectrum of therapeutic ncRNA tools de.. veloped from endogenous ncRNA as blueprints (Figure 2). .. .. Fundamentally different from DNA, RNAs are carrying information .. not only in their linear sequences of nucleotides (primary structure), .. .. but local nucleotide pairing creates secondary structures e.g. Hairpins, .. and interactions among distantly located sequences create tertiary .. .. structures.196 In fact, this structural versatility needs to be considered .. for RNAs as therapeutic tools as well as targets. The plethora of .. .. RNA types, sequences, and structures created by evolution is a treas.. ure trove of potential therapeutic tools and targets (Figure 2).
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Figure 4 Cardiovascular RNA drugs address multiple organ systems. RNA drugs do not need to address heart (A) or vasculature (B) directly. Primarily liver-targeted RNA drugs (C) are the currently most successful development in the cardiovascular field. Another group of strategies addresses the immune system (D), in particular monocytes-macrophages. Carrier denotes a synthetic nanoparticle and/or receptor ligand employed to deliver an RNA drug to its tissue target.29,31,33,34,155–160 ‘Carrier’ is bound to and serves to stabilize the RNA drug within the circulation, and to endow it with at least partial selectivity for the target cells, in order to minimize side effects. ‘AAV9’ denotes a cardiac-targeting recombinant adenoassociated viral vector containing a genome from which the therapeutic RNA sequence is continuously transcribed (for transcript types see Figure 5). apo(a), apolipoprotein (a); CCR2, chemokine C-C motif receptor 2; CHAST, Cardiac hypertrophy associated transcript; PCSK9, proprotein convertase subtilisin/kexin type 9; PLB, phospholamban.
Classification of non-coding RNA therapeutics Figure 5 provides an overview of currently available ncRNA therapeutics and their key properties: (i) sufficient stability or continuous in vivo production of therapeutic ncRNA, or the ability to redose if needed, (ii) high specificity of therapeutic ncRNA for the molecular target, (iii) proper targeting to the correct cells type or tissue by use of appropriate vectors plus transcriptional targeting, (iv) side effects induced by the therapeutic ncRNA itself or its delivery system need to be minimized, and (v) regulatability for certain applications.197 Overall, RNA therapeutics may be chemically synthesized nucleic acids delivered using chemical delivery systems,33,34,198–200 or produced in vivo by recombinant viral vectors targeting different tissues.201–209 Viral vectors have the capacity to act as long-term productive ‘RNA drug factories’, whereas chemical synthesis allows introduction of RNA modifications that cannot be generated biologically (details in Supplementary material online, Figure S3). Table 1
.. .. summarizes studies employing ncRNAs in clinical trials, preclinical .. animal models, or other experimental studies in vivo. .. .. .. .. .. .. Challenges in translation .. Clinical translation of these fascinating and far-reaching options faces .. .. challenges ranging from ncRNA drug design and delivery (Box 1) to .. regulatory issues (Box 2). Whereas animal studies are most valuable .. .. and indispensable for proof of principle, most of these studies use .. young otherwise healthy animals and thus rarely reflect the clinical .. .. reality. Taking HF as an example, few if any animal models mimic the .. human HF reality, a chronic systemic condition lasting years to dec.. .. ades. Models of chronic HF in animals are rare and only recently a .. model emerged which combines diabetes, obesity, hypertension, and .. .. leads to kidney dysfunction.210,211 On the solid foundation of animal .. studies, few pioneering clinical trials were successful in demonstrating .. . the technical and clinical feasibility of ncRNA therapeutic approaches.
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Table 1
Translational studies addressing the therapeutic potential of non-coding RNAs
Investigation
Strategy
Technology
Clinical Trials LDL-C reduction
Therapeutic RNAi targeting PCSK9 reduced LDL-C Synthetic siRNA via nanoparticle carrier
Reference
.................................................................................................................................................................................................................... 133
in non-human primates LDL-C reduction
RNAi drug targeting PCSK9 reduced LDL-C in vol- Synthetic siRNA via nanoparticle carrier unteers (intravenous siRNA administration in lipid
134
LDL-C reduction
RNAi drug targeting PCSK9 reduced LDL-C in vol- synthetic siRNA conjugated to GalNAC unteers (subcutaneous siRNA administration con-
LDL-C reduction
ASO targeting PCSK9 reduced LDL-C in non-human LNA-ASO via nanoparticle carrier primates
136
Lp(a) reduction
ASO targeting apolipoprotein(a) reduced Lp(a) in a 20 -MOE-ASO via nanoparticle carrier randomised, double-blind, placebo-controlled
137–139
Transthyretin suppression
Clinical RNAi therapy for transthyretin amyloidosis Synthetic siRNA via nanoparticle carrier inhibited hepatic synthesis of mutant TTR protein
140
Antiviral
RNAi-based antiviral treatment in non-human
Synthetic siRNA via nanoparticle carrier
141
LNA-ASO intracoronary injection (swine)
104,142
nanoparticles) 135
jugated to GalNAC)
clinical study
primates Experimental Research Vascular-targeted anti-miR
miR-92a inhibition of ischaemia/reperfusion injury, and improvement of re-endothelialization following vascular injury
Cardiac-targeted RNAi
Cardiotropic silencing of Ca2+ cycle regulator phos- Cardiac-targeted AAV9-vector-derived pholamban for the treatment of severe heart failure
Monocyte-targeted RNAi
Nanoparticle-encapsulated synthetic siRNA for silencing of monocytic CCR2
36
shRNA (rat) Non-viral, lipid nanoparticle-mediated siRNA 38 delivery targeting particularly monocytes (mouse)
RNAi imaging in vivo
PHD2-shRNA followed by a hypoxia response
Cardiac RNAi
element-containing promoter RNAi induced alloimmune tolerance in heart trans- siRNA (rat)
Imaging of RNAi in space and time (mouse) 143 144
plantation model TLR adaptor silencing Vasculature-targeted RNAi RNAi visualization
RNAi to silence chymase increased plaque stability Lentivirus-based RNAi Visualizing lipid-formulated siRNA release and target Development of strategies to improve the
Improvement of stem cells by RNAi
RNAi to enhance survival and function of transplanted cells
Enhancement of the efficacy of cardiovascu- 147 lar stem cell therapies
Allele-specific RNAi
Allele-specific RNAi for human induced pluripo-
Rescue of autosomal dominant-negative
knockdown
tency stem cell cardiomyocytes
145 146
delivery of candidate RNAi drugs
148
disorders
Safety issues Scientific and Regulatory Policy Committee:
149
Biotherapeutics Scientific and Regulatory Policy Committee:
150
Antisense Oligonucleotides Emerging technologies Gene silencing
siRNA vs miRNA for gene silencing
Comparison of therapeutic siRNAs and
144,148,151,152
miRNAs RNA engineering RNAi visualization
High-throughput cellular RNA device engineering Visualizing lipid-formulated siRNA release and target To facilitate development of rational stratknockdown
RNAi-based functional profiling
RNAi-based functional profiling of loci from genome-wide association studies
ASO, antisense oligonucleotide; LNA, locked nucleic acid; RNAi, RNA interference.
153 146
egies to improve the cytosolic delivery of candidate drugs RNAi to analyse 133 candidate genes in 56 loci identified by GWAS
154
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Translational perspectives of the non-coding genome
Box 1 Goals and challenges of non-coding RNA therapeutics research
• Required Knowledge
• Required Properties of RNA Drug
Posttranscriptional processing and intracellular kinetics (lncRNAs) Expression patterns in different cell types and organs Cellular function(s) may grossly differ between cell types (pleiotropism)
Biological function(s) derived from germline or tissue-specific animal models
• Patient Selection High unmet clinical need
High specificity of RNA drug - RNA target interaction High stability of RNA drug in the circulation and within target cells
• RNA Drug Delivery System Selective targeting to diseased organ or specific cell type (to avoid off-target effects at organ level)
Clinical phenotype allows for rapid and significant improvement
Clinical Aspects
Subcellular localization (miRs, lncRNAs)
Technological Aspects
Rationale for ncRNAs as Therapeutic Targets
Key characteristics of ncRNA-based drugs, clinical goals, and challenges to overcome in translation from preclinical research to clinical applications.
Patient population straightforward to define and identify Endpoints well defined and easily measurable with detectable effects in
