Developing microRNA therapeutics for cardiovascular disease






Eva VAN ROOIJ, PhD
Hubrecht Institute KNAW and
University Medical Center Utrecht
Utrecht, NETHERLANDS

Developing microRNA therapeutics for cardiovascular disease

by E. van Rooij, Netherlands

Despite a detailed understanding of the molecular and cellular processes governing cardiac function and contractility, cardiovascular disease remains the primary cause of morbidity and mortality worldwide. Although numerous treatment options show therapeutic benefit, such as statins, angiotensin-converting enzyme (ACE) inhibitors, and β-blockers, cardiovascular disease continues to increase in prevalence, underscoring the need for new therapeutic strategies. In recent years, prominent roles for microRNAs (miRNAs) have been uncovered in a variety of cardiovasculardisorders. miRNAs are short, single-stranded, noncoding RNAs that anneal with complementary sequences in messenger RNAs (mRNAs), thereby suppressing protein expression. Their known sequence and heightened functions under conditions of pathophysiological stress and disease make them attractive candidates for therapeutic manipulation. Lessons learned from antisense technologies catalyzed opportunities to therapeutically regulate miRNAs by using oligonucleotide chemistries, which by now have proven to be efficacious in targeting pathological miRNAs in animals and even humans. The aim of this review is to discuss the current approaches to target miRNAs, summarize potential therapeutic targets for cardiovascular disorders, and consider the challenges associated with this new therapeutic modality.

Medicographia. 2014;36:319-325 (see French abstract on page 325)



In the last decade, it has become increasingly clear that microRNAs (miRNAs) are relevant players during disease, including cardiovascular disorders. Because of their known and conserved sequence, antisense chemistries, known as anti-miRs, were rapidly developed to target disease-related miRNAs in vivo. By now, anti-miR chemistries have proven to be efficacious in targeting pathological miRNAs in animals and even humans. Here the current state of miRNA therapeutics is discussed and potential hurdles in developing these novel drugs will be contemplated.

miRNA therapeutics

miRNAs are small, noncoding RNAs that negatively influence gene expression in a sequence-dependent fashion. Since individual miRNAs engage numerous messenger RNA (mRNA) targets that are often encoding multiple components of complex intracellular networks, the manipulation of a miRNA can have a profound impact on cellular phenotypes (Figure 1).1 In the last few years, genetic gain- and loss-of-function approaches, as well as pharmacological modulation of individual miRNAs or miRNA families in animal disease models, have shown miRNAs to be important reg- ulators of many diseases, including cardiovascular disease.2,3 Their involvement in disease, combined with their pharmacological properties, has catalyzed efforts to develop miRNAs as novel drug targets.


Figure 1
Figure 1. MicroRNAs (miRNAs) regulate related targets.

miRNAs regulate gene expression in a sequence-dependent fashion. Since individual
miRNAs engage numerous messenger RNA (mRNA) targets that are often
encoding multiple components of complex intracellular networks, the manipulation
of a single miRNA can have a profound impact on cellular or disease phenotypes.
Abbreviations: H, hydrogen; O, oxygen; N, nitrogen; R, radical.
After reference 1: Montgomery and van Rooij. J Cardiovasc Pharmacol. 2011;
57:1-7. © 2011, Lippincott Williams & Wilkins.



Anti-miR chemistries
Based on antisense technologies, to date there are several tools available to selectively target miRNAs. These modified antisense oligonucleotides, anti-miRs, can reduce the levels of pathogenic or aberrantly expressed miRNAs,4,5 and have been shown to be efficacious in both animals and humans.3,6 Because miRNAs typically act as inhibitors of gene expression, anti-miRs will result in a derepression of the mRNAs that are normally targeted by the miRNA.4

There are several key requirements for anti-miRs to achieve effective pharmacological inhibition of disease-associated miRNAs, namely in vivo stability, specificity, and high binding affinity to the miRNA of interest. The modifications that are most commonly used are high-affinity 2’ sugar modifications, such as 2’-O-methyl (2’-O-Me), 2’-O-methoxyethyl (2’-MOE), 2’-fluoro (2’-F), or locked nucleic acid (LNA), in which the 2’- O-oxygen is bridged to the 4’ position via a methylene linker to form a rigid bicycle, locked into a C3’-endo (RNA) sugar conformation.5-7 Another chemical modification applied to confer oligonucleotide stability by nuclease resistance and facilitate cellular uptake is the balance between phosphodiester (PO) and phosphorothioate (PS) linkages between the nucleotides. PS backbone linkages, whereby sulfur replaces one of the nonbridging oxygen atoms in the phosphate group, are more resistant to nucleases than PO, thereby providing more stability to the oligonucleotide. In addition to increasing stability, the PS backbone modifications promote plasma protein binding, thus reducing clearance by glomerular filtration and urinary excretion, which facilitates tissue delivery of anti-miRs in vivo (Figure 2, page 336).3-5,8,9





In 2005, Krutzfeldt et al reported on the first mammalian in vivo study using modified cholesterol-conjugated oligonucleotides complementary to the mature miRNA sequence, called antagomirs, to inhibit miR-122, a liver-specific miRNA.4 Antagomirs are fully 2’-O-Me modified, harbor optimized PS modifications (2 at the 5’ end and 4 toward the 3’ end), and require a >19-nucleotide length for highest efficiency. Their high level of specificity is indicated by the fact that they can discriminate between single nucleotide mismatches of the targeted miRNA. Remarkably, these studies indicated that a single intravenous bolus injection of an antagomir is sufficient to inhibit the function of its target miRNA for weeks, for both specifically expressed miRNAs (like miR-122) and more broadly expressed miRNAs (like miR-16). These experiments also indicated that the anti-miR chemistries do not cross the bloodbrain barrier. These lines of evidence validated the efficacy of antagomirs and founded the basis for the use of antisense oligonucleotides to silence miRNAs in vivo.

More recently, striking data demonstrating the therapeutic power of LNA-modified anti-miRs have been reported in rodents (see Table I [page 337] for cardiovascular examples10-29), nonhuman primates,17,30 and even humans.6 Although all of the 2’ modifications improve nuclease resistance and increase duplex melting temperature (Tm),4 LNAs possess the highest affinity with an increase in Tm of +2°C to +8°C per introduced LNA modification against complementary RNA, and leads to the thermodynamically strongest duplex formation with complementary RNA known due to their high affinity.31,32 It should be noted that, unlike antagomirs, these molecules are unconjugated, indicating that cholesterol conjugation for this chemistry is not required for functional miRNA inhibition. As a consequence of the high binding affinity, biological activity is often already attained with shorter LNA-modified oligonucleotides. Several studies have reported that subcutaneous delivery of high-affinity 15- to 16-nucleotide LNA/DNA mixmers containing roughly 50% LNA bases targeting the 5’ region of the mature miRNA are sufficient to establish a functional effect in vivo. Even tiny 8-mer LNA anti-miRs with a complete PS backbone directed against the seed region of a miRNA (the region most important for target recognition) silence miRNAs in vivo with-out additional conjugation or formulation chemistries.10,31,33,34 To date, all development-stage anti-miR chemistries consist of unconjugated, PS-modified antisense molecules with various additional modifications such as 2’-MOE, 2’-F, or LNA/LNAlike conformationally restricted nucleotides.


Figure 2
Figure 2. Anti-miR chemistries.

Anti-miR chemistries currently use various high-affinity 2’ sugar modifications such as 2’-O-methyl (2’-O-Me), 2’-O-Methoxyethyl (2’-MOE), 2’-fluoro (2’-F), or locked nucleic acid (LNA) conformationally restricted nucleotides. To increase nuclease resistance, most anti-miR chemistries to date harbor phosphorothioate (PS) backbone linkages, whereby sulfur replaces one of the nonbridging oxygen atoms in the phosphate group. Anti-miRs containing cholesterol, conjugated via a 2’-O-Me linkage, named antagomirs, are fully complementary to the mature miRNA sequence with several PS moieties to increase stability. Several unconjugated phosphorothioated antisense molecules with various high-affinity 2’ sugar modifications such as 2’-MOE, 2’-F, or LNA are currently also being used. While all these modifications improve nuclease resistance and increase duplex melting temperature (Tm), the high duplex melting temperature of LNA-modified oligonucleotides enables efficient miRNA inhibition with shorter anti-miRs.
Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymine; U, uracil.
After reference 3: van Rooij and Olson. Nat Rev Drug Discov. 2012;11:860-872. © 2012, Nature Publishing Group.



Pharmacokinetic and -dynamic properties of anti-miRs
The size and charge of anti-miRs limit intestinal absorption, thereby preventing them from becoming good candidates for oral administration.35 Although relatively little is known about the mechanisms of cellular uptake, subcutaneous delivery warrants rapid uptake of anti-miR chemistries in many tissues.

Once inside cells,many modified anti-miRs are extremely stable with half-lives in the order of weeks.26 Additionally, while systemically delivered antagomirs appear to accumulate in a cytoplasmic compartment distinct from processing bodies and induce miRNA degradation by a RNA interference–independent pathway,4,9 LNA-modifiedanti-miRs seemto inhibitmiRNA function by sequestration of the mature miRNA,36,37 implying different modes of action for the different anti-miR chemistries.

For the heart, it is known that anti-miRs reach cardiomyocytes, fibroblasts, and vascular cell populations, but it will be of interest to determine whether some cell types are more efficiently targeted, and whether stress plays a role in cellular uptake and distribution. Additionally, since the currently used doses provide a vast excess of anti-miR copies relative to the miRNA present within the cell, the duration of effects suggests that there is a cellular reservoir that, over time, enables antimiR to inhibit newly formed miRNAs. Subcellular sites of antimiR storage, as well as kinetics of release, may vary depending on chemical modifications of the oligonucleotide. The impact of a miRNA on its target depends on the relative ratio of miRNA to mRNA target. miRNAs display a range of intracellular concentrations, with the most abundant miRNAs being expressed at up to tensof thousandsofcopies per cell.38 Strategies to inhibit miRNA function and thereby derepress expression of their targets are based on the presumption that relatively modest increases in targets are sufficient to evoke significant therapeutic benefits, since individual gene target regulation in response to miRNA modulation in general is relatively modest, with the average change being less than twofold following miRNA inhibition.39,40 The profound effect of some miRNAs suggest that it is the cumulative impact of small changes in expression of myriad targets versus pronounced changes in single targets that mediate the biological actions of miRNAs.

Although the pharmacological effect of an anti-miR is the summation of the derepression of genes that are related in function or contribute to a joint cellular event, it is thought that miRNA function becomes pronounced under conditions of injury or stress. In line with this statement, genetic analyses of miRNAs in mice have revealed relatively minor functions under conditions of homeostasis in controlled laboratory settings, while pronounced effects can be observed under appropriate stress conditions.41 The heightened function of a miRNA during stress can also be explained by changes in abundance of the miRNA, of its mRNA targets, or by differences in miRNA activity under stress,42 whereby both the severity and the type of cellular stress influence whether an mRNA is regulated by a miRNA (van Rooij, unpublished data).

Common toxicities with anti-miR therapeutics
A common source of toxicities with anti-miR chemistries can arise from the chemistry itself and depends on the chemical modifications that are used. PS oligonucleotides, for example, can inhibit the tenase complex in the intrinsic clotting cascade,43 activate the alternative pathway of the complement cascade,44 and activate innate immunity. Additionally, while LNA-containing oligonucleotides have the potential to improve potency for targeting miRNAs, some signs of hepatotoxicity have been observed with LNA-modified antisense oligonucleotides directed against mRNAs measured by serum transaminases, organ weight, and body weight.45 Further toxicity studies of chemically-modified miRNA inhibitors will be required to establish safety parameters for the different anti-miR chemistries, but will likely be chemistry- and even sequencedependent.


Table I
Table I. Therapeutic targeting of cardiovascular microRNAs (miRNAs).

Preclinical rodent and large animal studies have shown that anti-miR–mediated inhibition of specific miRNAs has therapeutic potential in different aspects of cardiovascular disease.
Abbreviations: HDL, high-density lipoprotein; LNA, locked nucleic acid; MI, myocardial infarction; VLDL, very low-density lipoprotein; 2’-F/MOE, 2’fluoro/methoxyethyl.
Modified from reference 3: van Rooij and Olson. Nat Rev Drug Discov. 2012;11:860-872. © 2012, Macmillan Publishers Limited. All rights reserved.



In contrast to many other therapeutic modalities, anti-miR drugs are designed knowing that they will affect all genes that are under the control of the target miRNA. While miRNAs often target many related genes involved in cellular processes, which are intended to be manipulated by the anti-miR therapeutic, a single miRNA will likely also target unrelated genes and possibly produce unexpected (sometimes undesired) changes in gene expression. The potential pleiotropy of miRNA action contrasts with the mechanistic basis of most classical drugs, which actwith maximal specificity against single cellular targets.

Potential sources of toxicity after administration of a miRNA inhibitor can result not only from toxicities induced by the chemistry or unwanted gene changes, but can also arise from effects of the anti-miR on off-target, nondiseased tissues. While some miRNAs have a very cell- or tissue-specific expression pattern, systemic inhibition of more broadly expressed miRNAs may have multiple effects in different tissues, confounding the interpretation of the responses to miRNA-based therapeutics and potentially causing unwanted effects outside the tissue of interest. Caution should be taken when using miRNA therapeutics for more chronic indications and local delivery options should be contemplated.

Potential therapeutic miRNA targets in cardiovascular disease

The rapidly growing knowledge on the functional relevance of miRNAs during heart disease, the shortage of effective therapies, and the ability to potently and specifically regulate mi- RNAs in vivo has catalyzed efforts to explore pharmacological manipulation of miRNAs for the treatment of heart disease.

By now, many preclinical rodent studies have shown effective cardiac delivery and miRNA inhibition using anti-miR chemistries, and have indicated the potent effects of miRNA inhibition under disease conditions (Table I).10-29 Two examples of miRNA targets that are now seriously being considered as clinical candidates for cardiovascular disease are outlined below.

Control of pathological cardiac remodeling by miR-208a
Myosin heavy chain (MHC) is the major contractile protein of striated muscles and α-MHC is the predominant myosin isoform expressed in the adult rodent heart. An intron of this gene encodes miR-208a which, like the host gene, is expressed specifically in the heart,46,4747 Therapeutic inhibition of miR-208a in a hypertension-driven model of heart failure by subcutaneous injection of LNA-modified anti-miR evokes similar benefits in settings of heart disease by potent and functional inhibition of the cardiomyocyte specific miR-208a.26 These findings provide proof-of-concept support for the potential therapeutic benefit of anti–miR-208a inhibitors in the setting of heart disease. Whether oligonucleotide inhibition of miR-208a confers cardiac benefits in forms of heart disease beyond diastolic dysfunction remains to be determined.

Gene profiling studies identified a cohort of predicted mRNA targets that are elevated in expression in response to anti– miR-208a inhibition in vivo, many of which have unknown functions in the heart.26 While such genes serve as sensitive biomarkers of anti-miR efficacy, their unknown functions underscore the challenges associated with establishing the mechanistic basis of therapeutic efficacy of this anti-miR.

Control of cardiomyocyte apoptosis and regeneration by the miR-15 family
The miR-15 family, which includes miR-15a, 15b, 16-1, 16-2, 195, and 497, is broadly expressed, and members have been implicated in cell-cycle arrest and cell survival in a variety of cell types, by regulating many antiapoptotic and cell-cycle genes.48 During heart disease, this family is upregulated in response to cardiac stress and myocardial infarction (MI), which cause death of cardiomyocytes and loss of pump function.49,50 A miRNA family is characterized by the fact that the members have a comparable seed sequence, the main region responsible for target recognition, but differ in their 3’ end. This implies that miRNA family members, in theory, should be able to target comparable mRNAs. However, the divergence in sequence between different members of miRNA families prevents their collective inhibition by delivery of single antisense oligonucleotide inhibitors. One approach to potentially overcome such miRNA redundancy is through the use of tiny LNA inhibitors (Figure 2),3 which target the conserved seed regions of miRNAs and thereby enable inhibition of coexpressed mi- RNA family members that may have redundant biological functions. A recent report showed that an 8-mer directed against the seed region of the miR-15 family was able to target multiple members of the miR-15 family, which in a model of ischemia reperfusion showed that miR-15 family inhibition reduced infarct size and improved cardiac function 2 weeks after ischemic damage.10

Whether inhibition of the miR-15 family exerts a cardioprotective effect through enhanced cardiomyocyte survival, or through improvement of the regenerative capacity of the heart, remains to be determined. However, due to the broad expression pattern of the miRNA family and its involvement in cell proliferation and survival, for more chronic treatment regimens, localized delivery strategies, like cardiac catheters or injectable hydrogel techniques, should be contemplated to prevent unwanted side effects. Recent data in a porcine model of ischemic injury additionally showed a potential advantage to deliver anti-miR to the heart using a catheter. miR-92a, a member of the miR-17-92 cluster, has been implicated in neoan-giogenesis following ischemic injury via the derepression of multiple proangiogenic factors, including integrin subunit α5, a direct target of miR-92a. More recently, catheter-based delivery, but not systemic delivery, of LNA-modified anti–miR-92a in a porcine model of ischemic injury showed a reduction in infarct size, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure, indicating relevance for more directed cardiac delivery approaches.22

Looking to the future

miRNA research has unveiled an unconventional disease mechanism that provides a unique opportunity to exploit an entirely new area of biology. Obviously, there are several significant advantages to miRNAs becoming a new class of drug target. Their small size, and known and conserved sequence, makes them attractive candidates from a development standpoint. Additionally, the direct downstream targets of a single miRNA are commonly related genes that function in a comparable cellular process or signaling cascade. This implies that targeting of a single miRNA will likely result in a dramatic effect, due to the combinatorial effect of gene expression changes in all these related downstream targets. Based on the obvious relevance and importance of miRNAs during disease, there is great enthusiasm for the continued exploration of miRNAs as new drug targets.

With the advent of miRNAs as viable therapeutic targets for many serious health conditions, many new companies, like miRagen Therapeutics, Regulus Therapeutics, and Mirna Therapeutics, have been established to translate the exciting scientific discoveries into real-world, commercial uses. At the same time, some older companies, which had an initial focus on the development of small interfering RNA (siRNA)–based compounds, have begun to explore their proprietary chemistries and delivery systems for the design of miRNA inhibitors and mimics. So far, Santaris Pharma, the Denmark-originated company that developed the LNA chemistry, has been the only company that has used an miRNA inhibitor in phase 2 clinical trials for the treatment of hepatitis C. Although so far no miRNA-based therapeutics for cardiovascular disorders have reached human trials, the promising results in numerous animal models of diseases, such as heart failure, cardiac hypertrophy, fibrosis, and hyperlipidemias, suggest that human data will soon be forthcoming.

While we are taking important steps forward in developing antimiR chemistries as a novel therapeutic, numerous challenges and questions remain in the path toward development of mi- RNA-based therapeutics in general. Currently, there are a lot of unknowns still surrounding the mode of action of the different anti-miR chemistries. Current data suggest that specific chemistries might affect cellular uptake and differ in the degree of blocking of the function of a miRNA. Their long-term effect (up to weeks or even months) indicates a high degree of stability, but also suggests the cell has a reservoir of anti-miR, that with time is slowly released into the cytoplasm to bind to or scavenge the newly formed copies of a miRNA. However, the precise mechanism is still unclear and more in-depth biochemistry will be required to gain more insight. Additionally, in most animal studies to date, the doses used are unlikely to be therapeutically feasible. Follow-up preclinical studies will have to guide appropriate dosing regimens to establish the lowest possible efficacious doses, while attempting to prevent unacceptable side effects.

Despite the unknowns, the interest surrounding miRNAs as novel therapeutic entities is tremendous. The anticipated success of these early forerunners will likely trigger the search for additional miRNA therapeutic targets, innovation in the areas of miRNA inhibitors and mimics, and will advance the search for techniques for efficient in vivo delivery of these therapeutics. While we are eagerly awaiting more efficacy data in human subjects, the next few years promise to provide many more insights into miRNA and anti-miR biology, and will hopefully further strengthen the enthusiasm for this new class of drugs.





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Keywords: anti-miR; cardiovascular disease; microRNA; miR mimic; oligonucleotide; therapeutics