Using microRNA as an Alternative Treatment for Hyperlipidemia and Cardiovascular Disease: cardio-miRs in the Pipeline

Abstract

It is now appreciated that over 90% of the human genome is comprised of non-coding RNAs that have the ability to affect other components of the genome and regulate gene expression. This has galvanized the development of RNA-based therapeutics for a myriad of diseases, including cancer, inflammatory conditions and cardiovascular disease. Several classes of RNA therapeutics are currently under clinical development, including anti-sense oligonucleotides, small interfering RNA and microRNA mimetics and inhibitors. The field of anti-sense technology saw a huge leap forward with the recent FDA approval of the first anti-sense therapy, directed against apolipoprotein B, for the treatment of familial hypercholesterolemia. In addition, recent progress in the development of approaches to inhibit microRNAs has helped to illuminate their roles in repressing gene networks, and also revealed their potential as therapeutic targets. In this review, we summarize these exciting opportunities in the field of drug discovery, with a focus on emerging therapeutics in the field of cardiovascular disease.

Introduction

Cardiovascular disease (CVD) is the number one cause of death globally. According to a 2013 report by the American Heart Association and the Center for Disease Control, one in four deaths in the US are due to conditions associated with CVD such as coronary artery disease (CAD), hypertension, Inflammatory heart disease and stroke¹, and the associated costs are $312.6 billion (US) each year for health care and loss of productivity. The statin class of lipid-lowering agents are widely used to treat CVD as well as to prevent the onset of disease. Although very effective at lowering LDL cholesterol, the outcomes of large-scale clinical trials have shown that statins reduce the risk of subsequent cardiovascular events by only 20%–40%. Furthermore, while statins are generally well-tolerated, side effects such as myopathy occur in approximately 10% of patients receiving treatment² and a proportion of patients are statin resistant (estimates of up to 20%) or intolerant³. Thus, there is a need for new therapies to reduce residual disease burden in these patient populations.

RNA-based therapeutics hold potential as new treatments for human disease. In the last decade there has been an explosion of knowledge in the RNA field. The completion of the recent ENCODE (Encyclopedia of DNA Elements) project, a multi-center effort whose goal was to generate a comprehensive list of functional elements in the human genome by sequencing RNA from a diverse range of sources, identified that over 90% of the human genome is comprised of non-coding RNA that have the ability to affect other components of the genome⁴. Using comparative genomics, integrative bioinformatic methods, and human curation, the ENCODE investigators identified such elements as binding sites for proteins that influence gene activity. Non-coding RNA species have numerous roles and serve to silence stretches of our chromosomes⁵. Several RNA-based therapeutics are currently under clinical development by biotechnology companies, including anti-sense oligonucleotides (ASOs), small interfering RNA (siRNA) and microRNA (miRNA) mimetics and inhibitors (Figure 1). In this review, we will summarize these exciting opportunities in the field of drug discovery, with a focus on emerging therapeutics in the field of cardiovascular disease.

Figure 1. RNA-based therapeutics currently under clinical development.

miRNAs are transcribed from the genome as primary transcripts, and processed by enzymes in the nucleus (Drosha/Pasha) and cytoplasm (Dicer) of the cell resulting in the generation of a mature 20–22 miRNA. The mature miRNA, in association with RISC, binds via its seed sequence to complementary target sites in the 3’UTR of target mRNAs. Oligonucleotide-based therapies targeting these pathways that are under clinical development include: 1. LNA or 2’F/MOE-modified anti-miRs, which act to sequester complementary mature miRNAs and prevent them from binding and repressing target mRNAs, leading to enhanced target gene expression; 2. miR sponges, which bind to 6–8 nucleotide seed sequences of mature miRNAs to also prevent them from binding to target mRNAs, thus leading to increased target gene expression; and 3. miR mimetics, which mimic endogenous miRNAs and act to target mRNAs resulting in a decrease mRNA expression.

Targeting individual genes using anti-sense oligonucleotides and siRNA

ASOs offer promise for the treatment of conditions in which the reduction of a single protein may be beneficial. ASOs are single stranded, highly specific, complementary sequences that target a single gene by interrupting translation of mRNA through the degradation of the transcript by an RNase H cleavage mechanism⁶. This prevents the target mRNA from being translated, thereby reducing the level of the protein by preventing it from reaching the ribosome^7,8. These oligonucleotides are generally 15–23 nucleotides in length and can be chemically modified with the insertion of a 2’methoxyethyl or 2’4’-constrained 2’O-ethyl into the ribose ring to increase their stability and reduce their susceptibility to DNases. ASOs delivered by intravenous (i.v.) infusion or subcutaneous injection accumulate predominantly in the kidney, liver, spleen, adipocytes and bone marrow, but do not cross the blood brain barrier.

The field of anti-sense technology saw a huge leap forward this year with the FDA approval of Mipomersen, an ASO targeting apolipoprotein B (ApoB), an essential component of LDL particles and related atherogenic lipoproteins. This first-in-class drug developed by Isis Pharmaceuticals, now renamed KYNAMRO, is currently approved for treatment of familial hypercholesterolemia (FH)^9–11, a genetic disorder of lipid metabolism that is characterized by the elevation of low-density lipoprotein cholesterol (LDL-C), and increased risk for premature coronary heart disease. KYNAMRO accumulates predominantly in the liver, where it silences apoB mRNA, thereby reducing hepatic apoB-100, and total and LDL-C, in a dose-and time-dependent manner. KYNAMRO therapy results in a 25–50% reduction in LDL-C levels in FH patients on maximally effective lipid therapies. Potential side effects of KYNAMRO and other ASOs include injection site reactions, as well as liver toxicity and hepatosteatosis following chronic use.

The approval of KYNAMRO will no doubt pave the way for other anti-sense drugs and there are currently eight open clinical trials examining the use of ASOs for the treatment of various conditions including cancers and atrial fibrillation (clinicaltrials.gov). Isis Pharmaceuticals has several other promising drug candidates in the pipeline for the treatment of various CVD disorders including an ASO directed against Apolipoprotein CIII (ApoCIII), which is currently being evaluated in a phase 2 clinical trial in patients with hypertriglyceridemia. ApoCIII is a component of very low density lipoproteins (VLDL) and inhibits lipoprotein and hepatic lipase, and acts to reduce hepatic uptake of triglycerides. Elevated levels of ApoCIII are found in patients with dyslipidemia, insulin resistance and metabolic syndrome. Another ASO being developed by Isis Pharmaceuticals targets C-reactive protein (CRP), a gene expressed in the liver that is elevated in response to inflammation. By targeting CRP, this anti-sense therapy may be beneficial for a range of diseases including inflammatory conditions such as Crohn’s disease and rheumatoid arthritis. Other ASOs in development for the treatment of CVD include ASOs targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) (Alnylam, ALN-PCS) and microsomal triglyceride transfer protein (MTTP) (Aegerion Pharmaceuticals – Lomitapide).

RNA interference (RNAi) is a highly conserved endogenous mechanism present in most eukaryotic cells that enables degradation of specific mRNAs. The exploitation of this mechanism using siRNA for therapeutic targeting of single genes attracted a considerable amount of attention in the last decade. Unlike ASOs which are single stranded oligonucleotides, siRNAs are short stretches of double stranded RNA (19–25 bp) from both exogenous and endogenous sources. SiRNAs, are incorporated into the RNA-induced silencing complex (RISC) found in the cytoplasm of the cell and bind to target mRNAs resulting in their degradation by a RISC-dependent mechanism¹². The length of complementarity between an siRNA and its gene target maximizes specificity and minimizes off target effects¹³. RNAi is thought to have evolved as a defense mechanism against invading viral RNA to inhibit the replication of the virus within a cell¹⁴. Despite their early promise, difficulties in the delivery of the siRNAs to the appropriate organ or tissue, as well as injection site reactions and interferon production, have hampered their clinical development¹⁵. Ongoing work is targeted at improving delivery systems for siRNAs to improve their effectiveness.

Targeting gene pathways by manipulating microRNAs

miRNAs are short (20–22 nucleotides) non-coding RNAs that post-transcriptionally regulate target gene expression. Originally discovered in C. elegans by Victor Ambros and Gary Ruvkun in 1993^16,17, there are now 25,141 identified mature miRNAs in 193 species¹⁸, including 2,042 mature miRNAs in humans. Each of these miRNAs has the potential to simultaneously target multiple mRNAs, and repress genes found in the same or similar pathways to alter biological networks¹⁹. Dysregulation of miRNAs in disease states can thus alter gene networks, and miRNA replacement therapy or anti-sense inhibition of miRNAs offer the potential to restore gene expression in the cell to the normal state. Furthermore, the characteristic ability of miRNAs to target gene networks, such as those controlling key cellular processes, including insulin signaling or cholesterol efflux, offers a new approach for the treatment of disease by modulating gene pathways rather than single targets.

microRNAs as fine tuners of gene expression

miRNAs are transcribed from intergenic or intronic regions of the genome as hairpin-containing primary RNA transcripts. Once transcribed, the primary miRNA (pri-miRNA) is processed by the nuclear RNA machinery Drosha and DGCR8/Pasha into an ~65 nucleotide precursor stem-loop structure (pre-miRNA) that contains the miRNA and its complementary strand within the stem. The pre-miRNA is exported into the cytoplasm of the cell and further processed by the Dicer enzyme into a 21–23 base-pair RNA duplex. One strand is selected as the leading strand and the other the lagging (star or *) strand²⁰. The mechanism of strand selection remains unclear but it is thought that thermodynamic properties of the strands lead to a greater susceptibility to degradation of one over the other²¹. While the leading strand is often the -5p strand, there are several examples of miRNAs where the 3p strand is more highly abundant, such as miR-27 (microRNA.org). Although the *strand was originally proposed to be degraded, it is becoming clear that both strands can be functional²². These small strands of RNA do not contain start and stop codons that would allow a ribosome to attach and translate it into a functional protein. Instead, they associate with the RISC complex which is composed of Argonaute proteins (Ago1/2) and GW182²³. RISC uses the strand of miRNA as a template to recognize messenger RNA (mRNA) that has a 6–8 nucleotide sequence complementary to the miRNA seed sequence (miRNA nucleotides 2 through 7 or 8). The binding of the miRNA to target mRNAs generally occurs in the 3’ untranslated region (UTR) of the mRNA but miRNAs can also bind to the coding region, as well as the 5’ UTR^24–26. The base-pairing of miRNA to mRNA ultimately leads to inhibition of protein translation and/or mRNA destabilization and degradation^27,28. There have been conflicting reports as to which comes first, translation inhibition or mRNA decay. Mammalian miRNAs have been reported to decrease target gene mRNA levels and subsequently affect translation²⁹. However, subsequent studies using Drosophila melanogaster and zebrafish as models demonstrated that the effects of miRNAs on translational repression precede the effects on mRNA target deadenylation or decay^30,31.

Predicting microRNA target genes

Current computational approaches estimate that more than 60% of human genes are targeted by miRNAs, and many of these interactions are highly conserved throughout evolution³². Different databases, such as Targetscan, Miranda, PicTar, PITA and miRBase have generated algorithms to predict miRNA/mRNA interactions based on sequence complementarity. Each algorithm considers several “rules” that predict the likelihood of a miRNA finding a successful mRNA binding partner. Nucleotide 1 of the miRNA should have an A nucleotide across from it on the mRNA strand near its polyA tail, nucleotides 2 through 8 (the seed sequence) should have perfect base-pairing, nucleotide 9 should have an A or a U across from it on the mRNA and nucleotides 13–16 of the miRNA should have good base pairing with its mRNA target^33–35. The databases also consider the degree of seed sequence conservation between various species, which can be an indicator of evolutionary significance. Despite these rules, each database uses a slightly different algorithm that results in a great deal of false-positives.

Using bioinformatics analysis such as Gene Ontology annotation can provide global insight into the biological function of a particular miRNA in a biological pathway where a miRNA may be acting as an “on/off” switch. Through their ability to regulate many proteins in a pathway at various steps, miRNAs can exert powerful control over processes like cellular differentiation and metabolism^36,37. Identifying legitimate targets of miRNAs has proven to be a daunting task. mRNA regulation by a miRNA only requires an interaction of 6–8 nucleotides and these seed sequences can be found in the genome on average approximately every 4 kilobases, thus a miRNA has the potential to regulate hundreds of genes. Artificially introducing or inhibiting miRNAs has proven useful in providing clues to their functions. Such miRNA overexpression and knockdown studies have revealed that the majority of miRNAs regulate a number of proteins at modest levels of less than 2-fold^19,38. However, these techniques are laborious and require verifying targets on a single gene basis. To overcome these limitations, techniques such as Argonaute HITS-CLIP (Ago-HITS-CLIP), miRNA-biotin pull-down and miRNP immunopurification experiments are now being used to experimentally determine the compendium of mRNAs interacting with a specific miRNA^39–42.

Therapeutic inhibition of microRNAs

Although miRNAs exert subtle effects on individual gene targets, the cumulative effect of miRNA targeting of multiple genes in a pathway is significant and produces measurable phenotypic results. The ability to target single miRNAs and alter the expression of gene networks provides a unique approach to drug development that moves beyond the “one-drug-one-target” mode of treatment. A promising approach is the use of anti-miR oligonucleotides that are chemically modified to enhance target affinity, stability and tissue uptake⁴³. Anti-miR oligonucleotides can be adapted with locked nucleic acids (LNAs) which have high binding efficiencies and improved stability with the addition of a methylene link between the 2′-oxygen and the 4′-carbon resulting in a “locked” position and reducing the flexibility of the ribose ring⁴⁴. Other chemical modifications such as 2’methoxyethyl (MOE) and 2’4’-constrained 2’O-ethyl (cEt), into the ribose sugar ring of a nucleotide have also improved the pharmacokinetics of ASO clinical candidates. Unlike their double-stranded counterparts, single stranded oligonucleotide inhibitors can be formulated in saline for subcutaneous or intravenous delivery and do not require lipid-based delivery systems. Cholesterol analogs have been added to anti-miRs in an attempt increase cellular uptake, and this promotes their incorporation into low and high density lipoproteins (LDL and HDL)^45–47. Preclinical studies in mice and non-human primates have shown that upon systemic delivery, these compounds rapidly leave the plasma and are taken up by multiple tissues, most prominently liver, spleen, kidney, adipose tissue and bone marrow^48,49. Once taken up by cells, the anti-miR forms a stable, high-affinity bond with the miRNA reducing the availability of the endogenous miRNA for binding to the 3’UTR of the mRNA target.

The use of chemically modified anti-miRs has proven therapeutically beneficial in mouse models of cardiac dysfunction, mouse tumor models and hepatitis C virus (HCV) infection in non-human primates^50,51. The most advanced miRNA inhibition program is directed against miR-122 which targets HCV RNA and facilitates viral replication in the host cell⁵². Studies using an LNA-modified ASO directed against miR-122 showed long-lasting suppression of HCV viremia in non-human primates, with no evidence of viral resistance or side effects⁵³. Santaris Pharma initiated a phase 2a study in humans to assess the safety and antiviral activity of this anti-miR, now called Miravirsen, in HCV patients who had never received treatment⁵⁴. Miravirsen demonstrated dose-dependent antiviral activity when given as a 4-week monotherapy that was maintained for more than 4 weeks after the end of therapy. Furthermore, HCV RNAs in four of nine patients treated with the highest doses of Miravirsen became undetectable during the study⁵⁵, demonstrating the potential of anti-miR therapeutics. Silencing and overexpressing miR-122 has also been shown to have an impact on plasma lipoproteins⁵⁶. Overexpression of miR-122 increases the expression of several cholesterol-synthesis genes including Hmgcrs1, Sqle and Dhrc7 thus increasing cholesterol synthesis⁵¹ and conversely inhibitors of miR-122 decrease hepatic expression of genes implicated in cholesterol synthesis and triglyceride metabolism, resulting in reduced plasma cholesterol. However, the direct targets of miR-122 that confer this effect on cholesterol synthesis and metabolism pathways remain elusive.

miRNA sponges or decoy transcripts are another approach for inhibiting miRNAs in the cell⁵⁷ and these molecules act as competitive inhibitors of the miRNA of interest. miRNA sponges contain multiple binding sites that are complementary to the seed sequence of a miRNA of interest. The sponge binds to the miRNA of interest so it can no longer bind to its targets. A potential disadvantage of the miRNA sponge approach is the difficulty in determining the dosage. If a miRNA is highly expressed, a potentially unfeasible dose of sponge may be needed to silence it, and also where there is an abundance of miRNA target genes, a much lower dose of sponge will be required to silence the miRNA. Notably, when vectors containing miRNA sponges were transfected into cells, miRNA target genes were as derepressed as when anti-miR oligonucleotides were used⁵⁸. miRNA sponges can be delivered using viral vectors, and their expression can be made to be inducible in a specific cell type or developmental stage by using specific promoters.

Delivery of microRNA mimetics

miRNA mimetics may be used therapeutically to overexpress a miRNA leading to the downregulation of its target genes. Mimetics could be used to reconstitute a miRNA that is downregulated during disease (many miRNAs are decreased in cancer⁵⁹), or to decrease gene pathways involved in the pathology of the disease. The delivery of therapeutic miRNA molecules in vivo faces many of the same challenges as siRNAs due to the double-stranded nature of the molecule. Drug delivery vehicles such as liposomes, polymeric micelles and lipoprotein-based drug carriers are being developed to deliver these oligonucleotides to cells. A model drug delivery vehicle should be non-toxic, biodegradable and avoid recognition by the host’s immune defense mechanisms. Interestingly, endogenous HDL particles were recently shown to be associated with miRNAs in the circulation and to deliver their miRNA cargo to other cells⁶⁰. Thus, lipoprotein-based drug carriers like HDL particles also hold potential to deliver miRNA mimetics to specific target tissues such as the liver. One of the biggest challenges associated with miRNA replacement technology is the ability to target miRNAs to a specific tissue. Multiple doses of a miRNA mimetic may be required to provide sufficient miRNA levels in target tissues to achieve sustained target repression.

Viruses are also being used as gene delivery carriers for miRNAs, including short hairpin RNAs that can be processed in the target cell into the mature miRNA. There are several examples of preclinical studies in mice using viral vectors to either overexpress or inhibit miRNAs⁶¹. In a study relevant to cardiovascular disease, lentiviral delivery was used to overexpress and inhibit miR-33, which targets ABCA1, a key gene involved in cellular cholesterol efflux. Lentiviral delivery of miR-33 or anti-miR-33 to the liver resulted in decreased and increased expression of hepatic ABCA1 respectively, and this correlated with changes in plasma levels of HDL-C⁶².

Subsequent studies using ASOs delivered subcutaneously showed similar results⁶³, and thus a viral delivery system seems unlikely to be used clinically. However, for delivery of miRNAs to other tissues, viral vectors may confer an advantage. Recent approaches to tailor miRNA delivery to a specific tissues such as cardiac muscle involves incorporating target sites for tissue-specific miRNAs, such as liver specific miR-122 and miR-192⁶⁴, which would silence the vector in the liver, but allow it to be functional in other tissues. In all such strategies, the dosing of mimetics must be carefully considered, as the effects of mimetics are dependent upon the availability of RISC complexes, which may become saturated with overexpression. There is also the possibility of competition between miRNAs and shRNAs for limiting cellular factors required for the processing of various small RNAs such as RISC and the exportin-5 protein used to transport RNA species from the nucleus to the cytoplasm.

The Cardio-miR Pipeline

Several biopharmaceutical companies are leading the discovery race toward RNA therapeutics to treat cardiovascular disease. miRagen Therapeutics’s lead program, MGN-9103, a LNA-modified ASO against a cardiac-specific miRNA, miR-208, has shown benefits in cardiac dysfunction and may have therapeutic potential in a variety of metabolic disorders that contribute to metabolic syndrome⁶⁵. Cardiac-specific pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance through the targeting of MED13, a component of the mediator complex which is responsible for bridging DNA bound transcription factors and RNA polymerase II ^66,67.

Regulus Therapeutics Inc. is developing an anti-miR oligonucleotide against miR-33a/b for the treatment of atherosclerosis⁶³. As described above, miR-33 targets genes involved in cellular cholesterol efflux and HDL metabolism, such ABCA1, as well as genes involved in fatty acid oxidation, including CROT, CPT1, and HADHB. Subcutaneous delivery of 2’F/MOE-modified anti-miR-33 to mouse and non-human primate models increased plasma HDL cholesterol levels by 35–50%⁶⁸. Furthermore, anti-miR-33 was shown to directly target macrophages in mouse atherosclerotic plaques, and to cause regression of atherosclerosis characterized by a 35% reduction in plaque size and decreases in macrophages and inflammatory gene expression⁶⁸. Whether these effects were due to increases in circulating HDL or direct effects on the plaque macrophages are yet to be determined. Notably, no adverse effects of anti-miR-33 were observed in preclinical models in which this anti-miR was delivered for up to 12 weeks, as assessed by body weight, liver enzymes, plasma cytokine levels, blood chemistry panels and blood counts. This suggests that anti-miR-33 treatment may be a promising therapeutic to treat dyslipidemias that promote atherosclerosis.

With the continued obesity epidemic there is an urgent need for new therapeutics for type 2 diabetes and its associated co-morbidities. The recent discovery of miR-103 and miR-107 as key regulators of insulin sensitivity and obesity identifies these miRNAs as potential therapeutic targets in the treatment of type 2 diabetes, obesity and associated metabolic diseases⁶⁹. These miRNAs were identified from a screen of miRNAs dysregulated in the livers of leptin-deficient (ob/ob) or diet-induced obese mice. Anti-miR-based silencing of these miR-103 and miR-107 in obese mice led to improved glucose homeostasis and insulin sensitivity through the targeting of caveolin-1 (Cav-1)⁶⁹. The upregulation of Cav-1 led to increased expression of the insulin receptor and insulin signaling, as well as decreased adipocyte size and enhanced insulin-stimulated glucose uptake. Thus, manipulation of miR-103/107 may offer a new therapeutic approach to treating insulin resistance, however more research is needed to understand the roles of these miRNAs in normal human physiology and disease.

Another area of promise for miRNA therapeutics is the treatment of fibrosis. Cardiac fibrosis is a condition where there is an abnormal thickening of the heart valves due to uncontrolled proliferation of fibroblasts, which can lead to heart failure. miR-29 is implicated in cardiac fibrosis and has been found to be downregulated following myocardial infarction in the region of the heart next to the infarct⁷⁰. This miRNA has a variety of targets involved in fibrosis such as collagens, fibrillins and elastin that are upregulated in response to MI and resulting fibrosis. Because miR-29 is decreased in response to cardiac injury, miRagen Therapeutics is developing a so-called pro-miR called MGN-4220 that targets multiple components of the fibrosis pathway.

Cardiac fibroblasts have elevated miR-21 levels leading to a decrease in its target mRNA, Sprouty-1 (Spry1), a negative regulator of ERK-MAPkinase activity and fibroblast growth factor-2 (FGF2) secretion⁷¹. Regulus Therapeutics Inc. and Sanofi Aventis are developing an ASO to miR-21 that would elevate Spry1 expression, reduce FGF2 and thus decrease fibroblast growth. Preclinical studies in a rodent model of Alport syndrome show that anti-miR-21 reduces the severity of kidney fibrosis and improves kidney function⁷². However, genetic deletion of miR-21 did not offer protection to mice undergoing cardiac stresses^73,74, suggesting potential miRNA redundancies, or off-target effects of the anti-miR, possibilities that need to be further investigated. Despite these conflicting reports, anti-miR-21 may have the potential to treat a variety of fibrotic conditions, including cardiac fibrosis.

A number of miRNAs found to be upregulated in the heart following myocardial infarction are also potential drug targets. The miR-15 family of miRNAs, including miR-15, miR-16, miR-195 and miR-497 has been implicated in cell cycle arrest and survival through the regulation of anti-apoptotic and cell cycle genes⁷⁵. These miRNAs are upregulated following myocardial infarction, causing loss of cardiomyocytes and cardiac pump function and overexpression of miR-195 in the heart was sufficient to cause heart failure⁷⁶. However, targeting a family of miRNAs that share targets genes can be challenging. Each of the miR-15 family members has a slightly different sequence and this divergence means that a single ASO will likely be ineffective at overcoming this redundancy. miRagen Therapeutics is developing an anti-miR towards miR-15/195 called MGN-1374 for post-MI remodeling of the heart. Interestingly, a recent study using an 8-mer (nucleotide) directed against the seed region of the miR-15 family was more effective in derepression of target genes than an LNA-modified 16-mer⁷⁵.

Several other miRNAs have been used in pre-clinical models of cardiac diseases, and show promise for future development. miR-155 has been implicated in viral myocarditis which results from cardiotropic viruses ^77,78. An LNA-anti-miR directed against miR-155 delivered to mice derepressed the target gene PU.1 and resulted in reduced infiltration of monocytes, decreased T-cell activation and diminished myocardial damage during myocarditis. Furthermore, miR-145 has been linked to pulmonary hypertension, which can lead to heart failure and miRagen Therapeutics is developing an anti-miR therapy that has shown potential in reducing pulmonary vascular remodeling in response to hypoxia⁷⁹. Genetic deletion of miR-145 resulted in excessive right ventrical remodeling and reduced blood pressure suggesting that timing and dosing need to be optimized for the anti-miR-145 therapy. Finally, the miR-34 family of miRNAs is upregulated in the heart in response to stress, including myocardial infarction. miRNA Therapeutics Inc. is developing an LNA-modified anti-miR against miR-34a that has demonstrated the ability to improve systolic pressure and increase angiogenesis and Akt activity ⁸⁰. These effects were accompanied by an increase in mir-34a target genes Notch1 and Semaphorin 4B.

The Future of the Pipeline

The health and financial burdens that cardiovascular disease presents to the global population is vast and daunting. It was once thought that cardiovascular tissues would be difficult to target with gene therapies. miRNA therapeutics offer hope for the development of novel treatments for CVD. The preclinical studies described in this review illustrate the early success of miRNA inhibitors and mimics. The recent FDA approval of Mipomersen/KYNAMRO, a first-in-class ASO inhibitor that targets apoB leading to the reduction of LDL-C for the treatment of homozygous familial hypercholesterolemia¹⁰, opens the door to other oligonucleotide-based therapies, bringing miRNA therapeutics one step closer to the clinic.

Table 1.

Current RNA-based therapeutic approaches

RNA Technology	Components	Target RNA	Method of Delivery	Mechanism
Anti-sense oligonucleotide (ASO)	Single-stranded oligonucleotide (15-23nt) with chemical modifications	mRNA	Subcutaneous or intravenous injection of ASO	Cleavage of target mRNA by RNase H
siRNA	Double-stranded RNA (19-25bp)	mRNA	Requires lipid-delivery vehicle	Cleavage of target mRNA by RISC
Anti-miR oligonucleotide	Single-stranded RNA (15-19nt) with chemical modifications	Mature miRNA	Subcutaneous or intravenous injection of oligo.	Sequesters mature miRNA inhibits from binding target mRNA
miR mimic	Double-stranded RNA (20-23bp)	mRNA	Requires lipid-delivery vehicle	Cleavage of target mRNA by RISC

Table 2.

The Cardio-miR pipeline

miRNA	Disease Indication	Drug Design	Target Tissue	Potential Target Gene	Functional Outcome	Company	Ref.
miR-122	HCV, hyperlipidemia	Miravirsen (LNA modified anti-sense oligonucleotide)	Liver	Hmgcrs1, Sqle and Dhrc7	Suppression of HCV viremia, reduces plasma cholesterol levels	SantarisPharma	51–56
miR-208	Obesity, diabetes, metabolic syndrome	MGN-9103 (LNA modified anti-sense oligonucleotide)	Cardiac-specific	MED13	Resistance to high-fat diet-induced obesity, improves systemic insulin sensitivity and glucose tolerance	miRagen Therapeutics	65–67
miR-33	Atherosclerosis	2’F/MOE-modified anti-sense oligonucleotide; anti-miR-33lentivirus	Liver	ABCA1	Raised HDL and induced regression of atherosclerotic plaques	Regulus Therapeutics, AstraZeneca	62,63,68
miR-103/107	Obesity, Diabetes	2′-OMe-modified anti-miR with phosphorothioate linkage and cholesterol linked	Liver	Caveolin-1	Improved glucose homeostasis,	Regulus Therapeutics, Alnylam	69
miR-29	Fibrosis	Mimetic molecule (lead strand - 2’ F nucleosides at every pyrimidine residue; lagging strand - 2 5’ terminal 2-OMe residues)	Heart	ELNF, BN1, COL1A1, COL1ACOL3A1	Inhibition of miR-29 induces fibrosis, overepxression leads to decrease in collagen genes and subsequent fibrosis	Miragen Therapeutics	70
miR-21	Fibrosis	2′-OMe-modified anti-miR with phosphate backbone	Kidney	PPARα, Spry1	Reduces severity of kidney fibrosis, improves renal function only in response to injury	Regulus Therapeutics, Sanofi Aventis	71–74
miR-15 family	Myocardial Infarction	MGN-1374 (LNA modified anti-sense oligonucleotide); 8-mer LNA oligonucleotide targeting seed region	Heart	Pdk4, SGK1, Bcl2, Arl2	post-MI remodeling	Miragen Therapeutics	75,76
miR-155	viral myocarditis	LNA-anti-miR-155	Cardiac Monocytes/ macrophage	PU.1	Reduced infiltration of cardiac monocytes and macrophages, T-cell activation and myocardial damage		77,78
miR-145	pulmonary hypertension	LNA-anti-miR-145	Lung and cardiac tissues		Potential in reducing pulmonary vascular remodeling in response to hypoxia	Miragen Therapeutics	79
miR-34a	Myocardial Infarction	LNA-anti-miR-34a	Cardiac tissues	Notch1, Sema4b	Improve systolic pressure and increase angiogenesis and Akt activity	miRNA Therapeutics Inc.	80