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Targeting and delivery of microRNA-targeting antisense oligonucleotides in cardiovascular diseases

      Highlights

      • MicroRNAs (miRNAs) drive the onset and/or progression of multiple cardiovascular diseases.
      • MiRNA-targeting antisense oligonucleotides (antagomirs) are a promising therapeutic approach to precisely inhibit miRNAs.
      • Viral or non-viral targeting enhances delivery of antagomirs to cardiac and vascular cells, decreasing systemic exposure.

      Abstract

      Discovered three decades ago, microRNAs (miRNAs) are now recognized as key players in the pathophysiology of multiple human diseases, including those affecting the cardiovascular system. As such, miRNAs have emerged as promising therapeutic targets for preventing the onset and/or progression of several cardiovascular diseases. Anti-miRNA antisense oligonucleotides or “antagomirs” precisely block the activity of specific miRNAs and are therefore a promising therapeutic strategy to repress pathological miRNAs. In this review, we describe advancements in antisense oligonucleotide chemistry that have significantly improved efficacy and safety. Moreover, we summarize recent approaches for the targeted delivery of antagomirs to cardiovascular tissues, highlighting major advantages as well as limitations of viral (i.e., adenovirus, adeno-associated virus, and lentivirus) and non-viral (i.e., liposomes, extracellular vesicles, and polymer nanoparticles) delivery systems. We discuss recent preclinical studies that use targeted antagomir delivery systems to treat three major cardiovascular diseases (atherosclerosis, myocardial infarction, and cardiac hypertrophy, including hypertrophy caused by hypertension), highlighting therapeutic results and discussing challenges that limit clinical applicability.

      Graphical abstract

      Keywords

      1. Micro-RNAs (miRNAs) as therapeutic targets

      1.1 Biogenesis and activity of miRNAs

      Only 1–2% of the mammalian genome encodes proteins, whereas the remainder is untranslated. These untranslated genomic regions contain numerous sequences that are transcribed into “noncoding RNAs” (ncRNAs). NcRNAs are classified based on length into small RNAs [18–200 bp; including “microRNAs” or “miRNAs”, “piRNAs”, “snoRNAs”, tRNA-derived fragments (“tRFs”), and likely others] and long RNAs (>200 bp; “long non-coding RNAs” or “lncRNAs”). MiRNAs are primarily involved in the posttranscriptional regulation of protein-coding genes [
      • O'Brien J.
      • Hayder H.
      • Zayed Y.
      • Peng C.
      Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.
      ]. Consistent with their roles in regulating critical biological processes, expression of individual miRNAs is tightly regulated in tissue- and developmental stage-specific patterns. For example, several miRNAs (e.g., miR-1, miR-133, and miR-499) are expressed during embryonic heart development, in which they regulate fundamental processes of cardiogenesis such as cardiomyocyte proliferation and differentiation [
      • Espinoza-Lewis R.A.
      • Wang D.Z.
      MicroRNAs in heart development.
      ]. As described in greater detail elsewhere [
      • O'Brien J.
      • Hayder H.
      • Zayed Y.
      • Peng C.
      Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.
      ], miRNA-encoding sequences are transcribed within the nucleus by RNA polymerase II to generate primary miRNAs (pri-miRNAs) that are first cleaved by Drosha, a class 2 ribonuclease III enzyme, to become precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported to the cytosol by exportin 5, where they are further cleaved by Dicer (also part of the ribonuclease III family) into double-stranded mature miRNAs. One of the miRNA strands is then loaded onto the RNA-induced silencing complex (RISC), guiding RISC to a target messenger RNA (mRNA). The “seed” sequence of the miRNA, located between nucleotides 2 and 7, binds to a complementary sequence in the 3′-untranslated region of a target mRNA, inhibiting mRNA translation [
      • O'Brien J.
      • Hayder H.
      • Zayed Y.
      • Peng C.
      Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.
      ]. Importantly, a single miRNA can target multiple mRNAs, simultaneously altering levels of several proteins in single or multiple biological pathways. For example, by repressing SOX6, miR-208 activates cellular signaling that stimulates proliferation while simultaneously inhibiting pro-apoptotic pathways by repressing CDKN1A [
      • Zhang X.-T.
      • Xu M.-G.
      Potential link between microRNA-208 and cardiovascular diseases.
      ]. MiR-223 also has a multifaceted role, regulating cholesterol metabolism via targeting SCARB1 (repressing high-density lipoprotein uptake) and by targeting HMGCR (lowering cholesterol biosynthesis) [
      • Vickers K.C.
      • Landstreet S.R.
      • Levin M.G.
      • Shoucri B.M.
      • Toth C.L.
      • Taylor R.C.
      • et al.
      MicroRNA-223 coordinates cholesterol homeostasis.
      ]. In the vascular wall, miR-155 modulates distinct biological processes that shape the endothelial phenotype. Through direct inhibition of NOS3, miR-155 represses vasorelaxation [
      • Sun H.X.
      • Zeng D.Y.
      • Li R.T.
      • Pang R.P.
      • Yang H.
      • Hu Y.L.
      • et al.
      Essential role of microRNA-155 in regulating endothelium-dependent vasorelaxation by targeting endothelial nitric oxide synthase.
      ], whereas it induces endothelial inflammation by targeting the transcription factor Bcl6 [
      • Nazari-Jahantigh M.
      • Wei Y.
      • Noels H.
      • Akhtar S.
      • Zhou Z.
      • Koenen R.R.
      • et al.
      MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages.
      ].

      1.2 Roles of miRNAs in cardiovascular homeostasis and disease

      MiRNAs regulate numerous molecular pathways that help maintain cardiovascular homeostasis. Accordingly, dysregulation of miRNAs can significantly alter cellular physiology, triggering the onset and progression of cardiovascular disease (CVD). Because the pathophysiological relevance of miRNAs in CVDs is discussed extensively in previous reports [
      • Romaine S.P.R.
      • Tomaszewski M.
      • Condorelli G.
      • Samani N.J.
      MicroRNAs in cardiovascular disease: an introduction for clinicians.
      ,
      • Lu Y.
      • Thavarajah T.
      • Gu W.
      • Cai J.
      • Xu Q.
      Impact of miRNA in atherosclerosis.
      ,
      • Melman Y.F.
      • Shah R.
      • Das S.
      MicroRNAs in heart failure: is the picture becoming less miRky?.
      ,
      • Ghafouri-Fard S.
      • Gholipour M.
      • Taheri M.
      Role of MicroRNAs in the pathogenesis of coronary artery disease.
      ,
      • Hosen M.R.
      • Goody P.R.
      • Zietzer A.
      • Nickenig G.
      • Jansen F.
      MicroRNAs as master regulators of atherosclerosis: from pathogenesis to novel therapeutic options.
      ] and to limit the scope of this review, herein we focus primarily on the role of miRNAs in three major CVDs: atherosclerosis, myocardial infarction (MI) and its complications (including arrhythmias and fibrosis), and cardiac hypertrophy, including hypertrophy caused by hypertension. A focus on these 3 CVDs also allows us to highlight major issues and approaches in targeting and delivery of antagomirs to cardiac and vascular cells. Because of space limitations, here we focus on a subset of miRNAs that are relevant to these 3 CVDs, selecting miRNAs that are clearly connected both with mRNA targets and with regulation of cardiovascular physiology and disease.
      MiRNAs regulate several biological processes that contribute to atherosclerosis (Table 1) [
      • Hosen M.R.
      • Goody P.R.
      • Zietzer A.
      • Nickenig G.
      • Jansen F.
      MicroRNAs as master regulators of atherosclerosis: from pathogenesis to novel therapeutic options.
      ]; some of these processes are specific to endothelial cells (EC). For example, miR-10a maintains EC homeostasis and helps prevent atherosclerosis by downregulating the NFKB1 activators MAP3K7 and BTRC, thereby reducing levels of EC adhesion molecules [e.g., E-selectin and vascular cell adhesion molecule-1 (VCAM1)]. MiR-10a also reduces expression of proinflammatory cytokines (e.g., IL6 and CCL2) [
      • Fang Y.
      • Shi C.
      • Manduchi E.
      • Civelek M.
      • Davies P.F.
      MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro.
      ]. Supporting an atheroprotective role for miR-10a, humans with coronary artery disease [
      • Infante T.
      • Forte E.
      • Punzo B.
      • Cademartiri F.
      • Cavaliere C.
      • Soricelli A.
      • et al.
      Correlation of circulating miR-765, miR-93-5p, and miR-433-3p to obstructive coronary heart disease evaluated by cardiac computed tomography.
      ] have lower levels of miR-10a both in serum and coronary artery endothelium [
      • Kuo J.T.
      • Tsai H.E.
      • Lin C.T.
      • Lee C.I.
      • Lee P.L.
      • Ruan Y.R.
      • et al.
      Low levels of MicroRNA-10a in cardiovascular endothelium and blood serum are related to human atherosclerotic disease.
      ]. MiR-126 also has atheroprotective activity, reducing atherosclerosis in mice by blocking the Notch1 inhibitor Dlk1 in EC, leading to increased EC proliferation that may improve vascular healing [
      • Schober A.
      • Nazari-Jahantigh M.
      • Wei Y.
      • Bidzhekov K.
      • Gremse F.
      • Grommes J.
      • et al.
      MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1.
      ]. Consistent with this mechanism, miR-126 expression in human carotid artery lesions correlates inversely with DLK1 positive EC [
      • Schober A.
      • Nazari-Jahantigh M.
      • Wei Y.
      • Bidzhekov K.
      • Gremse F.
      • Grommes J.
      • et al.
      MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1.
      ].
      Table 1Examples of miRNAs involved in CVDs.
      CVDmiRNATarget genesBiological role of miRNARef.
      AtherosclerosismiR-10aMAP3K7 and BTRCReduced expression of endothelial adhesion molecules and proinflammatory cytokines[
      • Fang Y.
      • Shi C.
      • Manduchi E.
      • Civelek M.
      • Davies P.F.
      MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro.
      ]
      miR-126Dlk1Enhanced proliferation of endothelial cells[
      • Schober A.
      • Nazari-Jahantigh M.
      • Wei Y.
      • Bidzhekov K.
      • Gremse F.
      • Grommes J.
      • et al.
      MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1.
      ]
      miR-155Hbp1Increased lipid uptake, synthesis of ROS, and foam cell formation[
      • Tian F.J.
      • An L.N.
      • Wang G.K.
      • Zhu J.Q.
      • Li Q.
      • Zhang Y.Y.
      • et al.
      Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.
      ]
      miR-33aABCA1Reduced cholesterol efflux and increased foam cell formation[
      • Price N.L.
      • Goedeke L.
      • Suarez Y.
      • Fernandez-Hernando C.
      miR-33 in cardiometabolic diseases: lessons learned from novel animal models and approaches.
      ]
      miR-145KLF5Prevents neointima formation[
      • Cheng Y.
      • Liu X.
      • Yang J.
      • Lin Y.
      • Xu D.Z.
      • Lu Q.
      • et al.
      MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation.
      ]
      miR-362ADAMTS1Prevents vascular smooth muscle cell proliferation and migration into the intima[
      • Li M.
      • Liu Q.
      • Lei J.
      • Wang X.
      • Chen X.
      • Ding Y.
      MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1.
      ]
      Myocardial infarction (MI)miR-1Kcnj2 and Gja1Decreased cardiac conduction and induction of arrhythmias[
      • Yang B.
      • Lin H.
      • Xiao J.
      • Lu Y.
      • Luo X.
      • Li B.
      • et al.
      The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
      ]
      miR-433Azin1Activates TGF-β signaling to induce cardiac fibrosis[
      • Tao L.
      • Bei Y.
      • Chen P.
      • Lei Z.
      • Fu S.
      • Zhang H.
      • et al.
      Crucial role of miR-433 in regulating cardiac fibrosis.
      ]
      miR-24Gata2 and Pak4Induction of apoptosis of endothelial cells and reduced neovascularization of the ischemic myocardium[
      • Fiedler J.
      • Jazbutyte V.
      • Kirchmaier B.C.
      • Gupta S.K.
      • Lorenzen J.
      • Hartmann D.
      • et al.
      MicroRNA-24 regulates vascularity after myocardial infarction.
      ]
      miR-134Kdm2aRepression of myocardial angiogenesis after MI[
      • Li X.
      • Wei C.
      • Zhang Z.
      • Jin Q.
      • Xiao X.
      MiR-134-5p regulates myocardial apoptosis and angiogenesis by directly targeting KDM2A after myocardial infarction.
      ]
      Hypertension and hypertensive heart diseasemiR-181a and miR-663RENDownregulation may lead to higher renin expression in the renal cortex of hypertensive patients[
      • Marques F.Z.
      • Campain A.E.
      • Tomaszewski M.
      • Zukowska-Szczechowska E.
      • Yang Y.H.
      • Charchar F.J.
      • et al.
      Gene expression profiling reveals renin mRNA overexpression in human hypertensive kidneys and a role for microRNAs.
      ]
      miR-143ACE2Downregulation may enhance ACE2 expression in the arteries of hypertensive patients[
      • Chen L.J.
      • Xu R.
      • Yu H.M.
      • Chang Q.
      • Zhong J.C.
      The ACE2/apelin signaling, MicroRNAs, and hypertension.
      ]
      miR-26Gata4Prevents pressure overload-induced cardiac hypertrophy[
      • Han M.
      • Yang Z.
      • Sayed D.
      • He M.
      • Gao S.
      • Lin L.
      • et al.
      GATA4 expression is primarily regulated via a miR-26b-dependent post-transcriptional mechanism during cardiac hypertrophy.
      ]
      miR-217PtenPromotes cardiac hypertrophy via AKT pathway activation[
      • Nie X.
      • Fan J.
      • Li H.
      • Yin Z.
      • Zhao Y.
      • Dai B.
      • et al.
      miR-217 promotes cardiac hypertrophy and dysfunction by targeting PTEN.
      ]
      miR-214Sirt3Enhances mitochondrial dysfunction and cardiac hypertrophy[
      • Ding Y.Q.
      • Zhang Y.H.
      • Lu J.
      • Li B.
      • Yu W.J.
      • Yue Z.B.
      • et al.
      MicroRNA-214 contributes to Ang II-induced cardiac hypertrophy by targeting SIRT3 to provoke mitochondrial malfunction.
      ]
      CVD: cardiovascular disease; ROS: reactive oxygen species; MI: myocardial infarction.
      MiRNAs also regulate atherogenesis via their activities in macrophages, including effects on lipid metabolism [
      • Ouimet M.
      • Ediriweera H.
      • Afonso M.S.
      • Ramkhelawon B.
      • Singaravelu R.
      • Liao X.
      • et al.
      microRNA-33 regulates macrophage autophagy in atherosclerosis.
      ]. For example, miR-155 (which is upregulated by Ox-LDL) accelerates atherosclerosis by targeting Hbp1, leading to enhanced lipid uptake in murine macrophages, increased generation of reactive oxygen species, and enhanced foam cell formation [
      • Tian F.J.
      • An L.N.
      • Wang G.K.
      • Zhu J.Q.
      • Li Q.
      • Zhang Y.Y.
      • et al.
      Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.
      ]. Circulating monocytes of CAD patients have elevated levels of miR-155, supporting an atherogenic role for miR-155 in humans [
      • Tian F.J.
      • An L.N.
      • Wang G.K.
      • Zhu J.Q.
      • Li Q.
      • Zhang Y.Y.
      • et al.
      Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.
      ]. Similarly, miR-33a contributes to atherogenesis by reducing macrophage ABCA1 expression, lowering cholesterol efflux, and sustaining foam cell formation [
      • Price N.L.
      • Goedeke L.
      • Suarez Y.
      • Fernandez-Hernando C.
      miR-33 in cardiometabolic diseases: lessons learned from novel animal models and approaches.
      ].
      The contribution of vascular smooth muscle cells (VSMCs) to atherogenesis depends on their phenotype, which is tightly regulated by miRNAs. For example, miR-145 maintains the differentiated state of VSMCs via inhibition of the transcription factor KLF5, preventing neointima formation in balloon-injured rat carotid arteries [
      • Cheng Y.
      • Liu X.
      • Yang J.
      • Lin Y.
      • Xu D.Z.
      • Lu Q.
      • et al.
      MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation.
      ]. MiR-145 also has biological roles outside the cardiovascular system. For example, inhibition of KLF5 by miR-145 represses proliferation and migration of cervical and nasopharyngeal carcinoma cells [
      • Cao H.
      • Pan G.
      • Tang S.
      • Zhong N.
      • Liu H.
      • Zhou H.
      • et al.
      miR-145-5p regulates the proliferation, migration and invasion in cervical carcinoma by targeting KLF5.
      ,
      • Yuan C.H.
      • Hsu W.C.
      • Huang A.M.
      • Yuan B.C.
      • Chen I.H.
      • Hsu C.A.
      • et al.
      MicroRNA-145-5p modulates Kruppel-like factor 5 and inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma.
      ]. MiR-362 also plays an atheroprotective role by inhibiting ADAMTS1, thereby repressing VSMC proliferation and migration into the intima [
      • Li M.
      • Liu Q.
      • Lei J.
      • Wang X.
      • Chen X.
      • Ding Y.
      MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1.
      ]. MiR-362 levels are decreased in plasma of CAD patients, consistent with an atheroprotective role [
      • Li M.
      • Liu Q.
      • Lei J.
      • Wang X.
      • Chen X.
      • Ding Y.
      MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1.
      ].
      MiRNAs play significant roles in the regulation of cardiac biology and disease, including ischemic heart disease and MI (Table 1) [
      • Colpaert R.M.W.
      • Calore M.
      MicroRNAs in cardiac diseases.
      ]. For example, muscle-enriched miR-1 impairs cardiac contractility by repressing Kcnj2 and Gja1 proteins that form K+ and gap junction channels, respectively, slowing cardiac conduction and promoting arrhythmias after MI in rats [
      • Yang B.
      • Lin H.
      • Xiao J.
      • Lu Y.
      • Luo X.
      • Li B.
      • et al.
      The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
      ]. Moreover, miR-1 is upregulated in the myocardium of patients with CAD and its levels are inversely correlated with levels of KCNJ2 and GJA1 proteins, suggesting a detrimental role for miR-1 in humans with ischemic heart disease [
      • Yang B.
      • Lin H.
      • Xiao J.
      • Lu Y.
      • Luo X.
      • Li B.
      • et al.
      The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
      ]. Cardiac remodeling after MI is altered by miRNAs such as miR-433, which—at least in part by inhibiting Azin1—increases Tgf-β1, leading to Smad3 activation and cardiac fibrosis after experimental MI in mice [
      • Tao L.
      • Bei Y.
      • Chen P.
      • Lei Z.
      • Fu S.
      • Zhang H.
      • et al.
      Crucial role of miR-433 in regulating cardiac fibrosis.
      ]. Interestingly, miR-433 is upregulated both in the plasma of patients with CAD and in the left ventricle of patients with nonischemic dilated cardiomyopathy, suggesting that miR-433 might contribute to both ischemic and non-ischemic left ventricular dysfunction [
      • Infante T.
      • Forte E.
      • Punzo B.
      • Cademartiri F.
      • Cavaliere C.
      • Soricelli A.
      • et al.
      Correlation of circulating miR-765, miR-93-5p, and miR-433-3p to obstructive coronary heart disease evaluated by cardiac computed tomography.
      ,
      • Tao L.
      • Bei Y.
      • Chen P.
      • Lei Z.
      • Fu S.
      • Zhang H.
      • et al.
      Crucial role of miR-433 in regulating cardiac fibrosis.
      ]. MiRNAs also regulate the myocardial angiogenic response to ischemia. For example, miR-24 is upregulated in ECs of ischemic mouse myocardium, and impairs neovascularization after experimental MI, in part by promoting EC apoptosis. These effects are probably mediated via direct inhibition of the transcription factors Gata2 and Pak4, which leads to decreased expression of Hmox1 (antioxidant enzyme) and activation of Bad (a pro-apoptotic protein), respectively [
      • Fiedler J.
      • Jazbutyte V.
      • Kirchmaier B.C.
      • Gupta S.K.
      • Lorenzen J.
      • Hartmann D.
      • et al.
      MicroRNA-24 regulates vascularity after myocardial infarction.
      ]. MiR-134 also inhibits myocardial angiogenesis after experimental MI in mice, likely by repressing Kdm2a, leading to reduced expression of vascular endothelial growth factor A [
      • Li X.
      • Wei C.
      • Zhang Z.
      • Jin Q.
      • Xiao X.
      MiR-134-5p regulates myocardial apoptosis and angiogenesis by directly targeting KDM2A after myocardial infarction.
      ]. Importantly, miR-134 also exerts an anti-angiogenic role in non-cardiac pathologies such as osteosarcoma [
      • Zhang L.
      • Lv Z.
      • Xu J.
      • Chen C.
      • Ge Q.
      • Li P.
      • et al.
      MicroRNA-134 inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway.
      ], raising the possibility that efforts to improve myocardial vascularization by targeting miR-134 could have harmful off-target effects.
      MiRNAs also contribute to the regulation of blood pressure and—by extension—to the pathogenesis of hypertension and hypertensive heart disease (Table 1). MiRNAs help regulate the renin-angiotensin-aldosterone system, which plays a central role in the pathogenesis of essential hypertension. Marques et al. identified miR-181a and miR-663 as repressors of renin and found decreased expression of both miRNAs in the renal cortex of hypertensive versus normotensive patients [
      • Marques F.Z.
      • Campain A.E.
      • Tomaszewski M.
      • Zukowska-Szczechowska E.
      • Yang Y.H.
      • Charchar F.J.
      • et al.
      Gene expression profiling reveals renin mRNA overexpression in human hypertensive kidneys and a role for microRNAs.
      ]. MiR-143 directly targets angiotensin converting enzyme 2, and miR-143 levels are lower in plasma of patients with essential hypertension [
      • Chen L.J.
      • Xu R.
      • Yu H.M.
      • Chang Q.
      • Zhong J.C.
      The ACE2/apelin signaling, MicroRNAs, and hypertension.
      ]. MiRNAs also regulate pathways that contribute to hypertension-induced cardiac hypertrophy. For example, miR-26 inhibits the transcription factor Gata4 (a well-established promoter of cardiomyocyte growth) and prevents cardiac hypertrophy in mice subjected to cardiac pressure overload [
      • Han M.
      • Yang Z.
      • Sayed D.
      • He M.
      • Gao S.
      • Lin L.
      • et al.
      GATA4 expression is primarily regulated via a miR-26b-dependent post-transcriptional mechanism during cardiac hypertrophy.
      ]. In turn, miR-217 promotes cardiac hypertrophy by targeting Pten, thereby activating the AKT signaling pathway [
      • Nie X.
      • Fan J.
      • Li H.
      • Yin Z.
      • Zhao Y.
      • Dai B.
      • et al.
      miR-217 promotes cardiac hypertrophy and dysfunction by targeting PTEN.
      ], and mir-214 enhances angiotensin II-induced cardiac hypertrophy in mice by targeting Sirt3, leading to mitochondrial dysfunction [
      • Ding Y.Q.
      • Zhang Y.H.
      • Lu J.
      • Li B.
      • Yu W.J.
      • Yue Z.B.
      • et al.
      MicroRNA-214 contributes to Ang II-induced cardiac hypertrophy by targeting SIRT3 to provoke mitochondrial malfunction.
      ].
      In summary, miRNAs are key regulators of cardiovascular pathophysiology, acting by downregulating components of critical pathways that either promote or prevent several CVDs, including atherosclerosis, MI and its sequelae, and hypertension and its sequelae. Accordingly, miRNAs are attractive targets for development of novel cardiovascular therapies. However, their roles in other pathologies such as cancer must be considered as miRNA inhibitory strategies are translated into human therapies.

      2. MiRNA-targeting therapeutics: structure, mechanism, chemical modifications, and toxicity

      2.1 Agomirs and antagomirs: structure and mechanism of action

      Therapies that target miRNAs aim either to increase the expression/abundance of miRNAs that prevent disease or to reduce the activity of miRNAs that cause disease. These therapies employ miRNA mimics or miRNA inhibitors, respectively. MiRNA mimics (or agomiRs) are synthetic double-stranded small RNA molecules that specifically match the sequence of endogenous mature miRNAs. MiRNA inhibitors are classified as either antisense oligonucleotides (ASOs) or miRNA sponges based on their molecular structure [
      • Diener C.
      • Keller A.
      • Meese E.
      Emerging concepts of miRNA therapeutics: from cells to clinic.
      ]. MiRNA-targeting ASOs or antagomirs are single-stranded small RNAs that bind complementarily to their miRNA targets, blocking interaction of these targets with their corresponding endogenous mRNA targets, thereby increasing (or “de-repressing”) expression of these mRNA targets. Similarly, miRNA sponges possess multiple miRNA binding sequences that can sequester multiple copies of either a single miRNA species or several different miRNA species. Accordingly, sponges are potentially a more potent approach for reducing levels of target miRNA. Although miRNA mimics (i.e., agomiRs) have shown potential in preclinical studies and are beginning to be tested clinically [
      • Hong D.S.
      • Kang Y.-K.
      • Borad M.
      • Sachdev J.
      • Ejadi S.
      • Lim H.Y.
      • et al.
      Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.
      ,
      • Gallant-Behm C.L.
      • Piper J.
      • Lynch J.M.
      • Seto A.G.
      • Hong S.J.
      • Mustoe T.A.
      • et al.
      A MicroRNA-29 mimic (remlarsen) represses extracellular matrix expression and fibroplasia in the skin.
      ], they have not yet been tested in clinical trials for treatment of CVDs. For this reason, and to limit the scope of this review, we will focus on miRNA inhibitors rather than agomiRs. Among miRNA inhibitors, we will discuss antagomirs rather than sponges because antagomirs are far more widely used both in preclinical studies and clinical trials. Moreover, because many advances in antagomir-based therapies were originally developed for mRNA-targeting ASOs, some of the studies cited herein involve mRNA-targeting ASOs.

      2.2 Chemical modification of ASO antagomirs

      Unmodified RNA molecules injected in vivo are quickly degraded by endogenous nucleases [
      • Pos O.
      • Biro O.
      • Szemes T.
      • Nagy B.
      Circulating cell-free nucleic acids: characteristics and applications.
      ]. Accordingly, unmodified ASO antagomirs administered via intravenous or subcutaneous injection have limited bioavailability and are unlikely to reach their miRNA targets. Moreover, ASOs have an overall negative charge that hampers cellular uptake, requiring injection of high doses to achieve biologically meaningful effects. To overcome these challenges, several chemical modifications are used to improve ASO stability, increase cellular uptake, and enhance affinity for target miRNAs. These modifications [
      • Hammond S.M.
      • Aartsma-Rus A.
      • Alves S.
      • Borgos S.E.
      • Buijsen R.A.M.
      • Collin R.W.J.
      • et al.
      Delivery of oligonucleotide-based therapeutics: challenges and opportunities.
      ] are applied to the backbone as well as the ribose and/or nucleobase of ASOs and include: (1) changes in the phosphodiester bonds such as incorporating a phosphorothioate internucleotide linkage to confer resistance to nuclease degradation and increase bioavailability; (2) modification of ribose to enhance ASO stability and affinity for target miRNA (e.g., 2′-O-methyl, 2′-O-methoxyethyl, and 2′-fluoro modifications); (3) construction of a locked nucleic acid [
      • Hong D.S.
      • Kang Y.-K.
      • Borad M.
      • Sachdev J.
      • Ejadi S.
      • Lim H.Y.
      • et al.
      Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.
      ] by placing a methyl bridge between the 2′-O and 4′ position of the ribose ring, thereby increasing the affinity of LNAs for their target miRNAs; and (4) substitution of the ribose-phosphate backbone with a pseudopeptide sequence [creating a peptide nucleic acid (PNA) molecule], or with a 6-membered morpholino ring and phosphorodiamidate linkages, generating a phosphorodiamidate morpholino oligomer (PMO). Both PNAs and PMOs are neutrally charged and are resistant to cleavage by nucleases [
      • Lima J.F.
      • Cerqueira L.
      • Figueiredo C.
      • Oliveira C.
      • Azevedo N.F.
      Anti-miRNA oligonucleotides: a comprehensive guide for design.
      ]. All the aforementioned chemical modifications of ASOs have been thoroughly described in previous reviews [
      • Hammond S.M.
      • Aartsma-Rus A.
      • Alves S.
      • Borgos S.E.
      • Buijsen R.A.M.
      • Collin R.W.J.
      • et al.
      Delivery of oligonucleotide-based therapeutics: challenges and opportunities.
      ,
      • Fu Y.
      • Chen J.
      • Huang Z.
      Recent progress in microRNA-based delivery systems for the treatment of human disease.
      ].

      2.3 Antagomir toxicity

      Despite the above advancements in ASO chemistry, toxicity remains a major barrier to clinical application. Because ASOs accumulate primarily in liver and kidney after intravenous or subcutaneous injection, they may have toxic effects in these organs, which may be exacerbated by high doses [
      • Geary R.S.
      • Norris D.
      • Yu R.
      • Bennett C.F.
      Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.
      ]. LNA-modified ASOs may induce hepatotoxicity, potentially by increasing hepatic expression of apoptotic proteins (e.g., caspase 3), although the underlying molecular mechanisms that drive hepatotoxicity remain unclear [
      • Swayze E.E.
      • Siwkowski A.M.
      • Wancewicz E.V.
      • Migawa M.T.
      • Wyrzykiewicz T.K.
      • Hung G.
      • et al.
      Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.
      ]. Moreover, circulating ASOs also alter platelet activity, causing thrombocytopenia in humans [
      • Crooke S.T.
      • Baker B.F.
      • Witztum J.L.
      • Kwoh T.J.
      • Pham N.C.
      • Salgado N.
      • et al.
      The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials.
      ]. In vitro studies suggest that thrombocytopenia is a consequence of platelet hyperactivation and aggregation that are stimulated via ASO activation of TLR-7, -8 and -9 [
      • Zaslavsky A.
      • Adams M.
      • Cao X.
      • Yamaguchi A.
      • Henderson J.
      • Busch-Ostergren P.
      • et al.
      Antisense oligonucleotides and nucleic acids generate hypersensitive platelets.
      ]. The toxicities of ASOs also include off-target effects caused by hybridization with RNA molecules other than their intended targets [
      • Bartoszewski R.
      • Sikorski A.F.
      Editorial focus: understanding off-target effects as the key to successful RNAi therapy.
      ]. For all of these reasons, extensive preclinical assessment of the toxicity of an ASO antagomir is required before it can be administered to humans.
      To diminish their toxicity while simultaneously enhancing their biological activity, ASOs can be chemically linked to biomolecules that target the ASOs to specific tissues or cell types. For example, ASOs can be targeted via conjugation to aptamers, single-stranded oligonucleotides (20–100 nt) that bind specific target proteins with high affinity. Accordingly, aptamers enhance delivery of ASO to cells that express a specific protein on their surfaces [
      • Tanaka K.
      • Okuda T.
      • Kasahara Y.
      • Obika S.
      Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide.
      ]. Similarly, conjugation of ASOs to N-acetylgalactosamine, a ligand of the liver-enriched asialoglycoprotein receptor (ASGR1), enhances ASO delivery to human hepatocytes [
      • Biessen E.A.L.
      • Van Berkel Tjc
      N-acetyl galactosamine targeting: paving the way for clinical application of nucleotide medicines in cardiovascular diseases.
      ,
      • Debacker A.J.
      • Voutila J.
      • Catley M.
      • Blakey D.
      • Habib N.
      Delivery of oligonucleotides to the liver with GalNAc: from research to registered therapeutic drug.
      ]. Lucas et al. used a different approach to achieve site-specific ASO activity: construction of a light-activatable ASO by attachment of photolabile protecting groups or “cages” to the ASO nucleobases (Fig. 1A) [
      • Lucas T.
      • Schafer F.
      • Muller P.
      • Eming S.A.
      • Heckel A.
      • Dimmeler S.
      Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice.
      ]. These cages prevent duplex formation with the target miRNA but can be removed by irradiation with 325 nm light. Intradermal injection of a caged anti-miR-92a antagomir in diabetic mice significantly reduced miR-92a levels in irradiated skin and improved wound healing similarly to conventional anti-miR-92a. However, mice injected with the light-activated anti-miR-92a had unchanged miR-92a expression in liver and kidney whereas mice injected with conventional anti-miR-92a had significantly lower expression of kidney miR-92a. The authors speculated that this approach could be used—likely with the aid of devices such as catheters or endoscopes—to achieve site-specific activity of antagomirs in internal organs while avoiding toxicity caused by systemic distribution of antagomirs.
      Fig. 1
      Fig. 1Schematic representation of targeted antagomir delivery systems and administration routes.
      (Upper panel): Antagomirs (pink) are administered as non-encapsulated ASOs (A), are expressed from viral vectors (B), or are delivered by non-viral nanoparticle (NP) carriers (C). Some NP carriers can be loaded with plasmids (not shown) that express the antagomirs after cell entry. (A) Non-encapsulated antagomirs can be linked to functional groups such as ligands (e.g., N-acetylgalactosamine) that bind specific cell surface receptors or to cholesterol or cell-permeable peptides that enhance cell entry. Alternatively, photolabile “cages” can be linked to the antagomir nucleobases, allowing site-specific activation of the antagomir by illumination. (B) Antagomirs can also be expressed from viral vectors, typically adenoviruses (Ad), adeno-associated viruses (AAV), and lentiviruses (LV). In these cases, an expression cassette containing the antagomir sequence (pink) is inserted into the viral genome, potentially allowing long-term antagomir expression from within transduced cells. (C) Synthetic antagomirs (pink) or antagomir-encoding plasmids (not shown) can be incorporated within lipid-based NPs (e.g., extracellular vesicles (EVs) or liposomes), or polymer-based NPs. Lower panel: Antagomirs are commonly administered via intravenous injection (D), subcutaneously (not shown), or by injection into the adventitia (E). Antagomirs can also be injected directly into the myocardium or applied to the epicardium using hydrogel scaffolds (F). Intracoronary administration (not shown) is also used to deliver antagomirs to the myocardium. This figure was partly generated using Servier Medical Art. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
      Modifications to ASOs that increase cellular uptake and bioavailability could also reduce toxicity by reducing their circulating half-life and systemic distribution and by allowing administration of lower doses. Conjugation of ASOs to cholesterol improves their cellular uptake and endosomal escape, leading to increased intracellular availability in several cell types including cardiomyocytes [
      • Wang S.
      • Allen N.
      • Prakash T.P.
      • Liang X.H.
      • Crooke S.T.
      Lipid conjugates enhance endosomal release of antisense oligonucleotides into cells.
      ]. Intraperitoneal administration of cholesterol-modified anti-miR-199b ASO effectively reduced miR-199b levels in mouse hearts, outperforming unconjugated ASOs [
      • Duygu B.
      • Juni R.
      • Ottaviani L.
      • Bitsch N.
      • Wit J.B.M.
      • de Windt L.J.
      • et al.
      Comparison of different chemically modified inhibitors of miR-199b in vivo.
      ]. Betts et al. attached an arginine-rich cell-penetrating peptide to uncharged ASOs, enhancing their delivery to murine hearts after intravenous injection [
      • Betts C.
      • Saleh A.F.
      • Arzumanov A.A.
      • Hammond S.M.
      • Godfrey C.
      • Coursindel T.
      • et al.
      Pip6-PMO, A new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment.
      ]. These are promising results; however, both cholesterol conjugation and attachment of a cell-penetrating peptide would likely enhance antagomir entry into both targeted and non-targeted cells, potentially increasing off-target effects and toxicity.
      The above-described advancements in the chemistry and targeting of ASOs have, in some cases, allowed ASOs to advance to clinical application [
      • Abplanalp W.T.
      • Fischer A.
      • John D.
      • Zeiher A.M.
      • Gosgnach W.
      • Darville H.
      • et al.
      Efficiency and target derepression of anti-miR-92a: results of a first in human study.
      ,
      • Taubel J.
      • Hauke W.
      • Rump S.
      • Viereck J.
      • Batkai S.
      • Poetzsch J.
      • et al.
      Novel antisense therapy targeting microRNA-132 in patients with heart failure: results of a first-in-human Phase 1b randomized, double-blind, placebo-controlled study.
      ]. However, clinical use of ASOs to treat CVDs remains limited, and use of miRNA-targeting ASOs (i.e., antagomirs) continues to be challenged by toxic effects and limited efficacy [
      • Bonneau E.
      • Neveu B.
      • Kostantin E.
      • Tsongalis G.J.
      • De Guire V.
      How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market.
      ].

      3. ASO delivery systems

      3.1 ASO delivery to cardiac or vascular cells

      Intravenous or subcutaneous administration of non-encapsulated ASOs/antagomirs remains the most common delivery method in preclinical experimental models and clinical studies. The most clinically successful ASO delivery approach to date uses subcutaneous injection of ASOs that are targeted to the liver by conjugation to N-acetylgalactosamine. These ASOs are taken up by the hepatocyte-expressed asialoglycoprotein receptor ASGR1, as described above. Targeted ASO/antagomir delivery to cardiac or vascular cells has proved more challenging because neither cardiac nor vascular cells express cell-type-specific receptors that internalize ligands similarly to ASGR1. Lack of cardiovascular equivalents to ASGR1 has prompted development of alternative delivery systems in which ASOs/antagomirs are either expressed by a viral vector that has tropism for specific cardiac or vascular cell types or are encapsulated in non-viral nanoparticles (NPs) that are constructed with a goal of targeting their ASO/antagomir cargo to one of these cell types [
      • Lu D.
      • Thum T.
      RNA-based diagnostic and therapeutic strategies for cardiovascular disease.
      ]. The viral vectors contain and express nucleotide sequences encoding the ASOs/antagomirs, whereas the NPs can carry either the ASOs/antagomirs themselves (as synthetic oligonucleotides) or can deliver plasmid constructs encoding ASOs/antagomirs, allowing longer-term expression as well as cell type-specific expression by using promoters that are exclusively expressed in a desired cell type. Use of either viral vectors or NPs to deliver ASO/antagomirs prevents their destruction by endogenous nucleases, facilitates cellular uptake, and reduces endosomal/lysosomal degradation. Therefore, both viral vectors and NPs can increase cell-type-specific delivery to cardiac and vascular cells while enhancing intracellular bioavailability. Specific viral vectors and NPs have both advantages and limitations that must be considered when designing a strategy for delivering ASOs to cardiac and vascular cells (Table 2). In addition, both viral vectors and NPs can be delivered via multiple routes including parenteral injection with systemic distribution, local injection in proximity to the target cells, and device-assisted local delivery. In this section, we discuss viral and non-viral delivery systems as well as strategies for delivery of ASOs/antagomirs to cardiac and vascular cells (Fig. 1).
      Table 2Advantages and disadvantages of ASO delivery systems.
      Delivery systemAdvantagesLimitations
      ViralAdenovirus (Ad)
      • High packaging capacity (∼37 kb)
      • Strong inflammatory response (lower in third-generation Ads)
      • High transduction rate of cardiac and vascular cells
      • Presence of neutralizing antibodies
      • Capsid modifications may enhance tropism towards cardiac and vascular cells
      • Inefficient targeting of cardiac and vascular cells
      • Non-integrative
      Adeno-associated virus (AAV)
      • Native tropism towards the cardiomyocytes (AAV1, 6, 8 and 9) and vascular wall cells (AAV1 and 5)
      • Inflammatory response
      • Capsid modifications may enhance tropism towards cardiovascular cells
      • Presence of neutralizing antibodies
      • Sufficient packaging capacity for antagomirs (∼4.7 kb)
      • Inefficient targeting of vascular cells
      • Mostly non-integrative
      Lentivirus (LV)
      • High packaging capacity (∼10 kb)
      • Potential insertional oncogenesis (for integrating LV vectors)
      • Long-term transgene expression
      • Limited in vivo cardiovascular cell transduction, although pseudotyping may enhance tropism towards cardiovascular cells
      • Low immunogenicity
      • Low prevalence of neutralizing antibodies
      Non-viralLiposomes
      • High loading capacity
      • Toxicity of cationic liposomes
      • Low immunogenicity
      • Transient transgene expression
      • Allow functionalization
      EVs
      • Highly biocompatible
      • Contain complex molecular cargo in addition to antagomirs
      • Efficient cellular uptake
      • Limited batch-to-batch reproducibility
      • Readily scalable production
      • Allow functionalization
      Polymer NPs
      • High loading capacity (especially dendrimers)
      • Toxicity of cationic polymers.
      • Biocompatibility (e.g., PLGA or chitosan)
      • Limited knowledge of the PK/PD properties
      • Allow functionalization
      NP: nanoparticle; EV: extracellular vesicle; PLGA: poly(lactic-co-glycolic acid); PK/PD: pharmacokinetic/pharmacodynamic.

      3.2 Viral vectors

      Adenoviruses (Ads) and adeno-associated viruses (AAVs) are the most commonly used viral vectors for delivering genes to cardiovascular cells (Fig. 1B). Both vectors can transduce quiescent cells such as ECs, VSMCs, or cardiomyocytes [
      • Kieserman J.M.
      • Myers V.D.
      • Dubey P.
      • Cheung J.Y.
      • Feldman A.M.
      Current landscape of heart failure gene therapy.
      ]. Ads are non-integrating double-stranded DNA vectors with high vascular-cell and cardiomyocyte transduction efficiency. First-generation Ads contain most of the native viral genome, have limited capacity for transgene insertion (∼7 kb), achieve only short-term transgene expression (1–2 weeks), and induce a strong inflammatory response in the vascular wall [
      • Newman K.D.
      • Dunn P.F.
      • Owens J.W.
      • Schulick A.H.
      • Virmani R.
      • Sukhova G.
      • et al.
      Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.
      ]. In contrast, 3rd generation or helper-dependent Ads lack all viral protein-coding sequences, have a large capacity for transgene insertion (up to ∼35 kb) [
      • Rincon M.Y.
      • VandenDriessche T.
      • Chuah M.K.
      Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.
      ], and achieve long-term (at least 48 weeks) transgene expression in preclinical models of vascular wall gene transfer, with a minimal local inflammatory response [
      • Wen S.
      • Graf S.
      • Massey P.G.
      • Dichek D.A.
      Improved vascular gene transfer with a helper-dependent adenoviral vector.
      ]. Serotype 5 Ads are typically used to deliver genes to cardiac and vascular cells. Importantly, effective transduction by serotype 5 relies on the presence of the Coxsackievirus and adenovirus receptor (CAR) on the surface of targeted cells. CAR expression is high in cardiomyocytes, but is also present at adequate levels in ECs and VSMCs [
      • Quax P.H.A.
      • Lamfers M.L.M.
      • Grimbergen J.M.
      • Teeling J.
      • Hoeben R.C.
      • van Nieuw Amerongen G.P.
      • et al.
      Effects of infection with recombinant adenovirus on human vascular endothelial and smooth muscle cells.
      ]. Other Ad serotypes, such as type 35, show higher tropism towards VSMCs, through interaction with the CD46 receptor, and may therefore be more suitable for transducing the arterial intima and media [
      • Parker A.L.
      • White K.M.
      • Lavery C.A.
      • Custers J.
      • Waddington S.N.
      • Baker A.H.
      Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.
      ]. More-precise targeting of Ads to ECs and VSMCs (and more-efficient transduction) may be achieved by modification of capsid proteins to target molecules that are abundant on the surfaces of these cell types, including heparan sulfate and angiotensin-converting enzyme [
      • Kibbe M.R.
      • Murdock A.
      • Wickham T.
      • Lizonova A.
      • Kovesdi I.
      • Nie S.
      • et al.
      Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.
      ,
      • Morecroft I.
      • White K.
      • Caruso P.
      • Nilsen M.
      • Loughlin L.
      • Alba R.
      • et al.
      Gene therapy by targeted adenovirus-mediated knockdown of pulmonary endothelial Tph1 attenuates hypoxia-induced pulmonary hypertension.
      ]. As an example of successful Ad-mediated antagomir delivery into cardiomyocytes, intramyocardial injection of Ads encoding anti-miR-327 and anti-miR-98 effectively depressed expression of their corresponding target miRNAs in vivo [
      • Yang Y.
      • Yang J.
      • Liu X.W.
      • Ding J.W.
      • Li S.
      • Guo X.
      • et al.
      Down-regulation of miR-327 alleviates ischemia/reperfusion-induced myocardial damage by targeting RP105.
      ,
      • Yang Y.
      • Ago T.
      • Zhai P.
      • Abdellatif M.
      • Sadoshima J.
      Thioredoxin 1 negatively regulates angiotensin II-induced cardiac hypertrophy through upregulation of miR-98/let-7.
      ].
      AAVs are single-stranded DNA vectors that can achieve long-term transgene expression. AAVs have lower immunogenicity than Ads, although their capacity for transgene insertion is limited to ∼4.7 kb [
      • Rincon M.Y.
      • VandenDriessche T.
      • Chuah M.K.
      Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.
      ]. However, for expression of an antagomir, 4.7 kb of packaging capacity is more than sufficient. Several AAV serotypes display high cardiac tropism, especially AAV1, AAV6, AAV8 and AAV9. Cardiomyocyte transduction by these AAVs is facilitated by binding of their capsids to proteins on the cardiomyocyte surface including the laminin receptor or N-linked sialic acid [
      • Ambrosi C.M.
      • Sadananda G.
      • Han J.L.
      • Entcheva E.
      Adeno-associated virus mediated gene delivery: implications for scalable in vitro and in vivo cardiac optogenetic models.
      ]. The ability of wild-type AAVs to transduce vascular cells appears relatively low in preclinical models, although some serotypes such as AAV1 and AAV5 are reported to transduce rabbit vascular media and adventitia in vivo [
      • Sen S.
      • Conroy S.
      • Hynes S.O.
      • McMahon J.
      • O'Doherty A.
      • Bartlett J.S.
      • et al.
      Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.
      ]. Modifications of the AAV2 and AAV9 capsids have improved their tropism towards ECs [
      • Bozoglu T.
      • Lee S.
      • Ziegler T.
      • Jurisch V.
      • Maas S.
      • Baehr A.
      • et al.
      Endothelial retargeting of AAV9 in vivo.
      ,
      • Liu Y.B.
      • Xu B.C.
      • Chen Y.T.
      • Yuan X.
      • Liu J.Y.
      • Liu T.
      • et al.
      Directed evolution of AAV accounting for long-term and enhanced transduction of cardiovascular endothelial cells in vivo.
      ,
      • Krolak T.
      • Chan K.Y.
      • Kaplan L.
      • Huang Q.
      • Wu J.
      • Zheng Q.
      • et al.
      A high-efficiency AAV for endothelial cell transduction throughout the central nervous system.
      ]. For example, Krolak et al. modified the AAV9 capsid, enhancing its capacity to transduce cultured human microvascular ECs by over 70-fold [
      • Krolak T.
      • Chan K.Y.
      • Kaplan L.
      • Huang Q.
      • Wu J.
      • Zheng Q.
      • et al.
      A high-efficiency AAV for endothelial cell transduction throughout the central nervous system.
      ]. Moreover, intravenous injection of the modified AAV9 yielded high in vivo transduction of arteries, veins and capillaries in murine brain and of aortic endothelium [
      • Krolak T.
      • Chan K.Y.
      • Kaplan L.
      • Huang Q.
      • Wu J.
      • Zheng Q.
      • et al.
      A high-efficiency AAV for endothelial cell transduction throughout the central nervous system.
      ]. These modified AAVs have great potential for improving the delivery of antagomirs to cardiac and vascular cells. Examples of the use of AAVs to deliver ASOs to cardiomyocytes include repression of miR-20b in murine myocardium after intravenous injection of an AAV encoding anti-miR-20b [
      • Qiu Y.
      • Cheng R.
      • Liang C.
      • Yao Y.
      • Zhang W.
      • Zhang J.
      • et al.
      MicroRNA-20b promotes cardiac hypertrophy by the inhibition of mitofusin 2-mediated inter-organelle Ca(2+) cross-talk.
      ] and injection of an AAV9 encoding anti-let-7i-5p into murine myocardium, which de-repressed cardiac expression of the let-7i-5p target genes E2f2 and Ccnd2 [
      • Hu Y.
      • Jin G.
      • Li B.
      • Chen Y.
      • Zhong L.
      • Chen G.
      • et al.
      Suppression of miRNA let-7i-5p promotes cardiomyocyte proliferation and repairs heart function post injury by targetting CCND2 and E2F2.
      ].
      Lentiviral vectors (LVs) are enveloped single-stranded RNA viruses with high packaging capacity (∼10 kb) that achieve long-term transgene expression in both quiescent and proliferating cells, in part due to integration of the LV genome into the host cell genome [
      • Rincon M.Y.
      • VandenDriessche T.
      • Chuah M.K.
      Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.
      ]. However, genomic integration of LVs can potentially lead to insertional oncogenesis [
      • Luis A.
      The old and the new: prospects for non-integrating lentiviral vector technology.
      ]. This limitation of LVs has been addressed by generation of non-integrating LVs. Non-integrating LVs achieve long-term transgene expression in non-dividing cells and—to a lesser extent—in dividing cells [
      • Luis A.
      The old and the new: prospects for non-integrating lentiviral vector technology.
      ,
      • Shaw A.
      • Cornetta K.
      Design and potential of non-integrating lentiviral vectors.
      ]. Moreover, LVs have relatively low immunogenicity, low prevalence of pre-existing neutralizing antibodies, and can be easily pseudotyped (i.e., their envelopes can be modified to include surface proteins from other viruses such as the vesicular stomatitis virus surface glycoprotein), altering their tropism and reducing recognition by the immune system [
      • Shaw A.
      • Cornetta K.
      Design and potential of non-integrating lentiviral vectors.
      ]. LVs can deliver antagomirs to ECs and VSMCs in vitro [
      • Zhuang Z.
      • Xiao q
      • Hu H.
      • Tian S.Y.
      • Lu Z.J.
      • Zhang T.Z.
      • et al.
      Down-regulation of microRNA-155 attenuates retinal neovascularization via the PI3K/Akt pathway.
      ,
      • Sun L.
      • Zhao M.
      • Zhang J.
      • Lv M.
      • Li Y.
      • Yang X.
      • et al.
      MiR-29b downregulation induces phenotypic modulation of vascular smooth muscle cells: implication for intracranial aneurysm formation and progression to rupture.
      ]. However, in vivo transduction efficiency of cardiac and vascular cells by LVs appears to be limited [
      • Bi L.
      • Wacker B.K.
      • Stamatikos A.
      • Sethuraman M.
      • Komandur K.
      • Dichek D.A.
      Jugular vein injection of high-titer lentiviral vectors does not transduce the aorta-brief report.
      ,
      • Merentie M.
      • Lottonen-Raikaslehto L.
      • Parviainen V.
      • Huusko J.
      • Pikkarainen S.
      • Mendel M.
      • et al.
      Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.
      ]. Intravenously injected LVs primarily transduce liver, spleen, and bone marrow [
      • Follenzi A.
      • Sabatino G.
      • Lombardo A.
      • Boccaccio C.
      • Naldini L.
      Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors.
      ]. Compared to Ads and the cardiotropic AAVs (e.g., AAV2 and AAV9), LVs inefficiently transduced murine cardiomyocytes after intramyocardial injection [
      • Merentie M.
      • Lottonen-Raikaslehto L.
      • Parviainen V.
      • Huusko J.
      • Pikkarainen S.
      • Mendel M.
      • et al.
      Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.
      ]. Whether intravenously injected LVs can transduce cells in the aorta is controversial [
      • Bi L.
      • Wacker B.K.
      • Stamatikos A.
      • Sethuraman M.
      • Komandur K.
      • Dichek D.A.
      Jugular vein injection of high-titer lentiviral vectors does not transduce the aorta-brief report.
      ,
      • Esteban V.
      • Mendez-Barbero N.
      • Jimenez-Borreguero L.J.
      • Roque M.
      • Novensa L.
      • Garcia-Redondo A.B.
      • et al.
      Regulator of calcineurin 1 mediates pathological vascular wall remodeling.
      ]. Redondo et al. reported highly efficient LV-mediated transgene expression throughout the murine aortic wall after intravenous LV injection [
      • Esteban V.
      • Mendez-Barbero N.
      • Jimenez-Borreguero L.J.
      • Roque M.
      • Novensa L.
      • Garcia-Redondo A.B.
      • et al.
      Regulator of calcineurin 1 mediates pathological vascular wall remodeling.
      ]; however, presence of the LV genome in the aorta was never documented. In contrast, we found that LV genomes and LV-mediated transgene expression were both undetectable in murine aortas 4 days after intravenous injection of high-titer LVs—including LVs supplied by Dr. Redondo—casting doubt on whether any cells in the aorta are transduced by intravenously injected LVs [
      • Bi L.
      • Wacker B.K.
      • Stamatikos A.
      • Sethuraman M.
      • Komandur K.
      • Dichek D.A.
      Jugular vein injection of high-titer lentiviral vectors does not transduce the aorta-brief report.
      ]. Pseudotyping is a potential strategy for increasing the in vivo transduction efficiency of LVs. For example, local perfusion of a LV pseudotyped with the bunyaviral Hantaan virus glycoprotein efficiently transduced rabbit carotids after balloon injury [
      • Gilbert J.
      • Qian Z.
      • Arsenault J.
      • Aquirre J.
      • Lemos J.
      • Park F.
      936. Vascular tropism of lentiviral vectors using bunyaviral-pseudotyped lentiviral vectors.
      ].
      In summary, viral vectors can be used to express therapeutic antagomirs within specific cardiac and vascular cells. Use of antagomir-expressing viral vectors eliminates the need for resource-intensive synthesis and purification of clinical-grade antagomirs. Moreover, a single viral vector injection could achieve long-term in vivo expression of an antagomir, leading to durable suppression of its miRNA target. A disadvantage of viral vector delivery systems is that it is challenging to turn them “off” if antagomir expression either becomes unnecessary or is toxic. Moreover, clinical application of viral vector-mediated gene therapy is still hampered by intense systemic innate and adaptive immune responses to viral vector proteins and by the existence of high levels of neutralizing antibodies to Ad and AAV vectors in many humans. These challenges highlight the need for development of non-viral vectors for delivering antagomirs to cardiac and vascular cells.

      3.3 Liposomes

      Liposomes are synthetic lipid bilayer particles that can encapsulate a variety of molecular cargoes, including antagomirs (Fig. 1C) [
      • Scheideler M.
      • Vidakovic I.
      • Prassl R.
      Lipid nanocarriers for microRNA delivery.
      ]. Because of their high biocompatibility, loading capacity, and relative ease of preparation, liposomes are the most widely used non-viral ASO delivery system. Liposomes are typically synthesized from cationic lipids that entrap ASOs by electrostatic force. Liposomes are often coated with polyethylene glycol (PEG), which confers resistance to degradation by the mononuclear phagocytic system and enhances circulation lifetime [
      • Scheideler M.
      • Vidakovic I.
      • Prassl R.
      Lipid nanocarriers for microRNA delivery.
      ]. The positive charge of liposomal lipids facilitates binding to the negatively charged cellular plasma membrane. Liposomes can also be targeted to specific cell types by covalent conjugation to antibodies or to peptides that bind specific cell-surface antigens. For example, intravenously injected PEGylated liposomes covalently attached to an angiotensin II type 1 receptor binding peptide specifically targeted infarcted myocardium in mice, whereas healthy hearts were not targeted by these liposomes [
      • Dvir T.
      • Bauer M.
      • Schroeder A.
      • Tsui J.H.
      • Anderson D.G.
      • Langer R.
      • et al.
      Nanoparticles targeting the infarcted heart.
      ]. Ko et al. extended this method by conjugating liposomes to both an anti-cardiac myosin antibody and a cell-penetrating peptide. These bivalent liposomes had enhanced uptake in rat ischemic cardiomyocytes after intravenous injection [
      • Ko Y.T.
      • Hartner W.C.
      • Kale A.
      • Torchilin V.P.
      Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.
      ]. Conjugation of liposomes to antibodies or ligands can also enhance liposome delivery to vascular wall cells including macrophages and ECs [
      • Kiaie N.
      • Gorabi A.M.
      • Penson P.E.
      • Watts G.
      • Johnston T.P.
      • Banach M.
      • et al.
      A new approach to the diagnosis and treatment of atherosclerosis: the era of the liposome.
      ,
      • Dhanasekara C.S.
      • Zhang J.
      • Nie S.
      • Li G.
      • Fan Z.
      • Wang S.
      Nanoparticles target intimal macrophages in atherosclerotic lesions.
      ,
      • Bruch G.E.
      • Fernandes L.F.
      • Bassi B.L.T.
      • Alves M.T.R.
      • Pereira I.O.
      • Frezard F.
      • et al.
      Liposomes for drug delivery in stroke.
      ]. For example, incorporation of an oxidized phospholipid (a ligand for the CD36 receptor) into liposome-like nanoparticles increased their accumulation in atherosclerotic mouse aortas, presumably by facilitating uptake by intimal macrophages [
      • Dhanasekara C.S.
      • Zhang J.
      • Nie S.
      • Li G.
      • Fan Z.
      • Wang S.
      Nanoparticles target intimal macrophages in atherosclerotic lesions.
      ]. Liposomes conjugated with ligands of the transferrin receptor (enriched in brain endothelium) cross the blood brain barrier via transferrin receptor-mediated endocytosis, facilitating ASO delivery to the brain [
      • Zhao H.
      • Bao X.J.
      • Wang R.Z.
      • Li G.L.
      • Gao J.
      • Ma S.H.
      • et al.
      Postacute ischemia vascular endothelial growth factor transfer by transferrin-targeted liposomes attenuates ischemic brain injury after experimental stroke in rats.
      ].
      Despite their overall biocompatibility, cationic liposomes can have both cytotoxic and immunogenic effects. These effects are caused by inclusion of synthetic components in liposomes and by their positive charge. These factors drive liposome accumulation in the liver and spleen, promoting complement activation and rapid clearance by the phagocytic system [
      • Pecot C.V.
      • Calin G.A.
      • Coleman R.L.
      • Lopez-Berestein G.
      • Sood A.K.
      RNA interference in the clinic: challenges and future directions.
      ]. These issues can be mitigated by use of liposomes composed of neutral or ionizable lipids (i.e., lipids that are positively charged at acidic pH—facilitating the incorporation of ASOs into liposomes—but are neutrally charged at physiological pH). For example, Fisher et al. generated neutral lipid NPs conjugated with a VSMC-targeting peptide. These NPs had less in vitro cytotoxicity compared to cationic liposomes [
      • Fisher R.K.
      • Mattern-Schain S.I.
      • Best M.D.
      • Kirkpatrick S.S.
      • Freeman M.B.
      • Grandas O.H.
      • et al.
      Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications.
      ].

      3.4 Extracellular vesicles

      Extracellular vesicles (EVs) are released from all cell types and resemble liposomes in that they consist of a lipid bilayer surrounding a molecular cargo (Fig. 1C). EVs are derived from either the plasma membrane or from intracellular multivesicular bodies; the latter are termed exosomes [
      • Witwer K.W.
      • Buzas E.I.
      • Bemis L.T.
      • Bora A.
      • Lasser C.
      • Lotvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ]. Endogenous EVs contribute to intercellular communication and because they are cell-derived products EVs are highly biocompatible. As with liposomes, EVs can be used in vivo as vehicles for delivering ASOs to target cells. EVs can be easily isolated from multiple body fluids (e.g., blood, urine or cerebrospinal fluid) using methods such as ultracentrifugation and/or size-exclusion chromatography [
      • Witwer K.W.
      • Buzas E.I.
      • Bemis L.T.
      • Bora A.
      • Lasser C.
      • Lotvall J.
      • et al.
      Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
      ]. Alternatively, EVs can be isolated from medium conditioned by cultured cells. This is an attractive approach for obtaining large amounts of high purity EVs because it avoids contamination of EV preparations by apolipoproteins or Tamm–Horsfall protein which are commonly co-isolated with EVs from blood and urine, respectively [
      • Saenz-Pipaon G.
      • Echeverria S.
      • Orbe J.
      • Roncal C.
      Urinary extracellular vesicles for diabetic kidney disease diagnosis.
      ].
      Incorporation of antagomirs into EVs can be achieved by i) direct transfection or ii) transfection of cultured cells with an antagomir-encoding plasmid followed by collection of cell-conditioned medium and purification of EVs. The yield of ASO-containing EVs can be increased by inclusion in the ASO sequence of a tetranucleotide (termed the X-motif) that favors incorporation of oligonucleotides into EVs [
      • Peterson M.F.
      • Otoc N.
      • Sethi J.K.
      • Gupta A.
      • Antes T.J.
      Integrated systems for exosome investigation.
      ,
      • Cai J.
      • Gehrau R.
      • Tu Z.
      • Leroy V.
      • Su G.
      • Shang J.
      • et al.
      MicroRNA-206 antagomiRenriched extracellular vesicles attenuate lung ischemiareperfusion injury through CXCL1 regulation in alveolar epithelial cells.
      ]. As with liposomes, EVs can be modified to target specific cell types via incorporation of surface-exposed ligands [
      • Fu Y.
      • Chen J.
      • Huang Z.
      Recent progress in microRNA-based delivery systems for the treatment of human disease.
      ]. For example, Kim et al. generated small EVs that were coated with a cardiac-targeting peptide and showed that incorporation of the peptide enhanced delivery of intravenously injected EVs to murine hearts [
      • Kim H.
      • Yun N.
      • Mun D.
      • Kang J.Y.
      • Lee S.H.
      • Park H.
      • et al.
      Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes.
      ]. Although EVs are promising vehicles for delivery of ASOs in vivo, their use as biopharmaceuticals is limited by the difficulty of generating biochemically similar batches of EVs [
      • Herrmann I.K.
      • Wood M.J.A.
      • Fuhrmann G.
      Extracellular vesicles as a next-generation drug delivery platform.
      ]. Heterogeneity of EV preparations complicates their use in preclinical studies and is a major regulatory barrier to their use in humans [
      • Herrmann I.K.
      • Wood M.J.A.
      • Fuhrmann G.
      Extracellular vesicles as a next-generation drug delivery platform.
      ].

      3.5 Polymer nanoparticles

      Polymer nanoparticles (pNPs) differ from liposomes and EVs by not encapsulating ASOs in lipid bilayers, but instead carrying ASOs as cargo attached to a polymeric scaffold (Fig. 1C). PNPs composed of poly(lactic-co-glycolic acid) (PLGA) (a polymer approved for human use by both the FDA and EMA) have emerged as attractive alternatives to liposomes and EVs. PNPs have advantages of high biocompatibility, biodegradability, and ease of chemical modification [
      • Dasgupta I.
      • Chatterjee A.
      Recent advances in miRNA delivery systems.
      ]. Chitosan, a natural cationic polysaccharide, is also used as a polymer scaffold for delivery of antagomirs [
      • Jiang F.
      • Yin F.
      • Lin Y.
      • Xia W.
      • Zhou L.
      • Pan C.
      • et al.
      The promotion of bone regeneration through CS/GP-CTH/antagomir-133a/b sustained release system.
      ]. PNPs are often constructed as dendrimers, hyperbranched structures with high ASO-loading capacity along with the potential to incorporate ligands that target pNP delivery to specific cell types such as cardiomyocytes [
      • Xue X.
      • Shi X.
      • Dong H.
      • You S.
      • Cao H.
      • Wang K.
      • et al.
      Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: an early targeting therapy for myocardial infarction in mice.
      ]. However, use of pNPs to deliver ASOs in vivo is limited by the complex procedures that are required for their synthesis and large-scale production and by limited knowledge of their pharmacokinetic and pharmacodynamic properties [
      • Gagliardi A.
      • Giuliano E.
      • Venkateswararao E.
      • Fresta M.
      • Bulotta S.
      • Awasthi V.
      • et al.
      Biodegradable polymeric nanoparticles for drug delivery to solid tumors.
      ].

      3.6 Route of administration

      The efficacy of ASO therapies—whether delivered by viral or nonviral vectors—can be enhanced by selection of an optimal route of administration. As mentioned above, parenteral injection—either intravenous or subcutaneous—is the most common route for administering ASOs [
      • Geary R.S.
      • Norris D.
      • Yu R.
      • Bennett C.F.
      Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.
      ], with similar in vivo biodistribution with both routes (Fig. 1D) [
      • Montgomery R.L.
      • Hullinger T.G.
      • Semus H.M.
      • Dickinson B.A.
      • Seto A.G.
      • Lynch J.M.
      • et al.
      Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure.
      ]. However, catheters can be used to improve delivery of antagomirs to the heart [
      • Hinkel R.
      • Penzkofer D.
      • Zuhlke S.
      • Fischer A.
      • Husada W.
      • Xu Q.F.
      • et al.
      Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.
      ]. For example, Hinkel et al. used both antegrade and retrograde catheter-based intracoronary approaches to deliver anti-miR-92a to cardiomyocytes. Both approaches increased delivery to ischemic myocardium compared to intravenous injection [
      • Hinkel R.
      • Penzkofer D.
      • Zuhlke S.
      • Fischer A.
      • Husada W.
      • Xu Q.F.
      • et al.
      Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.
      ]. ASOs may also be directly injected to the myocardium or embedded in hydrogel scaffolds (Fig. 1F) that are attached to the injured myocardium [
      • Fujita M.
      • Otani H.
      • Iwasaki M.
      • Yoshioka K.
      • Shimazu T.
      • Shiojima I.
      • et al.
      Antagomir-92a impregnated gelatin hydrogel microsphere sheet enhances cardiac regeneration after myocardial infarction in rats.
      ,
      • Kang J.Y.
      • Park H.
      • Kim H.
      • Mun D.
      • Park H.
      • Yun N.
      • et al.
      Human peripheral bloodderived exosomes for microRNA delivery.
      ]. To improve ASO delivery to cells of the vessel wall, ASOs (either free or encapsulated, as well as ASO-expressing viral vectors) could be injected into the arterial adventitia (Fig. 1E). Adventitial injection has been used to deliver viral vectors or non-encapsulated ASOs to the artery wall [
      • Simons M.
      • Edelman E.R.
      • DeKeyser J.L.
      • Langer R.
      • Rosenberg R.D.
      Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo.
      ,
      • Schneider D.B.
      • Sassani A.B.
      • Vassalli G.
      • Driscoll R.M.
      • Dichek D.A.
      Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors.
      ]. Theoretically, transduction of vascular cells or cardiomyocytes with gene-transfer vectors expressing exosome-targeted antagomirs could exploit the natural process of exosome-mediated cell-cell communication and deliver antagomirs to cells throughout the vessel wall or the heart [
      • Stamatikos A.
      • Knight E.
      • Vojtech L.
      • Bi L.
      • Wacker B.K.
      • Tang C.
      • et al.
      Exosome-mediated transfer of anti-miR-33a-5p from transduced endothelial cells enhances macrophage and vascular smooth muscle cell cholesterol efflux.
      ].
      In summary, numerous non-viral and viral vector-mediated approaches for in vivo antagomir delivery have shown promise in preclinical models. Delivery of antagomirs to hepatocytes using the non-viral ASGR1-targeted system seems clinically viable; however, no delivery systems yet achieve comparable results with cardiomyocytes or vascular cells. Ideal delivery systems would incorporate ligands or cis-acting regulators of transcription that ensure efficient and cell-type-specific delivery and expression. If a viral vector is used, it should have low immunogenicity, a low prevalence of pre-existing immunity, and—to avoid genotoxicity—it should be non-integrating. Both viral and non-viral vectors must be biocompatible with substantial bioavailability. An optimal delivery platform would be modifiable to permit both transient and durable antagomir expression/persistence, adjusted according to therapeutic needs. Moreover, the potential for subcutaneous versus intravenous administration expedites clinical implementation, with oral administration a distant goal. Finally, to address regulatory and financial concerns, an antagomir-delivery platform must be amenable to production methods that are standardized, scalable, and economically viable.

      4. Use of miRNA-targeted ASOs for treatment of cardiovascular diseases

      Here we discuss several illustrative examples of recent preclinical and clinical studies that show the promise of antagomirs for treating three major CVDs (Table 3): i) atherosclerosis, ii) MI and its complications, and iii) cardiac hypertrophy, including hypertrophy driven by hypertension. These examples may not be comprehensive, and we regret any significant omissions.
      Table 3Delivery of antagomirs in preclinical models of CVDs.
      Cardiovascular disease (species)Targeting approach and delivery system (route)Target miRNAFindingsRef.
      Atherosclerosis (mouse)Anti-αvβ3 integrin polymer NPs (IV)miR-33aReduced lesion size and increased M2 macrophages[
      • Li C.
      • Dou Y.
      • Chen Y.
      • Qi Y.
      • Li L.
      • Han S.
      • et al.
      Site‐specific MicroRNA‐33 antagonism by pH‐responsive nanotherapies for treatment of atherosclerosis via regulating cholesterol efflux and adaptive immunity.
      ]
      Atherosclerosis (mouse)Lentivirus (RO)miR-33aIncreased hepatic ABCA1 protein and increased plasma HDL[
      • Rayner K.J.
      • Suarez Y.
      • Davalos A.
      • Parathath S.
      • Fitzgerald M.L.
      • Tamehiro N.
      • et al.
      MiR-33 contributes to the regulation of cholesterol homeostasis.
      ]
      Atherosclerosis (human and bovine cells)Ad (in vitro study)miR-33aIncreased ABCA1 protein and ApoA1-mediated cholesterol efflux[
      • Stamatikos A.
      • Knight E.
      • Vojtech L.
      • Bi L.
      • Wacker B.K.
      • Tang C.
      • et al.
      Exosome-mediated transfer of anti-miR-33a-5p from transduced endothelial cells enhances macrophage and vascular smooth muscle cell cholesterol efflux.
      ]
      Atherosclerosis (mouse)Anti-VCAM1 liposomes (IV)miR-712Reduced lesion size and Timp3 expression[
      • Kheirolomoom A.
      • Kim C.W.
      • Seo J.W.
      • Kumar S.
      • Son D.J.
      • Gagnon M.K.
      • et al.
      Multifunctional nanoparticles facilitate molecular targeting and miRNA delivery to inhibit atherosclerosis in ApoE(-/-) mice.
      ]
      MI (rat)Anti-cardiac troponin I liposomes (IV)miR-1Repressed arrhythmias[
      • Liu M.
      • Li M.
      • Sun S.
      • Li B.
      • Du D.
      • Sun J.
      • et al.
      The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy.
      ]
      MI (mouse)Anti-AT1R dendrimers (IV)miR-1Reduced apoptotic cells, fibrosis, and infarct size[
      • Xue X.
      • Shi X.
      • Dong H.
      • You S.
      • Cao H.
      • Wang K.
      • et al.
      Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: an early targeting therapy for myocardial infarction in mice.
      ]
      MI (pig)Polymer NP (IC)miR-92aEnhanced angiogenesis[
      • Bellera N.
      • Barba I.
      • Rodriguez-Sinovas A.
      • Ferret E.
      • Asin M.A.
      • Gonzalez-Alujas M.T.
      • et al.
      Single intracoronary injection of encapsulated antagomir-92a promotes angiogenesis and prevents adverse infarct remodeling.
      ]
      MI (rat)Gelatin hydrogel microsphere sheet (local device)miR-92aEnhanced angiogenesis and cardiomyogenesis, reduced infarct size, and improved left ventricle function[
      • Fujita M.
      • Otani H.
      • Iwasaki M.
      • Yoshioka K.
      • Shimazu T.
      • Shiojima I.
      • et al.
      Antagomir-92a impregnated gelatin hydrogel microsphere sheet enhances cardiac regeneration after myocardial infarction in rats.
      ]
      MI (mouse)EVs (IM)miR-21Reduced fibrosis and improved cardiac function[
      • Kang J.Y.
      • Park H.
      • Kim H.
      • Mun D.
      • Park H.
      • Yun N.
      • et al.
      Human peripheral bloodderived exosomes for microRNA delivery.
      ]
      MI (mouse)AAV9 (IM)let-7i-5pReduced infarct size and enhanced cardiomyocyte proliferation[
      • Hu Y.
      • Jin G.
      • Li B.
      • Chen Y.
      • Zhong L.
      • Chen G.
      • et al.
      Suppression of miRNA let-7i-5p promotes cardiomyocyte proliferation and repairs heart function post injury by targetting CCND2 and E2F2.
      ]
      Hypertension (rat)LV (IV)miR-20bReduced blood pressure, artery wall thickness, and vascular fibrosis[
      • Xu M.
      • Yu T.
      MiR-20b-5p contributes to the dysfunction of vascular smooth muscle cells by targeting MAGI3 in hypertension.
      ]
      Cardiac hypertrophy (mouse)AAV9 (IV)miR-20bDecreased left ventricle thickness[
      • Qiu Y.
      • Cheng R.
      • Liang C.
      • Yao Y.
      • Zhang W.
      • Zhang J.
      • et al.
      MicroRNA-20b promotes cardiac hypertrophy by the inhibition of mitofusin 2-mediated inter-organelle Ca(2+) cross-talk.
      ]
      Cardiac hypertrophy (mouse)Liposome-like NP (IV)miR-23aReduced left ventricle thickness and improved cardiac function[
      • Kopechek J.A.
      • McTiernan C.F.
      • Chen X.
      • Zhu J.
      • Mburu M.
      • Feroze R.
      • et al.
      Ultrasound and microbubble-targeted delivery of a microRNA inhibitor to the heart suppresses cardiac hypertrophy and preserves cardiac function.
      ]
      Cardiac hypertrophy (mouse)Cholesterol-coated polymer NP (IV)miR-182Repressed cardiomyocyte growth and improved cardiac function[
      • Zhi Y.
      • Xu C.
      • Sui D.
      • Du J.
      • Xu F.J.
      • Li Y.
      Effective delivery of hypertrophic miRNA inhibitor by cholesterol-containing nanocarriers for preventing pressure overload induced cardiac hypertrophy.
      ]
      AT1R: angiotensin II type 1 receptor; AAV: adeno-associated virus; Ad: adenovirus; CVD: cardiovascular disease; NP: nanoparticle; VCAM1: vascular cell adhesion molecule 1; IC: intracoronary; IV: intravenous; RO: retro-orbital; IM: intramyocardial; EVs: extracellular vesicles; MI: myocardial infarction.

      4.1 Atherosclerosis

      Preclinical data suggest that miR-33a has several pathogenic activities within atherosclerotic plaques including suppression of cholesterol efflux, inhibition of lipid droplet catabolism, reduction of efferocytosis, and maintenance of a proinflammatory immune cell profile [
      • Ouimet M.
      • Ediriweera H.
      • Afonso M.S.
      • Ramkhelawon B.
      • Singaravelu R.
      • Liao X.
      • et al.
      microRNA-33 regulates macrophage autophagy in atherosclerosis.
      ,
      • Afonso M.S.
      • Sharma M.
      • Schlegel M.
      • van Solingen C.
      • Koelwyn G.J.
      • Shanley L.C.
      • et al.
      miR-33 silencing reprograms the immune cell landscape in atherosclerotic plaques.
      ,
      • Rayner K.J.
      • Suarez Y.
      • Davalos A.
      • Parathath S.
      • Fitzgerald M.L.
      • Tamehiro N.
      • et al.
      MiR-33 contributes to the regulation of cholesterol homeostasis.
      ]. Accordingly, targeting of miR-33a with ASOs is atheroprotective in both mice and non-human primates. Systemic administration of anti-miR-33a increases circulating high-density lipoprotein-cholesterol in mice and non-human primates and reduces the size and lipid content of murine atherosclerotic plaques [
      • Rayner K.J.
      • Esau C.C.
      • Hussain F.N.
      • McDaniel A.L.
      • Marshall S.M.
      • van Gils J.M.
      • et al.
      Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides.
      ,
      • Rayner K.J.
      • Sheedy F.J.
      • Esau C.C.
      • Hussain F.N.
      • Temel R.E.
      • Parathath S.
      • et al.
      Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.
      ]. However, long-term systemic delivery of anti-miR-33a in mice fed a high-fat diet alters hepatic metabolism, leading to hypertriglyceridemia and hepatic steatosis [
      • Goedeke L.
      • Salerno A.
      • Ramirez C.M.
      • Guo L.
      • Allen R.M.
      • Yin X.
      • et al.
      Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice.
      ]. These toxic effects of hepatic miR-33a inhibition highlight the need for a delivery system that could target anti-miR-33a to plaque cells and minimize its activity at other sites.
      As an initial step in developing vessel-wall-targeted miR33a inhibition, our group generated a helper-dependent adenoviral vector encoding an anti-miR33a antagomir fused to the X-motif (see above). We added the X-motif to promote loading of the antagomir into exosomes that are released from transduced cells. Medium conditioned by transduced EC contained anti-miR-33a-loaded exosomes and unconcentrated conditioned medium efficiently delivered anti-miR-33a to macrophages and VSMCs in vitro, decreasing miR-33a levels, increasing ABCA1 protein, and promoting ApoAI-mediated cholesterol efflux in both of these cell types [
      • Stamatikos A.
      • Knight E.
      • Vojtech L.
      • Bi L.
      • Wacker B.K.
      • Tang C.
      • et al.
      Exosome-mediated transfer of anti-miR-33a-5p from transduced endothelial cells enhances macrophage and vascular smooth muscle cell cholesterol efflux.
      ]. We anticipate that exosomes released by transduced arterial endothelial cells (which are efficiently transduced by adenoviral vectors in vivo using an intraluminal dwell approach, in which vectors are incubated in the lumen of a segment of blood vessel that is transiently isolated from the circulation with ligatures or clamps) [
      • Lemarchand P.
      • Jones M.
      • Yamada I.
      • Crystal R.G.
      In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors.
      ,
      • Schulick A.H.
      • Dong G.
      • Newman K.D.
      • Virmani R.
      • Dichek D.A.
      Endothelium-specific in vivo gene transfer.
      ] would transfer the antagomir to neighboring vascular cells including macrophages and VSMCs, stimulating cholesterol efflux and reducing plaque size and lipid content, while favorably altering the plaque immune cell population [
      • Stamatikos A.
      • Knight E.
      • Vojtech L.
      • Bi L.
      • Wacker B.K.
      • Tang C.
      • et al.
      Exosome-mediated transfer of anti-miR-33a-5p from transduced endothelial cells enhances macrophage and vascular smooth muscle cell cholesterol efflux.
      ]. Use of vectors that achieve durable transgene expression (e.g., helper-dependent Ad, AAV, or lentivirus) could provide long-term protection from atherosclerosis. Recently, Li et al. developed anti-miR-33a-loaded polymer NPs coated with an αvβ3 integrin-binding ligand designed to target the NPs to plaque ECs and macrophages [
      • Li C.
      • Dou Y.
      • Chen Y.
      • Qi Y.
      • Li L.
      • Han S.
      • et al.
      Site‐specific MicroRNA‐33 antagonism by pH‐responsive nanotherapies for treatment of atherosclerosis via regulating cholesterol efflux and adaptive immunity.
      ]. Analysis of atherosclerotic mice after 8 weeks of injections showed that the NPs accumulated within the aortic plaque, reduced lesion size, and promoted an anti-inflammatory macrophage phenotype. Importantly, no long-term adverse effects were reported in blood, liver, kidneys, spleen, and lungs [
      • Li C.
      • Dou Y.
      • Chen Y.
      • Qi Y.
      • Li L.
      • Han S.
      • et al.
      Site‐specific MicroRNA‐33 antagonism by pH‐responsive nanotherapies for treatment of atherosclerosis via regulating cholesterol efflux and adaptive immunity.
      ]. This approach is promising, but the need for weekly injections and issues of cost are barriers to clinical translation.
      Expression of the atherogenic miR-712 is upregulated in ECs by disturbed blood flow. MiR-712 downregulates expression of the metalloprotease inhibitor Timp3, leading to increased activity of TIMP3 targets including matrix metalloproteinases and disintegrin and metalloproteases as well as increased endothelial inflammation and permeability [
      • Son D.J.
      • Kumar S.
      • Takabe W.
      • Kim C.W.
      • Ni C.W.
      • Alberts-Grill N.
      • et al.
      The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.
      ]. Subcutaneous injection of a miR-712 antagomir in mice prevented these atherogenic effects of miR-712, leading to reduced aortic atherosclerosis. However, this antagomir also repressed miR-712 expression in non-vascular tissues (e.g., spleen), potentially causing off-target effects [
      • Son D.J.
      • Kumar S.
      • Takabe W.
      • Kim C.W.
      • Ni C.W.
      • Alberts-Grill N.
      • et al.
      The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.
      ]. To avoid off-target effects outside the vasculature, Kheirolomoom et al. constructed an LNA-modified anti-miR-712 with a phosphorothioate backbone, encapsulated the antagomirs in cationic lipid NPs coupled to a Vcam1-targeting peptide, and injected the NPs intravenously in mice [
      • Kheirolomoom A.
      • Kim C.W.
      • Seo J.W.
      • Kumar S.
      • Son D.J.
      • Gagnon M.K.
      • et al.
      Multifunctional nanoparticles facilitate molecular targeting and miRNA delivery to inhibit atherosclerosis in ApoE(-/-) mice.
      ]. The NPs were efficiently delivered to aortic ECs, de-repressed expression of Timp3, and reduced atherosclerosis. Importantly, the effective dose of the encapsulated antagomir was lower than the effective dose of the naked antagomir [
      • Kheirolomoom A.
      • Kim C.W.
      • Seo J.W.
      • Kumar S.
      • Son D.J.
      • Gagnon M.K.
      • et al.
      Multifunctional nanoparticles facilitate molecular targeting and miRNA delivery to inhibit atherosclerosis in ApoE(-/-) mice.
      ]. Moreover, injection of the NPs did not alter miR-712 levels in non-vascular tissues including spleen, liver, kidneys, and bone marrow. These data suggest that encapsulation of antagomirs in targeted NPs may reduce systemic toxicity [
      • Kheirolomoom A.
      • Kim C.W.
      • Seo J.W.
      • Kumar S.
      • Son D.J.
      • Gagnon M.K.
      • et al.
      Multifunctional nanoparticles facilitate molecular targeting and miRNA delivery to inhibit atherosclerosis in ApoE(-/-) mice.
      ].

      4.2 Myocardial infarction (MI) and its complications

      MiR-1 is a muscle-enriched miRNA that is upregulated in ischemic human and murine hearts and impairs cardiac function after MI by lowering the expression of proteins involved in ion trafficking (e.g., Kcnj2 and Gja1) and apoptosis suppression (e.g., Prkce and Bcl2). Accordingly, elevated miR-1 expression after MI promotes arrhythmias and cardiomyocyte death [
      • Yang B.
      • Lin H.
      • Xiao J.
      • Lu Y.
      • Luo X.
      • Li B.
      • et al.
      The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
      ,
      • Pan Z.
      • Sun X.
      • Ren J.
      • Li X.
      • Gao X.
      • Lu C.
      • et al.
      miR-1 exacerbates cardiac ischemia-reperfusion injury in mouse models.
      ]. Intramyocardial injection of anti-miR-1 antagomirs reduced the incidence of ventricular arrhythmias in a rat MI model, and systemic injection of anti-miR-1 decreased infarct size in a mouse model [
      • Yang B.
      • Lin H.
      • Xiao J.
      • Lu Y.
      • Luo X.
      • Li B.
      • et al.
      The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
      ,
      • Pan Z.
      • Sun X.
      • Ren J.
      • Li X.
      • Gao X.
      • Lu C.
      • et al.
      miR-1 exacerbates cardiac ischemia-reperfusion injury in mouse models.
      ]. Unfortunately, off-target effects in other tissues expressing miR-1, particularly skeletal muscle, were not evaluated. To improve targeting of ischemic myocardium and prevent off-target effects, Liu et al. encapsulated anti-miR-1 in liposomes coated with anti-cardiac troponin I antibody [
      • Liu M.
      • Li M.
      • Sun S.
      • Li B.
      • Du D.
      • Sun J.
      • et al.
      The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy.
      ]. Intravenous injection of the liposomes after experimental MI in rats efficiently targeted the ischemic heart, upregulated the ion channel proteins Kcnj2 and Gja1 and reduced arrhythmias. Importantly, the encapsulated antagomirs had low cytotoxicity in vitro and were taken up preferentially in ischemic myocardium versus skeletal muscle after intravenous injection [
      • Liu M.
      • Li M.
      • Sun S.
      • Li B.
      • Du D.
      • Sun J.
      • et al.
      The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy.
      ]. Xue et al. used PEGylated dendrimers coated with an anti-angiotensin type 1 receptor-binding peptide to target anti-miR-1 to hypoxic murine myocardium after intravenous injection. Anti-miR-1 reduced infarct size and prevented cardiomyocyte apoptosis, potentially by de-repression of Prkce and Bcl2 [
      • Xue X.
      • Shi X.
      • Dong H.
      • You S.
      • Cao H.
      • Wang K.
      • et al.
      Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: an early targeting therapy for myocardial infarction in mice.
      ]. The dendrimers had low cytotoxicity in vitro, although their distribution and potential side effects in non-cardiac tissues were not fully evaluated [
      • Xue X.
      • Shi X.
      • Dong H.
      • You S.
      • Cao H.
      • Wang K.
      • et al.
      Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: an early targeting therapy for myocardial infarction in mice.
      ].
      MiR-92a represses expression of several pro-angiogenic factors (e.g., ITGA5 and KLF4), thereby limiting revascularization of ischemic myocardium. Accordingly, suppression of miR-92a could promote myocardial revascularization [
      • Iaconetti C.
      • Polimeni A.
      • Sorrentino S.
      • Sabatino J.
      • Pironti G.
      • Esposito G.
      • et al.
      Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury.
      ,
      • Bonauer A.
      • Carmona G.
      • Iwasaki M.
      • Mione M.
      • Koyanagi M.
      • Fischer A.
      • et al.
      MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.
      ]. Preclinical studies show that inhibition of miR-92a enhances angiogenesis and tissue vascularization in murine MI and hindlimb ischemia models, in part by de-repressing Itga5 [
      • Bonauer A.
      • Carmona G.
      • Iwasaki M.
      • Mione M.
      • Koyanagi M.
      • Fischer A.
      • et al.
      MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.
      ]. In addition, Bellera et al. injected PLGA NP-encapsulated anti-miR-92a into coronary arteries of pigs with experimental myocardial ischemia and found that it repressed miR-92a for at least 10 days [
      • Bellera N.
      • Barba I.
      • Rodriguez-Sinovas A.
      • Ferret E.
      • Asin M.A.
      • Gonzalez-Alujas M.T.
      • et al.
      Single intracoronary injection of encapsulated antagomir-92a promotes angiogenesis and prevents adverse infarct remodeling.
      ]. Moreover, these NPs were preferentially retained in the vessels of the injured myocardium and promoted angiogenesis. No NP accumulation was detected in other tissues, including liver, spleen, and lungs. Higher doses of NPs were associated with adverse effects on the heart; however, long-term effects were not evaluated [
      • Bellera N.
      • Barba I.
      • Rodriguez-Sinovas A.
      • Ferret E.
      • Asin M.A.
      • Gonzalez-Alujas M.T.
      • et al.
      Single intracoronary injection of encapsulated antagomir-92a promotes angiogenesis and prevents adverse infarct remodeling.
      ]. Fujita et al. delivered anti-miR-92a to infarcted rat hearts by direct attachment of a gelatin hydrogel microsphere sheet impregnated with anti-miR-92a. This novel approach promoted angiogenesis and cardiomyogenesis in the infarct border zone, leading to improved left ventricular function [
      • Fujita M.
      • Otani H.
      • Iwasaki M.
      • Yoshioka K.
      • Shimazu T.
      • Shiojima I.
      • et al.
      Antagomir-92a impregnated gelatin hydrogel microsphere sheet enhances cardiac regeneration after myocardial infarction in rats.
      ]. These studies prompted initiation of the first clinical study that tested angiogenic antagomirs in humans [
      • Abplanalp W.T.
      • Fischer A.
      • John D.
      • Zeiher A.M.
      • Gosgnach W.
      • Darville H.
      • et al.
      Efficiency and target derepression of anti-miR-92a: results of a first in human study.
      ]. In this study, intravenous injection of an LNA-modified anti-miR-92a reduced plasma miR-92a levels and increased plasma levels of proteins encoded by miR-92a target genes, including the pro-angiogenic ITGA5.
      The role of miR-21 in MI is controversial [
      • Surina S.
      • Fontanella R.A.
      • Scisciola L.
      • Marfella R.
      • Paolisso G.
      • Barbieri M.
      miR-21 in human cardiomyopathies.
      ]; however, several groups have identified miR-21 as a target for interventions that aim to decrease cardiac fibrosis after MI. These studies are based on the premise that miR-21 promotes cardiac fibrosis by at least 2 pathways [
      • O'Brien J.
      • Hayder H.
      • Zayed Y.
      • Peng C.
      Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.
      ]: inhibition of Smad7, which in turn de-represses pro-fibrotic TGF-β signaling via Smad2/3 [
      • Yuan J.
      • Chen H.
      • Ge D.
      • Xu Y.
      • Xu H.
      • Yang Y.
      • et al.
      Mir-21 promotes cardiac fibrosis after myocardial infarction via targeting Smad7.
      ], and [
      • Espinoza-Lewis R.A.
      • Wang D.Z.
      MicroRNAs in heart development.
      ] inhibition of Pten, leading to increased Akt1 phosphorylation and upregulation of Mmp2, which helps drive cardiac fibrosis [
      • Roy S.
      • Khanna S.
      • Hussain S.R.
      • Biswas S.
      • Azad A.
      • Rink C.
      • et al.
      MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue.
      ]. Kang et al. loaded peripheral blood-derived EVs with anti-miR-21 and incubated the EVs with mouse and rat cardiomyocyte cell lines. Cells incubated with the EVs had lower levels of miR-21 along with higher levels of miR-21 targets (e.g., Smad7 and Pten) [
      • Kang J.Y.
      • Park H.
      • Kim H.
      • Mun D.
      • Park H.
      • Yun N.
      • et al.
      Human peripheral bloodderived exosomes for microRNA delivery.
      ]. Moreover, intramyocardial injection of these EVs in a mouse MI model significantly reduced cardiac fibrosis (i.e., decreased cardiac collagen types I and III) and increased myocardial levels of the miR-21 targets Smad7 and Pten [
      • Kang J.Y.
      • Park H.
      • Kim H.
      • Mun D.
      • Park H.
      • Yun N.
      • et al.
      Human peripheral bloodderived exosomes for microRNA delivery.
      ].
      Let-7i-5p, one of the most abundant miRNAs in the heart, inhibits cardiomyocyte proliferation by repressing the cell cycle regulators E2f2 and Ccnd2 [
      • Hu Y.
      • Jin G.
      • Li B.
      • Chen Y.
      • Zhong L.
      • Chen G.
      • et al.
      Suppression of miRNA let-7i-5p promotes cardiomyocyte proliferation and repairs heart function post injury by targetting CCND2 and E2F2.
      ]. Accordingly, suppression of let-7i-5p could promote cardiac regeneration after MI. Hu et al. injected an AAV9 vector encoding an anti-let-7i-5p antagomir into infarcted mouse hearts and observed increased levels of myocardial E2f2 and Ccnd2, enhanced cardiomyocyte proliferation in the infarct border zone, and reduced infarct size [
      • Hu Y.
      • Jin G.
      • Li B.
      • Chen Y.
      • Zhong L.
      • Chen G.
      • et al.
      Suppression of miRNA let-7i-5p promotes cardiomyocyte proliferation and repairs heart function post injury by targetting CCND2 and E2F2.
      ].

      4.3 Cardiac hypertrophy

      Several miRNAs play roles in regulating cardiomyocyte growth and hypertrophy. For example, miR-20b is increased in hypertrophic hearts of hypertensive mice and promotes cardiac hypertrophy by downregulating Mfn2. Mfn2 regulates Ca2+ trafficking between the sarcoplasmic reticulum and mitochondria, and reduced cardiomyocyte Mfn2 levels lead to increased cytoplasmic Ca2+ and activation of calcium-induced hypertrophic signaling pathways [
      • Qiu Y.
      • Cheng R.
      • Liang C.
      • Yao Y.
      • Zhang W.
      • Zhang J.
      • et al.
      MicroRNA-20b promotes cardiac hypertrophy by the inhibition of mitofusin 2-mediated inter-organelle Ca(2+) cross-talk.
      ]. MiR-20b may also promote cardiac hypertrophy indirectly by stimulating proliferation and migration of VSMCs and increasing their synthesis of collagen and fibronectin leading to arterial stiffness and hypertension. The actions of miR-20b in VSMCs likely occur via inhibition of Magi3, although the precise mechanism is uncertain [
      • Xu M.
      • Yu T.
      MiR-20b-5p contributes to the dysfunction of vascular smooth muscle cells by targeting MAGI3 in hypertension.
      ].
      Several preclinical studies show that antagomirs can prevent cardiac hypertrophy. To prevent hypertrophy in hypertensive mice, Qiu et al. constructed an AAV9 vector expressing anti-miR-20b and injected it intravenously. AAV9-anti-miR-20b restored cardiac Mfn2 expression, decreased left ventricle wall thickness, and lowered levels of plasma markers of cardiac hypertrophy [i.e., atrial natriuretic peptide (Nppa) and brain natriuretic peptide (Nppb)] [
      • Qiu Y.
      • Cheng R.
      • Liang C.
      • Yao Y.
      • Zhang W.
      • Zhang J.
      • et al.
      MicroRNA-20b promotes cardiac hypertrophy by the inhibition of mitofusin 2-mediated inter-organelle Ca(2+) cross-talk.
      ]. Unfortunately, no data regarding potential adverse effects of the anti-miR-20b-expressing vector were reported. MiR-23a and miR-182 also promote cardiac hypertrophy, and both have been successfully targeted by antagomirs. MiR-23a is upregulated in cardiomyocytes by calcineurin and promotes cardiac hypertrophy by inhibiting the anti-hypertrophic protein Ring1 [
      • Lin Z.
      • Murtaza I.
      • Wang K.
      • Jiao J.
      • Gao J.
      • Li P.F.
      miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy.
      ]. Accordingly, knockdown of miR-23a by intravenously injected 2′-O-methyl modified antagomirs attenuated hypertension-induced hypertrophy in mice [
      • Lin Z.
      • Murtaza I.
      • Wang K.
      • Jiao J.
      • Gao J.
      • Li P.F.
      miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy.
      ]. Similarly, intravenous injection of cationic liposome-like NPs coated with anti-miR-23a downregulated miR-23a expression in mouse myocardium, reduced cardiac hypertrophy, and improved cardiac function [
      • Kopechek J.A.
      • McTiernan C.F.
      • Chen X.
      • Zhu J.
      • Mburu M.
      • Feroze R.
      • et al.
      Ultrasound and microbubble-targeted delivery of a microRNA inhibitor to the heart suppresses cardiac hypertrophy and preserves cardiac function.
      ]. However, biodistribution and adverse effects of these NPs were not analyzed.
      MiR-182 targets both the anti-hypertrophic Foxo3 transcription factor and the Bcat2 enzyme (inhibitors of the calcineurin and AKT/mTORC1 pathways, respectively), leading to accelerated cardiomyocyte growth [
      • Ni Y.G.
      • Berenji K.
      • Wang N.
      • Oh M.
      • Sachan N.
      • Dey A.
      • et al.
      Foxo transcription factors blunt cardiac hypertrophy by inhibiting calcineurin signaling.
      ,
      • Li N.
      • Hwangbo C.
      • Jaba I.M.
      • Zhang J.
      • Papangeli I.
      • Han J.
      • et al.
      miR-182 modulates myocardial hypertrophic response induced by angiogenesis in heart.
      ,
      • Zhi Y.
      • Xu C.
      • Sui D.
      • Du J.
      • Xu F.J.
      • Li Y.
      Effective delivery of hypertrophic miRNA inhibitor by cholesterol-containing nanocarriers for preventing pressure overload induced cardiac hypertrophy.
      ]. To block the hypertrophy-promoting effects of miR-182, Zhi et al. loaded cholesterol-coated polymer NPs with anti-miR-182 ASOs and injected the NPs intravenously in mice. The NPs efficiently delivered the ASOs to cardiomyocytes in vitro and to murine hearts in vivo, increasing myocardial Foxo3, reducing cardiomyocyte size, and improving cardiac function [
      • Zhi Y.
      • Xu C.
      • Sui D.
      • Du J.
      • Xu F.J.
      • Li Y.
      Effective delivery of hypertrophic miRNA inhibitor by cholesterol-containing nanocarriers for preventing pressure overload induced cardiac hypertrophy.
      ]. Importantly, this delivery system had low cytotoxicity in vitro, and did not alter the histology of other organs such as liver and kidney when injected into mice. However, these promising findings should be interpreted with caution because inhibition of miR-182 in murine hearts was not verified, and other reports indicate that miR-182 expression is not upregulated in mouse myocardium subjected to pressure overload [
      • Li N.
      • Hwangbo C.
      • Jaba I.M.
      • Zhang J.
      • Papangeli I.
      • Han J.
      • et al.
      miR-182 modulates myocardial hypertrophic response induced by angiogenesis in heart.
      ].

      5. Conclusions and future perspectives

      MiRNAs play fundamental roles in the pathogenesis of multiple CVDs. Antagomirs have emerged as a promising therapeutic strategy to inhibit miRNA activity and show substantial promise in preclinical models. To date, two antagomirs (i.e., anti-miR-92a and anti-miR-132) have been used in clinical trials for treatment of patients with CVDs [
      • Abplanalp W.T.
      • Fischer A.
      • John D.
      • Zeiher A.M.
      • Gosgnach W.
      • Darville H.
      • et al.
      Efficiency and target derepression of anti-miR-92a: results of a first in human study.
      ,
      • Taubel J.
      • Hauke W.
      • Rump S.
      • Viereck J.
      • Batkai S.
      • Poetzsch J.
      • et al.
      Novel antisense therapy targeting microRNA-132 in patients with heart failure: results of a first-in-human Phase 1b randomized, double-blind, placebo-controlled study.
      ]. Anti-miR-132 has advanced to a phase II clinical trial for treating MI patients with reduced left ventricular ejection fraction. However, despite significant improvements in antagomir chemistry and targeting, advancement of miRNA-targeted antagomirs into the clinic still faces numerous challenges. Inefficient targeting of cardiovascular cells (i.e., cardiomyocytes, endothelial cells, and smooth muscle cells) and accumulation of antagomirs in non-targeted organs reduces efficacy and increases toxicity in vivo. Encapsulation of antagomirs into targeted delivery systems (or expression of antagomirs by tissue-tropic viral vectors) has enhanced the therapeutic potential of antagomirs, reducing delivery to non-targeted cell types and improving both efficacy and safety in preclinical models of CVDs. However, scalability and cost are persistent challenges for both viral and non-viral vector systems. Further work is needed to improve the pharmacokinetic and pharmacodynamic properties of viral as well as non-viral vectors that are used to deliver antagomirs, for example by selecting optimal dosing and administration routes, by further improving targeting of specific cardiovascular cell types and by decreasing immunogenicity. Finally, approaches that exploit endogenous cell-cell communication mechanisms (e.g., exosomes) should be further explored.

      Declaration of competing interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      This work was supported by a grant from the National Heart Lung and Blood Institute (R01HL114541) and by the John L. Locke Jr. Charitable Trust. Dr. Dichek is a member of the Institute for Stem Cell and Regenerative Medicine at the University of Washington.

      References

        • O'Brien J.
        • Hayder H.
        • Zayed Y.
        • Peng C.
        Overview of MicroRNA biogenesis, mechanisms of actions, and circulation.
        Front. Endocrinol. 2018; 9: 402
        • Espinoza-Lewis R.A.
        • Wang D.Z.
        MicroRNAs in heart development.
        Curr. Top. Dev. Biol. 2012; 100: 279-317
        • Zhang X.-T.
        • Xu M.-G.
        Potential link between microRNA-208 and cardiovascular diseases.
        Journal of Xiangya Medicine. 2021; 6: 12
        • Vickers K.C.
        • Landstreet S.R.
        • Levin M.G.
        • Shoucri B.M.
        • Toth C.L.
        • Taylor R.C.
        • et al.
        MicroRNA-223 coordinates cholesterol homeostasis.
        Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 14518-14523
        • Sun H.X.
        • Zeng D.Y.
        • Li R.T.
        • Pang R.P.
        • Yang H.
        • Hu Y.L.
        • et al.
        Essential role of microRNA-155 in regulating endothelium-dependent vasorelaxation by targeting endothelial nitric oxide synthase.
        Hypertension. 2012; 60: 1407-1414
        • Nazari-Jahantigh M.
        • Wei Y.
        • Noels H.
        • Akhtar S.
        • Zhou Z.
        • Koenen R.R.
        • et al.
        MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages.
        J. Clin. Invest. 2012; 122: 4190-4202
        • Romaine S.P.R.
        • Tomaszewski M.
        • Condorelli G.
        • Samani N.J.
        MicroRNAs in cardiovascular disease: an introduction for clinicians.
        Heart. 2015; 101: 921-928
        • Lu Y.
        • Thavarajah T.
        • Gu W.
        • Cai J.
        • Xu Q.
        Impact of miRNA in atherosclerosis.
        Arterioscler. Thromb. Vasc. Biol. 2018; 38
        • Melman Y.F.
        • Shah R.
        • Das S.
        MicroRNAs in heart failure: is the picture becoming less miRky?.
        Circ Heart Fail. 2014; 7: 203-214
        • Ghafouri-Fard S.
        • Gholipour M.
        • Taheri M.
        Role of MicroRNAs in the pathogenesis of coronary artery disease.
        Front Cardiovasc Med. 2021; 8632392
        • Hosen M.R.
        • Goody P.R.
        • Zietzer A.
        • Nickenig G.
        • Jansen F.
        MicroRNAs as master regulators of atherosclerosis: from pathogenesis to novel therapeutic options.
        Antioxidants Redox Signal. 2020; 33: 621-644
        • Fang Y.
        • Shi C.
        • Manduchi E.
        • Civelek M.
        • Davies P.F.
        MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro.
        Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 13450-13455
        • Infante T.
        • Forte E.
        • Punzo B.
        • Cademartiri F.
        • Cavaliere C.
        • Soricelli A.
        • et al.
        Correlation of circulating miR-765, miR-93-5p, and miR-433-3p to obstructive coronary heart disease evaluated by cardiac computed tomography.
        Am. J. Cardiol. 2019; 124: 176-182
        • Kuo J.T.
        • Tsai H.E.
        • Lin C.T.
        • Lee C.I.
        • Lee P.L.
        • Ruan Y.R.
        • et al.
        Low levels of MicroRNA-10a in cardiovascular endothelium and blood serum are related to human atherosclerotic disease.
        Cardiol. Res. Pract. 2021; (2021)1452917
        • Schober A.
        • Nazari-Jahantigh M.
        • Wei Y.
        • Bidzhekov K.
        • Gremse F.
        • Grommes J.
        • et al.
        MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1.
        Nat. Med. 2014; 20: 368-376
        • Ouimet M.
        • Ediriweera H.
        • Afonso M.S.
        • Ramkhelawon B.
        • Singaravelu R.
        • Liao X.
        • et al.
        microRNA-33 regulates macrophage autophagy in atherosclerosis.
        Arterioscler. Thromb. Vasc. Biol. 2017; 37: 1058-1067
        • Tian F.J.
        • An L.N.
        • Wang G.K.
        • Zhu J.Q.
        • Li Q.
        • Zhang Y.Y.
        • et al.
        Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.
        Cardiovasc. Res. 2014; 103: 100-110
        • Price N.L.
        • Goedeke L.
        • Suarez Y.
        • Fernandez-Hernando C.
        miR-33 in cardiometabolic diseases: lessons learned from novel animal models and approaches.
        EMBO Mol. Med. 2021; 13e12606
        • Cheng Y.
        • Liu X.
        • Yang J.
        • Lin Y.
        • Xu D.Z.
        • Lu Q.
        • et al.
        MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation.
        Circ. Res. 2009; 105: 158-166
        • Cao H.
        • Pan G.
        • Tang S.
        • Zhong N.
        • Liu H.
        • Zhou H.
        • et al.
        miR-145-5p regulates the proliferation, migration and invasion in cervical carcinoma by targeting KLF5.
        OncoTargets Ther. 2020; 13: 2369-2376
        • Yuan C.H.
        • Hsu W.C.
        • Huang A.M.
        • Yuan B.C.
        • Chen I.H.
        • Hsu C.A.
        • et al.
        MicroRNA-145-5p modulates Kruppel-like factor 5 and inhibits cell proliferation, migration, and invasion in nasopharyngeal carcinoma.
        BMC Mol Cell Biol. 2022; 23: 28
        • Li M.
        • Liu Q.
        • Lei J.
        • Wang X.
        • Chen X.
        • Ding Y.
        MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1.
        Biochem. Biophys. Res. Commun. 2017; 493: 270-276
        • Colpaert R.M.W.
        • Calore M.
        MicroRNAs in cardiac diseases.
        Cells. 2019; 8: 737
        • Yang B.
        • Lin H.
        • Xiao J.
        • Lu Y.
        • Luo X.
        • Li B.
        • et al.
        The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2.
        Nat. Med. 2007; 13: 486-491
        • Tao L.
        • Bei Y.
        • Chen P.
        • Lei Z.
        • Fu S.
        • Zhang H.
        • et al.
        Crucial role of miR-433 in regulating cardiac fibrosis.
        Theranostics. 2016; 6: 2068-2083
        • Fiedler J.
        • Jazbutyte V.
        • Kirchmaier B.C.
        • Gupta S.K.
        • Lorenzen J.
        • Hartmann D.
        • et al.
        MicroRNA-24 regulates vascularity after myocardial infarction.
        Circulation. 2011; 124: 720-730
        • Li X.
        • Wei C.
        • Zhang Z.
        • Jin Q.
        • Xiao X.
        MiR-134-5p regulates myocardial apoptosis and angiogenesis by directly targeting KDM2A after myocardial infarction.
        Int. Heart J. 2020; 61: 815-821
        • Zhang L.
        • Lv Z.
        • Xu J.
        • Chen C.
        • Ge Q.
        • Li P.
        • et al.
        MicroRNA-134 inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway.
        FEBS J. 2018; 285: 1359-1371
        • Marques F.Z.
        • Campain A.E.
        • Tomaszewski M.
        • Zukowska-Szczechowska E.
        • Yang Y.H.
        • Charchar F.J.
        • et al.
        Gene expression profiling reveals renin mRNA overexpression in human hypertensive kidneys and a role for microRNAs.
        Hypertension. 2011; 58: 1093-1098
        • Chen L.J.
        • Xu R.
        • Yu H.M.
        • Chang Q.
        • Zhong J.C.
        The ACE2/apelin signaling, MicroRNAs, and hypertension.
        Int. J. Hypertens. 2015; (2015)896861
        • Han M.
        • Yang Z.
        • Sayed D.
        • He M.
        • Gao S.
        • Lin L.
        • et al.
        GATA4 expression is primarily regulated via a miR-26b-dependent post-transcriptional mechanism during cardiac hypertrophy.
        Cardiovasc. Res. 2012; 93: 645-654
        • Nie X.
        • Fan J.
        • Li H.
        • Yin Z.
        • Zhao Y.
        • Dai B.
        • et al.
        miR-217 promotes cardiac hypertrophy and dysfunction by targeting PTEN.
        Mol. Ther. Nucleic Acids. 2018; 12: 254-266
        • Ding Y.Q.
        • Zhang Y.H.
        • Lu J.
        • Li B.
        • Yu W.J.
        • Yue Z.B.
        • et al.
        MicroRNA-214 contributes to Ang II-induced cardiac hypertrophy by targeting SIRT3 to provoke mitochondrial malfunction.
        Acta Pharmacol. Sin. 2021; 42: 1422-1436
        • Diener C.
        • Keller A.
        • Meese E.
        Emerging concepts of miRNA therapeutics: from cells to clinic.
        Trends Genet. 2022; 38: 613-626
        • Hong D.S.
        • Kang Y.-K.
        • Borad M.
        • Sachdev J.
        • Ejadi S.
        • Lim H.Y.
        • et al.
        Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours.
        Br. J. Cancer. 2020; 122: 1630-1637
        • Gallant-Behm C.L.
        • Piper J.
        • Lynch J.M.
        • Seto A.G.
        • Hong S.J.
        • Mustoe T.A.
        • et al.
        A MicroRNA-29 mimic (remlarsen) represses extracellular matrix expression and fibroplasia in the skin.
        J. Invest. Dermatol. 2019; 139: 1073-1081
        • Pos O.
        • Biro O.
        • Szemes T.
        • Nagy B.
        Circulating cell-free nucleic acids: characteristics and applications.
        Eur. J. Hum. Genet. 2018; 26: 937-945
        • Hammond S.M.
        • Aartsma-Rus A.
        • Alves S.
        • Borgos S.E.
        • Buijsen R.A.M.
        • Collin R.W.J.
        • et al.
        Delivery of oligonucleotide-based therapeutics: challenges and opportunities.
        EMBO Mol. Med. 2021; 13e13243
        • Lima J.F.
        • Cerqueira L.
        • Figueiredo C.
        • Oliveira C.
        • Azevedo N.F.
        Anti-miRNA oligonucleotides: a comprehensive guide for design.
        RNA Biol. 2018; 15: 338-352
        • Fu Y.
        • Chen J.
        • Huang Z.
        Recent progress in microRNA-based delivery systems for the treatment of human disease.
        ExRNA. 2019; 1
        • Geary R.S.
        • Norris D.
        • Yu R.
        • Bennett C.F.
        Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.
        Adv. Drug Deliv. Rev. 2015; 87: 46-51
        • Swayze E.E.
        • Siwkowski A.M.
        • Wancewicz E.V.
        • Migawa M.T.
        • Wyrzykiewicz T.K.
        • Hung G.
        • et al.
        Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.
        Nucleic Acids Res. 2007; 35: 687-700
        • Crooke S.T.
        • Baker B.F.
        • Witztum J.L.
        • Kwoh T.J.
        • Pham N.C.
        • Salgado N.
        • et al.
        The effects of 2'-O-methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials.
        Nucleic Acid Therapeut. 2017; 27: 121-129
        • Zaslavsky A.
        • Adams M.
        • Cao X.
        • Yamaguchi A.
        • Henderson J.
        • Busch-Ostergren P.
        • et al.
        Antisense oligonucleotides and nucleic acids generate hypersensitive platelets.
        Thromb. Res. 2021; 200: 64-71
        • Bartoszewski R.
        • Sikorski A.F.
        Editorial focus: understanding off-target effects as the key to successful RNAi therapy.
        Cell. Mol. Biol. Lett. 2019; 24: 69
        • Tanaka K.
        • Okuda T.
        • Kasahara Y.
        • Obika S.
        Base-modified aptamers obtained by cell-internalization SELEX facilitate cellular uptake of an antisense oligonucleotide.
        Mol. Ther. Nucleic Acids. 2021; 23: 440-449
        • Biessen E.A.L.
        • Van Berkel Tjc
        N-acetyl galactosamine targeting: paving the way for clinical application of nucleotide medicines in cardiovascular diseases.
        Arterioscler. Thromb. Vasc. Biol. 2021; 41: 2855-2865
        • Debacker A.J.
        • Voutila J.
        • Catley M.
        • Blakey D.
        • Habib N.
        Delivery of oligonucleotides to the liver with GalNAc: from research to registered therapeutic drug.
        Mol. Ther. 2020; 28: 1759-1771
        • Lucas T.
        • Schafer F.
        • Muller P.
        • Eming S.A.
        • Heckel A.
        • Dimmeler S.
        Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice.
        Nat. Commun. 2017; 815162
        • Wang S.
        • Allen N.
        • Prakash T.P.
        • Liang X.H.
        • Crooke S.T.
        Lipid conjugates enhance endosomal release of antisense oligonucleotides into cells.
        Nucleic Acid Therapeut. 2019; 29: 245-255
        • Duygu B.
        • Juni R.
        • Ottaviani L.
        • Bitsch N.
        • Wit J.B.M.
        • de Windt L.J.
        • et al.
        Comparison of different chemically modified inhibitors of miR-199b in vivo.
        Biochem. Pharmacol. 2019; 159: 106-115
        • Betts C.
        • Saleh A.F.
        • Arzumanov A.A.
        • Hammond S.M.
        • Godfrey C.
        • Coursindel T.
        • et al.
        Pip6-PMO, A new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment.
        Mol. Ther. Nucleic Acids. 2012; 1: e38
        • Abplanalp W.T.
        • Fischer A.
        • John D.
        • Zeiher A.M.
        • Gosgnach W.
        • Darville H.
        • et al.
        Efficiency and target derepression of anti-miR-92a: results of a first in human study.
        Nucleic Acid Therapeut. 2020; 30: 335-345
        • Taubel J.
        • Hauke W.
        • Rump S.
        • Viereck J.
        • Batkai S.
        • Poetzsch J.
        • et al.
        Novel antisense therapy targeting microRNA-132 in patients with heart failure: results of a first-in-human Phase 1b randomized, double-blind, placebo-controlled study.
        Eur. Heart J. 2021; 42: 178-188
        • Bonneau E.
        • Neveu B.
        • Kostantin E.
        • Tsongalis G.J.
        • De Guire V.
        How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market.
        EJIFCC. 2019; 30: 114-127
        • Lu D.
        • Thum T.
        RNA-based diagnostic and therapeutic strategies for cardiovascular disease.
        Nat. Rev. Cardiol. 2019; 16: 661-674
        • Kieserman J.M.
        • Myers V.D.
        • Dubey P.
        • Cheung J.Y.
        • Feldman A.M.
        Current landscape of heart failure gene therapy.
        J. Am. Heart Assoc. 2019; 8e012239
        • Newman K.D.
        • Dunn P.F.
        • Owens J.W.
        • Schulick A.H.
        • Virmani R.
        • Sukhova G.
        • et al.
        Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.
        J. Clin. Invest. 1995; 96: 2955-2965
        • Rincon M.Y.
        • VandenDriessche T.
        • Chuah M.K.
        Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.
        Cardiovasc. Res. 2015; 108: 4-20
        • Wen S.
        • Graf S.
        • Massey P.G.
        • Dichek D.A.
        Improved vascular gene transfer with a helper-dependent adenoviral vector.
        Circulation. 2004; 110: 1484-1491
        • Quax P.H.A.
        • Lamfers M.L.M.
        • Grimbergen J.M.
        • Teeling J.
        • Hoeben R.C.
        • van Nieuw Amerongen G.P.
        • et al.
        Effects of infection with recombinant adenovirus on human vascular endothelial and smooth muscle cells.
        Fibrinolysis. 1996; 10: 71-74
        • Parker A.L.
        • White K.M.
        • Lavery C.A.
        • Custers J.
        • Waddington S.N.
        • Baker A.H.
        Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.
        Gene Ther. 2013; 20: 1158-1164
        • Kibbe M.R.
        • Murdock A.
        • Wickham T.
        • Lizonova A.
        • Kovesdi I.
        • Nie S.
        • et al.
        Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.
        Arch. Surg. 2000; 135: 191-197
        • Morecroft I.
        • White K.
        • Caruso P.
        • Nilsen M.
        • Loughlin L.
        • Alba R.
        • et al.
        Gene therapy by targeted adenovirus-mediated knockdown of pulmonary endothelial Tph1 attenuates hypoxia-induced pulmonary hypertension.
        Mol. Ther. 2012; 20: 1516-1528
        • Yang Y.
        • Yang J.
        • Liu X.W.
        • Ding J.W.
        • Li S.
        • Guo X.
        • et al.
        Down-regulation of miR-327 alleviates ischemia/reperfusion-induced myocardial damage by targeting RP105.
        Cell. Physiol. Biochem. 2018; 49: 1049-1063
        • Yang Y.
        • Ago T.
        • Zhai P.
        • Abdellatif M.
        • Sadoshima J.
        Thioredoxin 1 negatively regulates angiotensin II-induced cardiac hypertrophy through upregulation of miR-98/let-7.
        Circ. Res. 2011; 108: 305-313
        • Ambrosi C.M.
        • Sadananda G.
        • Han J.L.
        • Entcheva E.
        Adeno-associated virus mediated gene delivery: implications for scalable in vitro and in vivo cardiac optogenetic models.
        Front. Physiol. 2019; 10: 168
        • Sen S.
        • Conroy S.
        • Hynes S.O.
        • McMahon J.
        • O'Doherty A.
        • Bartlett J.S.
        • et al.
        Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.
        J. Gene Med. 2008; 10: 143-151
        • Bozoglu T.
        • Lee S.
        • Ziegler T.
        • Jurisch V.
        • Maas S.
        • Baehr A.
        • et al.
        Endothelial retargeting of AAV9 in vivo.
        Adv. Sci. 2022; 9e2103867
        • Liu Y.B.
        • Xu B.C.
        • Chen Y.T.
        • Yuan X.
        • Liu J.Y.
        • Liu T.
        • et al.
        Directed evolution of AAV accounting for long-term and enhanced transduction of cardiovascular endothelial cells in vivo.
        Mol Ther Methods Clin Dev. 2021; 22: 148-161
        • Krolak T.
        • Chan K.Y.
        • Kaplan L.
        • Huang Q.
        • Wu J.
        • Zheng Q.
        • et al.
        A high-efficiency AAV for endothelial cell transduction throughout the central nervous system.
        Nat Cardiovasc Res. 2022; 1: 389-400
        • Qiu Y.
        • Cheng R.
        • Liang C.
        • Yao Y.
        • Zhang W.
        • Zhang J.
        • et al.
        MicroRNA-20b promotes cardiac hypertrophy by the inhibition of mitofusin 2-mediated inter-organelle Ca(2+) cross-talk.
        Mol. Ther. Nucleic Acids. 2020; 19: 1343-1356
        • Hu Y.
        • Jin G.
        • Li B.
        • Chen Y.
        • Zhong L.
        • Chen G.
        • et al.
        Suppression of miRNA let-7i-5p promotes cardiomyocyte proliferation and repairs heart function post injury by targetting CCND2 and E2F2.
        Clin. Sci. 2019; 133: 425-441
        • Luis A.
        The old and the new: prospects for non-integrating lentiviral vector technology.
        Viruses. 2020; 12
        • Shaw A.
        • Cornetta K.
        Design and potential of non-integrating lentiviral vectors.
        Biomedicines. 2014; 2: 14-35
        • Zhuang Z.
        • Xiao q
        • Hu H.
        • Tian S.Y.
        • Lu Z.J.
        • Zhang T.Z.
        • et al.
        Down-regulation of microRNA-155 attenuates retinal neovascularization via the PI3K/Akt pathway.
        Mol. Vis. 2015; 21: 1173-1184
        • Sun L.
        • Zhao M.
        • Zhang J.
        • Lv M.
        • Li Y.
        • Yang X.
        • et al.
        MiR-29b downregulation induces phenotypic modulation of vascular smooth muscle cells: implication for intracranial aneurysm formation and progression to rupture.
        Cell. Physiol. Biochem. 2017; 41: 510-518
        • Bi L.
        • Wacker B.K.
        • Stamatikos A.
        • Sethuraman M.
        • Komandur K.
        • Dichek D.A.
        Jugular vein injection of high-titer lentiviral vectors does not transduce the aorta-brief report.
        Arterioscler. Thromb. Vasc. Biol. 2021; 41: 1149-1155
        • Merentie M.
        • Lottonen-Raikaslehto L.
        • Parviainen V.
        • Huusko J.
        • Pikkarainen S.
        • Mendel M.
        • et al.
        Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.
        Gene Ther. 2016; 23: 296-305
        • Follenzi A.
        • Sabatino G.
        • Lombardo A.
        • Boccaccio C.
        • Naldini L.
        Efficient gene delivery and targeted expression to hepatocytes in vivo by improved lentiviral vectors.
        Hum. Gene Ther. 2002; 13: 243-260
        • Esteban V.
        • Mendez-Barbero N.
        • Jimenez-Borreguero L.J.
        • Roque M.
        • Novensa L.
        • Garcia-Redondo A.B.
        • et al.
        Regulator of calcineurin 1 mediates pathological vascular wall remodeling.
        J. Exp. Med. 2011; 208: 2125-2139
        • Gilbert J.
        • Qian Z.
        • Arsenault J.
        • Aquirre J.
        • Lemos J.
        • Park F.
        936. Vascular tropism of lentiviral vectors using bunyaviral-pseudotyped lentiviral vectors.
        Mol. Ther. 2005; 11: S362
        • Scheideler M.
        • Vidakovic I.
        • Prassl R.
        Lipid nanocarriers for microRNA delivery.
        Chem. Phys. Lipids. 2020; 226104837
        • Dvir T.
        • Bauer M.
        • Schroeder A.
        • Tsui J.H.
        • Anderson D.G.
        • Langer R.
        • et al.
        Nanoparticles targeting the infarcted heart.
        Nano Lett. 2011; 11: 4411-4414
        • Ko Y.T.
        • Hartner W.C.
        • Kale A.
        • Torchilin V.P.
        Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.
        Gene Ther. 2009; 16: 52-59
        • Kiaie N.
        • Gorabi A.M.
        • Penson P.E.
        • Watts G.
        • Johnston T.P.
        • Banach M.
        • et al.
        A new approach to the diagnosis and treatment of atherosclerosis: the era of the liposome.
        Drug Discov. Today. 2020; 25: 58-72
        • Dhanasekara C.S.
        • Zhang J.
        • Nie S.
        • Li G.
        • Fan Z.
        • Wang S.
        Nanoparticles target intimal macrophages in atherosclerotic lesions.
        Nanomedicine. 2021; 32102346
        • Bruch G.E.
        • Fernandes L.F.
        • Bassi B.L.T.
        • Alves M.T.R.
        • Pereira I.O.
        • Frezard F.
        • et al.
        Liposomes for drug delivery in stroke.
        Brain Res. Bull. 2019; 152: 246-256
        • Zhao H.
        • Bao X.J.
        • Wang R.Z.
        • Li G.L.
        • Gao J.
        • Ma S.H.
        • et al.
        Postacute ischemia vascular endothelial growth factor transfer by transferrin-targeted liposomes attenuates ischemic brain injury after experimental stroke in rats.
        Hum. Gene Ther. 2011; 22: 207-215
        • Pecot C.V.
        • Calin G.A.
        • Coleman R.L.
        • Lopez-Berestein G.
        • Sood A.K.
        RNA interference in the clinic: challenges and future directions.
        Nat. Rev. Cancer. 2011; 11: 59-67
        • Fisher R.K.
        • Mattern-Schain S.I.
        • Best M.D.
        • Kirkpatrick S.S.
        • Freeman M.B.
        • Grandas O.H.
        • et al.
        Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications.
        J. Surg. Res. 2017; 219: 136-144
        • Witwer K.W.
        • Buzas E.I.
        • Bemis L.T.
        • Bora A.
        • Lasser C.
        • Lotvall J.
        • et al.
        Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.
        J. Extracell. Vesicles. 2013; 220360
        • Saenz-Pipaon G.
        • Echeverria S.
        • Orbe J.
        • Roncal C.
        Urinary extracellular vesicles for diabetic kidney disease diagnosis.
        J. Clin. Med. 2021; 10: 2046
        • Peterson M.F.
        • Otoc N.
        • Sethi J.K.
        • Gupta A.
        • Antes T.J.
        Integrated systems for exosome investigation.
        Methods. 2015; 87: 31-45
        • Cai J.
        • Gehrau R.
        • Tu Z.
        • Leroy V.
        • Su G.
        • Shang J.
        • et al.
        MicroRNA-206 antagomiRenriched extracellular vesicles attenuate lung ischemiareperfusion injury through CXCL1 regulation in alveolar epithelial cells.
        J. Heart Lung Transplant. 2020; 39: 1476-1490
        • Kim H.
        • Yun N.
        • Mun D.
        • Kang J.Y.
        • Lee S.H.
        • Park H.
        • et al.
        Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes.
        Biochem. Biophys. Res. Commun. 2018; 499: 803-808
        • Herrmann I.K.
        • Wood M.J.A.
        • Fuhrmann G.
        Extracellular vesicles as a next-generation drug delivery platform.
        Nat. Nanotechnol. 2021; 16: 748-759
        • Dasgupta I.
        • Chatterjee A.
        Recent advances in miRNA delivery systems.
        Methods Protoc. 2021; 4
        • Jiang F.
        • Yin F.
        • Lin Y.
        • Xia W.
        • Zhou L.
        • Pan C.
        • et al.
        The promotion of bone regeneration through CS/GP-CTH/antagomir-133a/b sustained release system.
        Nanomedicine. 2020; 24102116
        • Xue X.
        • Shi X.
        • Dong H.
        • You S.
        • Cao H.
        • Wang K.
        • et al.
        Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: an early targeting therapy for myocardial infarction in mice.
        Nanomedicine. 2018; 14: 619-631
        • Gagliardi A.
        • Giuliano E.
        • Venkateswararao E.
        • Fresta M.
        • Bulotta S.
        • Awasthi V.
        • et al.
        Biodegradable polymeric nanoparticles for drug delivery to solid tumors.
        Front. Pharmacol. 2021; 12601626
        • Montgomery R.L.
        • Hullinger T.G.
        • Semus H.M.
        • Dickinson B.A.
        • Seto A.G.
        • Lynch J.M.
        • et al.
        Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure.
        Circulation. 2011; 124: 1537-1547
        • Hinkel R.
        • Penzkofer D.
        • Zuhlke S.
        • Fischer A.
        • Husada W.
        • Xu Q.F.
        • et al.
        Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.
        Circulation. 2013; 128: 1066-1075
        • Fujita M.
        • Otani H.
        • Iwasaki M.
        • Yoshioka K.
        • Shimazu T.
        • Shiojima I.
        • et al.
        Antagomir-92a impregnated gelatin hydrogel microsphere sheet enhances cardiac regeneration after myocardial infarction in rats.
        Regen Ther. 2016; 5: 9-16
        • Kang J.Y.
        • Park H.
        • Kim H.
        • Mun D.
        • Park H.
        • Yun N.
        • et al.
        Human peripheral bloodderived exosomes for microRNA delivery.
        Int. J. Mol. Med. 2019; 43: 2319-2328
        • Simons M.
        • Edelman E.R.
        • DeKeyser J.L.
        • Langer R.
        • Rosenberg R.D.
        Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo.
        Nature. 1992; 359: 67-70
        • Schneider D.B.
        • Sassani A.B.
        • Vassalli G.
        • Driscoll R.M.
        • Dichek D.A.
        Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors.
        J. Vasc. Surg. 1999; 29: 543-550
        • Stamatikos A.
        • Knight E.
        • Vojtech L.
        • Bi L.
        • Wacker B.K.
        • Tang C.
        • et al.
        Exosome-mediated transfer of anti-miR-33a-5p from transduced endothelial cells enhances macrophage and vascular smooth muscle cell cholesterol efflux.
        Hum. Gene Ther. 2020; 31: 219-232
        • Afonso M.S.
        • Sharma M.
        • Schlegel M.
        • van Solingen C.
        • Koelwyn G.J.
        • Shanley L.C.
        • et al.
        miR-33 silencing reprograms the immune cell landscape in atherosclerotic plaques.
        Circ. Res. 2021; 128: 1122-1138
        • Rayner K.J.
        • Suarez Y.
        • Davalos A.
        • Parathath S.
        • Fitzgerald M.L.
        • Tamehiro N.
        • et al.
        MiR-33 contributes to the regulation of cholesterol homeostasis.
        Science. 2010; 328: 1570-1573
        • Rayner K.J.
        • Esau C.C.
        • Hussain F.N.
        • McDaniel A.L.
        • Marshall S.M.
        • van Gils J.M.
        • et al.
        Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides.
        Nature. 2011; 478: 404-407
        • Rayner K.J.
        • Sheedy F.J.
        • Esau C.C.
        • Hussain F.N.
        • Temel R.E.
        • Parathath S.
        • et al.
        Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.
        J. Clin. Invest. 2011; 121: 2921-2931
        • Goedeke L.
        • Salerno A.
        • Ramirez C.M.
        • Guo L.
        • Allen R.M.
        • Yin X.
        • et al.
        Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice.
        EMBO Mol. Med. 2014; 6: 1133-1141
        • Lemarchand P.
        • Jones M.
        • Yamada I.
        • Crystal R.G.
        In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors.
        Circ. Res. 1993; 72: 1132-1138
        • Schulick A.H.
        • Dong G.
        • Newman K.D.
        • Virmani R.
        • Dichek D.A.
        Endothelium-specific in vivo gene transfer.
        Circ. Res. 1995; 77: 475-485
        • Li C.
        • Dou Y.
        • Chen Y.
        • Qi Y.
        • Li L.
        • Han S.
        • et al.
        Site‐specific MicroRNA‐33 antagonism by pH‐responsive nanotherapies for treatment of atherosclerosis via regulating cholesterol efflux and adaptive immunity.
        Adv. Funct. Mater. 2020; 302002131
        • Son D.J.
        • Kumar S.
        • Takabe W.
        • Kim C.W.
        • Ni C.W.
        • Alberts-Grill N.
        • et al.
        The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.
        Nat. Commun. 2013; 4: 3000
        • Kheirolomoom A.
        • Kim C.W.
        • Seo J.W.
        • Kumar S.
        • Son D.J.
        • Gagnon M.K.
        • et al.
        Multifunctional nanoparticles facilitate molecular targeting and miRNA delivery to inhibit atherosclerosis in ApoE(-/-) mice.
        ACS Nano. 2015; 9: 8885-8897
        • Pan Z.
        • Sun X.
        • Ren J.
        • Li X.
        • Gao X.
        • Lu C.
        • et al.
        miR-1 exacerbates cardiac ischemia-reperfusion injury in mouse models.
        PLoS One. 2012; 7e50515
        • Liu M.
        • Li M.
        • Sun S.
        • Li B.
        • Du D.
        • Sun J.
        • et al.
        The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy.
        Biomaterials. 2014; 35: 3697-3707
        • Iaconetti C.
        • Polimeni A.
        • Sorrentino S.
        • Sabatino J.
        • Pironti G.
        • Esposito G.
        • et al.
        Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury.
        Basic Res. Cardiol. 2012; 107: 296
        • Bonauer A.
        • Carmona G.
        • Iwasaki M.
        • Mione M.
        • Koyanagi M.
        • Fischer A.
        • et al.
        MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.
        Science. 2009; 324: 1710-1713
        • Bellera N.
        • Barba I.
        • Rodriguez-Sinovas A.
        • Ferret E.
        • Asin M.A.
        • Gonzalez-Alujas M.T.
        • et al.
        Single intracoronary injection of encapsulated antagomir-92a promotes angiogenesis and prevents adverse infarct remodeling.
        J. Am. Heart Assoc. 2014; 3e000946
        • Surina S.
        • Fontanella R.A.
        • Scisciola L.
        • Marfella R.
        • Paolisso G.
        • Barbieri M.
        miR-21 in human cardiomyopathies.
        Front Cardiovasc Med. 2021; 8767064
        • Yuan J.
        • Chen H.
        • Ge D.
        • Xu Y.
        • Xu H.
        • Yang Y.
        • et al.
        Mir-21 promotes cardiac fibrosis after myocardial infarction via targeting Smad7.
        Cell. Physiol. Biochem. 2017; 42: 2207-2219
        • Roy S.
        • Khanna S.
        • Hussain S.R.
        • Biswas S.
        • Azad A.
        • Rink C.
        • et al.
        MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue.
        Cardiovasc. Res. 2009; 82: 21-29
        • Xu M.
        • Yu T.
        MiR-20b-5p contributes to the dysfunction of vascular smooth muscle cells by targeting MAGI3 in hypertension.
        J. Mol. Histol. 2022; 53: 187-197
        • Lin Z.
        • Murtaza I.
        • Wang K.
        • Jiao J.
        • Gao J.
        • Li P.F.
        miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy.
        Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 12103-12108
        • Kopechek J.A.
        • McTiernan C.F.
        • Chen X.
        • Zhu J.
        • Mburu M.
        • Feroze R.
        • et al.
        Ultrasound and microbubble-targeted delivery of a microRNA inhibitor to the heart suppresses cardiac hypertrophy and preserves cardiac function.
        Theranostics. 2019; 9: 7088-7098
        • Ni Y.G.
        • Berenji K.
        • Wang N.
        • Oh M.
        • Sachan N.
        • Dey A.
        • et al.
        Foxo transcription factors blunt cardiac hypertrophy by inhibiting calcineurin signaling.
        Circulation. 2006; 114: 1159-1168
        • Li N.
        • Hwangbo C.
        • Jaba I.M.
        • Zhang J.
        • Papangeli I.
        • Han J.
        • et al.
        miR-182 modulates myocardial hypertrophic response induced by angiogenesis in heart.
        Sci. Rep. 2016; 621228
        • Zhi Y.
        • Xu C.
        • Sui D.
        • Du J.
        • Xu F.J.
        • Li Y.
        Effective delivery of hypertrophic miRNA inhibitor by cholesterol-containing nanocarriers for preventing pressure overload induced cardiac hypertrophy.
        Adv. Sci. 2019; 61900023