Modulation of Polycystic Kidney Disease by non-coding RNAs

Abstract

PURPOSE OF REVIEW:

microRNAs (miRNAs) are a class of small, evolutionarily conserved, non-coding RNAs (ncRNAs) that function as inhibitors of post-transcriptional mRNA expression. They are implicated in the pathogenesis of numerous diseases, including many common kidney conditions. In this review, we focus on how miRNAs impact autosomal dominant polycystic kidney disease (ADPKD) progression. We also discuss the feasibility of the emerging novel antisense oligonucleotides (ASOs) drug class, which includes anti-miRNA drugs, for the treatment of ADPKD.

RECENT FINDINGS:

Aberrant miRNA expression is observed in multiple PKD murine models and human ADPKD samples. Gain and loss-of-function studies have directly linked dysregulated miRNA activity to kidney cyst growth. The most comprehensively studied miRNA in PKD is the miR-17 family, which promotes PKD progression through the rewiring of cyst metabolism and by directly inhibiting PKD1 and PKD2 expression. This discovery has led to the development of an anti-miR-17 drug for ADPKD treatment. Other miRNAs such as miR-21, miR-193, and miR-214 are also known to regulate cyst growth by modulating cyst epithelial apoptosis, proliferation, and interstitial inflammation.

SUMMARY:

miRNAs have emerged as novel pathogenic regulators of ADPKD progression. Anti-miR-based drugs represent a new therapeutic modality to treat ADPKD patients.

INTRODUCTION

The vast majority of the genome contains regions that do not code for proteins (1). Historically termed as the ‘junk DNA,’ it is now known that a significant portion of this DNA undergoes transcription to give rise to non-coding RNAs (ncRNAs) with pivotal functions (1). These RNAs, as the name suggests, do not encode for proteins but rather remain as RNAs during their lifetime and regulate gene/mRNA expression and mRNA metabolism.

ncRNAs are of two types - long ncRNAs (IncRNAs) that are >200 nucleotides in length and short ncRNAs of <30 nucleotides in length (2). There are three major classes of short ncRNAs - miRNAs, siRNAs, and piRNAs (3). Amongst the ncRNAs, miRNAs the most are widely studied. Compelling evidence supports the idea that miRNAs are novel regulators of disease pathogenesis (4, 5). First, aberrant miRNA expression/function is observed in numerous diseases such as cancer, tissue fibrosis, congenital and developmental conditions, and various monogenetic disorders including polycystic kidney disease (PKD). Second, gain and loss-of-function approaches causally link abnormal activity of many miRNAs to the progression of these diseases. Third, the pathogenic miRNAs contribute to the dysregulation of large gene networks in various diseases. Finally, these observations have led to the realization that miRNAs are novel drug targets for the treatment of many complex diseases. Indeed, multiple miRNA-based drugs have now advanced to human clinical trials for the treatment of cancer, tissue fibrosis, and two monogenic kidney diseases – Alport syndrome and autosomal dominant polycystic kidney disease (ADPKD).

PKD is amongst the most common human monogenic disease caused primarily by mutations in PKD1 or PKD2. The clinical hallmark of this disorder is the massive bilateral kidney enlargement due to innumerable cysts. The cysts are lined by hyperproliferative and hypersecretory cyst epithelial cells, which cause uncontrolled cyst growth and expansion, ultimately resulting in kidney failure. Several signaling pathways, including cAMP, mTOR, and cMyc have been known for a long time to drive PKD progression (6). Recent work indicates that miRNA-mediated signaling is a key new driver of PKD pathogenesis (7–10). This review focuses on the progress made so far in our understanding of the role of miRNAs in PKD with an emphasis on the therapeutic targeting of these ncRNAs in PKD treatment.

miRNA biogenesis and mechanism of action

miRNAs are ~22 nucleotides double-stranded RNA molecules and were first discovered in 1993 in C.elegans as regulators of larvae patterning (11, 12). Since this initial discovery, it has become clear that miRNAs are integral to virtually all aspects of mammalian biology ranging from the initial stages of embryo formation, stem cell biology and organ development to immune response, tissue injury/repair, aging, and disease progression (13, 14).

miRNAs are produced either from intragenic (located within a protein-coding gene) or intergenic loci (15, 16). The miRNAs encoded from intergenic loci have independent promoters and therefore function as free-standing miRNA genes. In contrast, the intragenic miRNAs are typically located in the introns of known protein-coding genes, although some miRNAs are generated from the exonic regions. While most intragenic miRNAs are co-transcribed with the host genes and are eventually spliced out of the mRNA product, some intragenic miRNAs have their independent regulatory elements and are transcribed separately from their host genes. Nearly half of all human miRNAs are encoded as polycistronic transcripts that generate multiple miRNAs (17).

Biogenesis of miRNAs begins in the nucleus where RNA Polymerase II/III binds to miRNA genes and transcribes a long single-stranded RNA (Figure 1) (18). The formation of a mature miRNA from this long transcript occurs through the canonical pathway or the non-canonical pathway of miRNA biogenesis. In the canonical pathway, transcription of a miRNA gene results in a long single-stranded RNA termed as the primary-miRNA (pri-miRNA). The pri-miRNAs fold into a secondary structure of double-stranded RNA (dsRNA) and hairpin loops. This substrate is cleaved into smaller pre-miRNAs by the microprocessor complex consisting of an RNA binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) and a ribonuclease III enzyme, Drosha. The pre-miRNAs translocate to the cytoplasm with the help of the exportin 5 (XPO5)/RanGTP complex. Once in the cytoplasm, the pre-miRNAs are processed by the RNase III endonuclease, Dicer, to form a ~22 nucleotides long duplex miRNA. This small double-stranded RNA is known as the mature miRNA duplex. The next step in miRNA biogenesis is the formation of the miRNA-induced silencing complex (miRISC), a multiprotein complex including the Argonaute (AGO) family of proteins. The miRNA duplex is transferred to a member of the AGO protein family. AGO chooses a “guide” strand based on the thermodynamic stability of the strand overhangs. The other strand becomes the “passenger strand,” which is usually degraded. The guide strand incorporation into AGO proteins prepares the miRISC for mRNA silencing (Figure 1). The less common biogenesis pathways are Drosha independent or Dicer independent pathways (19). In these pathways, miRNA substrates that are ready for loading on to the miRISC are produced via transcription without the need for post-transcriptional processing by Drosha or Dicer.

Figure 1. Biogenesis of miRNAs.

Transcription miRNA genes by RNA Polymerase II enzyme creates a long RNA transcript called primary-miRNA (pri-miRNA). The pri-miRNA folds into a secondary structure comprised of double-stranded RNA (dsRNA) and hairpin loops. The microprocessor complex (DROSHA and DGCR8) cleaves the pri-miRNA into a shorter pre-miRNAs. The pre-miRNAs are exported out of the nucleus by Exportin-5. In the cytoplasm, the RNAse III enzyme DICER processes the pre-miRNAs to form the 22~ nucleotide double-stranded mature miRNA. The mature miRNA guides the catalytic activity of Argonaute proteins, thus creating the RNA induced silencing complex (RISC).

miRNA-mediated repression of target mRNAs occurs in the miRISC. The miRISC scans target mRNAs to find sequences that are complementary to the miRNA seed sequence. The seed sequence refers to a stretch of 2-8 nucleotides at the 5’ end of a miRNA. Once Watson-Crick base pairing between the target mRNAs and the miRNA occurs, depending on the degree of complementarity, the mRNA targets are either degraded or translationally inhibited via the catalytic activity of the miRISC (20). miRNAs that have identical seed sequences target the same mRNAs and, therefore, are categorized as belonging to one family. The miRNA-binding sequence on the target mRNAs is termed the miRNA response elements (MREs) and is usually located in the 3’ UTR of mRNAs. Multiple mRNAs can have the same MREs and, thus, can be regulated by the same miRNA/miRNA family.

miRNAs in kidney development and homeostasis

The embryonic kidney consists of two precursor tissues: the metanephric mesenchyme and the ureteric bud (21–23). Molecular signals from the ureteric bud induce the nephron progenitor cells of the metanephric mesenchyme to undergo differentiation into renal vesicles, which eventually give rise to glomeruli and nephrons (21,22). Conversely, the metanephric mesenchyme induces branching morphogenesis of the ureteric bud, which differentiates into the collecting duct network (23). During this process, the nascent nephrons and the collecting duct branches connect via a common tubular lumen and undergo remarkable morphogenesis, eventually attaining proper segmentation, tubule length, and luminal diameter (24–26). This reciprocal cross-talk between the metanephric mesenchyme and the ureteric bud is repeated innumerable times to produce ~1 million nephrons and collecting duct branches (27, 28).

Functional Dicer/Drosha gene knockout studies in distinct lineage-specific cell types of the embryonic kidney show that the miRNA pathway is critical for every stage of kidney development (29, 30). Removal of miRNAs from the metanephric mesenchyme causes premature depletion of nephron progenitors leading to renal agenesis (31–33). Similarly, the ureteric bud fails to undergo branching in the absence of miRNAs, also causing renal agenesis (31). We have found that miRNAs are required for nascent nephron and collecting duct maturation following initial stages of nephrogenesis(34). The loss of miRNAs from the elongating renal tubules disrupts morphogenesis and causes tubular dilation producing a PKD-like phenotype. Surprisingly, we found that during tubule elongation, miRNAs regulate the dosage of many PKD genes, including PKD1 and HNF-1β. The loss-of-function of these miRNAs causes the tubules to form cysts.

miRNAs also play critical roles in maintaining kidney homeostasis. For example, loss of the miRNA biogenesis pathway from podocytes produces proteinuria, glomerulosclerosis, and kidney failure (35–38). The homeostatic Sanction of miRNAs in postnatal renal tubules has been a bit surprising. The absence of miRNAs from postnatal proximal tubules does not affect their histology or function (39). In contrast, we found that postnatal collecting ducts lacking miRNAs evoke an injury-like response, which culminates into kidney fibrosis, kidney failure, and death (40). Thus, while miRNAs are dispensable for proximal tubule maintenance, they are essential for the normal functioning of collecting ducts. These observations also implicate miRNAs and collecting ducts in the progression of tubulointerstitial fibrosis, the final common histological feature of all forms of chronic kidney diseases.

miRNAs: new regulators of PKD pathogenesis

Numerous miRNAs are aberrantly expressed in murine and human forms of ADPKD. However, only a few have been causally linked to ADPKD progression. In the following sections, we discuss two cyst-promoting (miR-21 and miR-17) and cyst-suppressing (miR-193 and miR-214) miRNAs.

miR-21

miR-21 is an evolutionary conserved and an extensively studied oncogenic miRNA (41). The miR-21 gene is located within the protein-coding TMEM49 gene; however, it is transcribed independently via its own promoter (42) (Figure 2). Multiple genomic regions in TMEM49 introns function as putative promoter regions of miR-21 (43, 44). miR-21 is broadly expressed in many cell types during development and, this includes the kidney epithelium and podocytes. Surprisingly, despite the widespread expression pattern, miR-21 null mice are viable with a normal lifespan indicating that this miRNA is dispensable for development (45). Instead, its physiological function may involve maintaining tissue homeostasis. Indeed, this microRNA is amongst the most commonly upregulated miRNA in response to tissue injury (46), where it is thought to mediate repair and regeneration. However, sustained miR-21 activation is maladaptive, and, rather than aiding repair, it can propagate injury and tissue fibrosis.

Figure 2. Mechanism by which miR-21 and miR-17~92 promote cyst growth.

A. The cAMP/CREB pathway promotes miR-21 transcription via its unique promoter located in the intron of Tmem49. The mature miR-21 inhibits PDCD4 activity resulting in reduced apoptosis of cystic epithelial cells. miR-21 also inhibits PPARa mediated oxidative phosphorylation. RG-012 is an anti-miR that inhibits miR-21 activity. B. The miR-17 miRNA family is derived from the miR-17~92 and two other paralogous miRNA clusters. c-Myc promotes the transcription of the miR-17~92 cluster. The miR-17 family consists of miR-17, miR-20a, miR-20b, miR-106a, miR-106b and miR-93. These miRNAs have the same seed sequence and directly inhibit PKD1, PKD2, and PPARa. RGLS4326 is an anti-miR drug that inhibits the miR-17 family.

With regards to diseases, miR-21 is activated in multiple types of cancers including glioblastoma, chronic lymphocytic leukemia, lymphoma and lung cancer, where it functions as a pro-survival, anti-apoptotic oncogenic miRNA (41, 47, 48). Considering the similarities in the aberrant signaling cascades between cancer and PKD, it is not surprising that miR-21 is also upregulated in the cyst epithelia in both murine and human forms of ADPKD (9, 49). In keeping with what’s known about miR-21 gene regulation, we found that miR-21 activation in ADPKD is indeed mediated via its unique 419-bp conserved promoter region in the 10^th intron of Tmem49 gene (9). The most potent activator of miR-21 in the context of cancer or fibrosis is the TGF-β/SMAD pathway (50–52). In contrast, and perhaps, pointing to some level of specificity, miR-21 is activated in ADPKD models via the cAMP/CREB pathway (9). The cAMP signaling cascade is a well-known driver of cyst growth. In fact, tolvaptan, a vasopressin receptor 2 (V2R) antagonist and the only FDA-approved drug for ADPKD treatment, mediates its efficacy by inhibiting cAMP in collecting ducts (53, 54). We found that cAMP signaling transactivates the miR-21 gene via conserved CREB motifs in the miR-21 promoter region. Mozavaptan, another selective V2R antagonist that also inhibits the cAMP pathway, reduces miR-21 transcription in ADPKD mouse models, further supporting the idea that miR-21 is cAMP/CREB responsive miRNA (9). These observations also suggest that part of V2R antagonist-mediated therapeutic efficacy may be due to a reduction in miR-21 levels.

The validated targets of miR-21 are well-known players of survival and inhibitors of apoptosis (55). Many of these targets including Pdcd4, Jagl, Pten, Spry, Cdc25a and Ppara are expressed in the kidney. As a direct causal link, we observed that miR-21 deletion induces cyst epithelial apoptosis and thereby attenuates PKD progression in the Pkd2-KO mouse model. Thus, similar to cancer, miR-21 functions as a pro-survival signal in PKD. miR-21 appears to mediate this effect by directly repressing the tumor suppressor Pdcd4 (programmed cell death 4), which - as the name suggests - mediates cell death. The role of Pdcd4 in PKD has not been studied, but at least two lines of evidence indicate that it is likely to be a significant contributor to cyst pathogenesis. First, a subset of Pdcd4−/− mice develops spontaneous kidney cysts. Second, Pdcd4 is targeted for proteasomal degradation by mTOR signaling, which in turn is known to aggravate cyst. Thus, miR-21-mediated inhibition of Pdcd4 may represent an entirely new mechanism for cyst growth.

miR-21 has emerged as a novel drug target for a second kidney monogenic disorder called Alport syndrome (56, 57). This condition is caused due to the mutations of COL4A3, COL4A4, or COL4A5 genes and is characterized by glomerular basement membrane defects causing proteinuria, hematuria, and eventually tubulointerstitial fibrosis and kidney failure (58–61). miR-21 genetic deletion or complementary pharmaceutical inhibition using anti-miR-21 compounds attenuates kidney fibrosis progression and improves the survival of Alport murine models (56). Human patients with Alport syndrome show elevated levels of miR-21 in damaged glomeruli and their miR-21 expression level co-relates with kidney function (57). An anti-miR-21 drug known as RG-012 has already entered phase 2 clinical trials for the treatment of Alport syndrome (62). Interestingly, miR-21 mediates its pathogenic effects through direct repression of Ppara and inhibition of fatty acid oxidation (FAO) (9, 63). As expected, RG-012 reverses these effects. Coincidently, reduced Ppara and FAO are also linked to ADPKD progression (64). Thus, in addition to inhibiting cyst epithelial apoptosis, miR-21 may also repress Ppara/FAO in ADPKD, providing a second mechanism for its pathogenic effects. More importantly, RG-012 may provide therapeutic benefit in ADPKD.

miR-17~92 cluster

The miR-17~92 cluster was amongst the first miRNAs found to be amplified in cancerous tissues earning it the moniker ‘oncomir-1’ (65). This finding sparked an intense interest in understanding its role in development and disease. miR-17~92 is a polycistronic miRNA cluster, which produces six individual miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-l, and miR-92a) (66, 67). The cluster is located within a 7 kb non-protein coding gene MIR17HG or C13orf25 (Figure 2) and is evolutionarily conserved amongst vertebrates (68, 69). Interestingly, during evolution, this cluster has undergone genomic duplication producing two paralogous clusters known as miR-106b~25 and miR-106a~363 (70). These closely related clusters collectively produce 15 miRNAs, each belonging to one of four seed families: miR-17, miR-18, miR-19, or miR-25 family (67). Having the same seed sequence implies that they have the same putative mRNA targets.

Amongst the three clusters, miR-17~92 is the most abundantly expressed in diverse cell types. Absence of miR-17~92 causes perinatal lethality with developmental defects in the bone, heart, lungs, and B cells in mice whereas deletion of the miR-106a~363 or miR-106b~25 clusters does not produce any obvious abnormality (71–74). Thus, miR-17~92 is essential, but the other two paralogous clusters are dispensable for embryonic development. miR-17~92 is also required for kidney development. Conditional miR-17~92 deletion in the mouse nephron progenitors impairs nephrogenesis (75, 76). Consistent with its role in development, germline microdeletions involving miR-17~92 underlie Feingold syndrome, a developmental genetic disease primarily characterized by short stature and skeletal abnormalities (72). However, the expression of these miRNAs declines with maturation. Accordingly, inducible deletion of miR-17~92 in adult mice does not impact their lifespan or general well-being, other than a mild reduction in mature hematopoietic lineages. Moreover, miR-17~92 deletion in renal tubules subsequent to nephrogenesis, also, has no impact.

We have found that the miR-17~92 miRNA cluster is transactivated in multiple orthologous models of PKD and in human ADPKD (7, 8). In fact, the miR-17 family alone accounts for > 2.7% of the total dysregulated miRNA pool in ADPKD. The oncogene c-Myc primarily mediates this upregulation. The miR-17~92 gene has many conserved Myc binding sites. c-Myc binding to the miR-17~92 promoter is both sufficient and necessary for miR-17~92 upregulation in ADPKD. Transgenic c-Myc upregulation is known to cause PKD in mice. Consistent with the c-Myc → miR-17~92 pathogenic axis in ADPKD, transgenic upregulation of miR-17~92 in wild-type kidney tubules is also sufficient to produce kidney cysts. More importantly, genetic deletion of the miR-17~92 cluster markedly attenuates disease progression and improves renal function and survival of multiple ADPKD mouse models (7, 8). These proof-of-principle genetic studies have conclusively established miR-17~92 as a novel drug target for ADPKD.

Therapeutically targeting miR-17~92 is complicated. First, as mentioned earlier, this cluster produces miRNAs belonging to four families (67). It will be difficult to develop one drug that simultaneously inhibits all four families. Second, recent studies suggest functional specialization among the various miRNA families derived from this cluster. For example, the miR-19 family drives oncogenic transformation in lymphoma models, whereas the other families appear to play no role in this process. On the other hand, miR-17 family regulates skeletal development whereas miR-18, miR-19, or miR-25 have little impact (71). Thus, it is essential to identify the family that is the crucial pathogenic driver in diseases where these clusters are amplified. To move a step closer to drug development, we recently dissected the role of various miR-17~92 cluster families in ADPKD pathogenesis (77). We designed LNA-modifed anti-miRs against the individual miRNA families of the six-member miR-17~92 cluster. Interesting, treating Pkd1-KO mice with anti-miR-17, but not anti-miR-18, anti-miR-19 or anti-miR-25 LNAs, attenuated cyst growth. Thus, the miR-17 family appears to be the primary driver of disease progression in ADPKD. These observations have provided the scientific rationale for targeting the miR-17 family for ADPKD treatment. Indeed, recently, we developed a novel anti-miR-17 drug called RGLS4326 for clinical testing in humans with ADPKD (discussed in detail in subsequent sections).

miR-17 is a pro-proliferative miRNA (78). The validated targets of miR-17 regulate almost all molecular pathways of cellular growth and proliferation (65, 79). Accordingly, its deletion or pharmaceutical inhibition reduces cyst proliferation (7, 8, 77). Mechanistically, two major molecular themes are proposed to explain the pathogenic effects of miR-17 in ADPKD. The first theme involves the rewiring of cyst metabolism. It is now well established that similar to many types of cancers, cyst epithelial metabolism is reprogrammed - not so much to produce ATP - but rather to maximize the usage of nutrient carbon for building blocks needed by dividing cells, such as nucleotides and lipids (80–82). The TCA cycle and oxidative phosphorylation pathways, which maximize ATP production, are downregulated in ADPKD. Conversely, aerobic glycolysis, which is an inefficient way to generate ATP but produces ample carbon for anabolic biosynthetic purposes, is activated. miR-17 appears to aid in this metabolic transition. We have reported that miR-17 directly represses PPARA, thereby reducing the ATP generating mitochondrial pathways (8, 10). Accordingly, miR-17~92 deletion or anti-miR-17 treatment upregulates PPARA and improves the mitochondrial function of cyst epithelia. Moreover, activating PPARA function independent of miR-17 is sufficient to attenuate cyst growth indicating that the c-Myc → miR-17 —| PPARA axis is a novel mechanism for cyst growth. However, the metabolic footprint of miR-17 in ADPKD is likely to be much larger than its effects of PPARA. miR-17 was recently shown to enhance glycolysis and glutaminolysis in c-Myc⁺ lymphoma cells suggesting that the activation of these metabolic pathways in ADPKD could also be, in part, due to miR-17.

The second more intriguing theme involves the direct repression of the ADPKD genes PKD1 and PKD2 by miR-17. The 3’-UTRs of PKD1 and PKD2 mRNAs contain evolutionarily conserved miR-17 binding sites. RNA immunoprecipitation assays have demonstrated direct miR-17 binding to PKD1/2 mRNAs. This physical interaction leads to a reduction in PKD1/2 mRNA expression and translation. Conversely, deleting miR-17 or anti-miR-17 treatment improves PKD1/2 expression. Thus, miR-17 may aggravate cyst growth by directly lowering the PKD1/2 gene dosage. This mode of action is especially likely to be relevant in the setting of hypomorphic mutations, where, cysts grow because PKD1/2 dosage reduces below a critical threshold. High miR-17 levels in this context will be expected to further lower PKD1/2 dosage and thereby aggravate cyst growth. While tolvaptan or other drugs under development for ADPKD target downstream pathways, anti-miR-17 treatment may mediate some of its efficacy by improving PKD1/2 expression.

Cyst suppressing miRNAs:

miR193-3p is a tumor suppressor miRNA (83–86). During ADPKD progression, its activity is suppressed (87). Parallel microarray analysis for miRNA and mRNA expression on cell lines derived from normal kidneys and human ADPKD kidneys has shown that miR193b-3p is one of two significantly downregulated miRNAs. This miRNA functions by inhibiting several factors that regulate cell proliferation, such as Erb-B2 Receptor Tyrosine Kinase 4 (ErbB4) (88–91). ErbB4 activity is increased in ADPKD cells resulting in increased cell division (87, 90). Moreover, levels of ErbB4 in urinary extracellular vesicles from ADPKD patients correlates with disease progression. Thus, enhancing miR-193b-3p expression could be one way of reducing cyst epithelial proliferation.

The second cyst suppressing miRNA is miR-214. It is derived from the long non-coding RNA called Dnm3os (Dynamin 3 opposite strand) that is produced by antisense transcription of the 14^th intron of the Dnm3 gene (92). Interestingly, Dnm3os and miR-214 are primarily expressed in the stromal cells during embryonic kidney development, and Dnm3os expression diminishes in the adult kidney. In ADPKD kidneys, Dnm3os and miR-214 are activated in the stromal cells surrounding the cysts suggesting that it may function to modulate the cyst microenvironment. Surprisingly, despite its upregulation, the deletion of miR-214 is associated with aggravated interstitial inflammation and cyst growth. miR-214 appears to suppress interstitial inflammation via repression of the TLR4 pathway. Thus, miR-214 is a protective compensatory signal produced in the cyst microenvironment that functions to restrain cyst-associated inflammation.

Pharmacological modulation of miRNA activity

RNA-based drugs predate the discovery of miRNAs by a couple of decades. The RNA-based approach is applied in two ways: first, is the antisense oligonucleotide (ASO)-based approach (93–96). ASOs bind to target mRNAs and physically prevent ribosomes from translating the mRNA. In this way, the ASOs act as steric inhibitors of mRNA translation. ASOs can also be designed such that upon binding to target mRNAs, they recruit RNAase H, which degrades the mRNA (97). The second approach is the siRNA-based approach (98, 99). This method takes advantage of the fact that each cell in our body has endogenous siRNA machinery. These are double-stranded RNA drugs, which are processed by Dicer to produce the active, single-stranded siRNA. The single-stranded siRNA is then loaded on the RISC, which catalyzes the reaction to inhibit target mRNAs.

Extra-cellular RNA is quickly degraded by nucleases and can evoke an intense immune reaction if administered systemically. However, RNAs can be chemically modified to overcome these issues (Figure 3). RNAs are composed of three chemical units: the phosphate backbone, a ribose sugar, and the nitrogenous nucleotide base, each of which can be modified to impart ‘drug-like’ properties (100). Amongst the most common modifications are: 1) Locked nucleic acid (LNA): this modification locks the ribose ring in a conformation that is ideal for Watson-Crick binding (101). The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the oxygen at the second position with carbon in the fourth position. This modification stabilizes binding to target RNA. 2) 2’-Fluro, 2’-O-methyl and 2’-O-methoxyethyl (2’-MOE): these are chemical modifications in the 2’ hydroxyl group of the sugar moiety (102). They confer stability against nucleases and also increase the stability of ASO-target RNA interaction. 3) Phosphorothioate (PS), thiophosphoroamidate, and phosphorodiamidate morpholino (PMO): these are modifications in the phosphodiester linkage (103, 104). Because these are analogs of RNA, they are not readily hydrolyzed by ribonucleases leading to their stabilization and enhanced biological activity.

Figure 3.

Unmodified RNA is quickly degraded by nucleases and can evoke an intense immune reaction if administered systemically. However, the phosphate backbone, a ribose sugar, and the nitrogenous nucleotide base of RNAs can be chemically modified to overcome these issues. Some of the common chemical modifications used to impart ‘drug-like’ properties are shown.

RNA drugs are hydrophilic, have a relatively large molecular weight, and the chemical modifications discussed above render them stable for long periods if adequately refrigerated. These drugs are dissolved in normal saline and administered systemically (intravenous and subcutaneous) or locally (skin, eyes, brain, lungs). They are quickly cleared from the plasma (3-5hrs) and accumulate primarily in the liver, kidney, spleen, and macrophages (97). The drugs are taken up by cells via non-specific endocytosis pathways, where they can last for up to 2-3 weeks (93, 105). Some ASOs are loosely protein-bound, which prevents them from being freely filtered via the kidney. Eventually, these drugs are degraded by nucleases and excreted via urine and bile. The properties of the ASOs, including biochemical modifications, the length, the charge, and hydrophobicity have a profound impact on drug delivery.

The ASO technique has been applied to inhibit miRNAs as well (106–109). Several approaches are available to harness the therapeutic potential of miRNAs (Figure 4). These technologies fall into three basic categories: 1) anti-miRNA oligonucleotides (AMOs), 2) miRNA mimics, and 3) target site blockers (TSBs). Anti-miRNA oligonucleotides are short (9-25 nucleotides) single-strand synthetic RNA molecules that inhibit endogenous mature miRNAs (Figure 3). They contain nucleotide sequences that are complementary to the target miRNA and operate by Watson-Crick base pairing with the miRNA. Drugs RGLS4326 and RG-012 are examples of AMOs. miRNA mimics are double-stranded synthetic mature miRNAs. They are synthesized to replace or supplement the function of endogenous miRNAs and are chemically modified to facilitate preferential engagement of the active miRNA strand with the miRISC. TSBs are oligonucleotides that are designed to block miRNAs from recognizing MREs in specific target mRNAs. This is achieved by designing oligonucleotides that selectively bind only to the MRE in target mRNAs of interest (Figure 4). In this way, the TSBs stabilize a specific target mRNA without affecting the rest of the miRNA-regulated mRNA network.

Figure 4. Strategies to modulate miRNA activity.

There are three main categories for pharmacologically modulating miRNA activity. A. miRNA mimics are synthetic versions of endogenous miRNAs. They amplify or restore miRNA activity. B. Anti-miR oligonucleotides are ASOs that repress the endogenous miRNA(s). They have complementary sequences to the miRNA of interest. Therefore, they bind to the mature miRNA, sequestering them away from the target mRNAs. Thus, the target mRNA network is de-repressed. C. Target site blockers (TSBs) are ASO that prevent the repression of a specific mRNA target of miRNA. They function by blocking miRNAs from binding to their target mRNAs.

miRNA-based therapeutics for PKD

The first miRNA drug to be tested in humans was Mirvirasen, an LNA-modifed anti-miR-122, for the treatment of Hepatitis C (HCV) (110). In a phase 2A clinical trial, this drug demonstrated remarkable efficacy in suppressing HCV (111). Although a follow-up clinical trial has not yet been performed, Miravirasen has provided the initial clinical proof of concept efficacy for the miRNA-based therapeutics platform. Several other miRNA-based drugs have now entered human clinical development for diverse diseases and indications. Cobomarsen is an LNA-modifed anti-miR-155 ASO, which is under development (Phase 2 clinical trials) for the treatment of various types of hematological malignancies, including cutaneous T cell lymphoma (CTCL), adult T cell lymphoma/leukemia, and chronic lymphocytic leukemia . The therapeutic potential of Cobomarsen relies on the findings that miR-155 is critical for the differentiation and proliferation of blood and lymph cells (112). Another drug miRNA drug in phase 2 clinical trials is a miR-29 mimic called Remlarsen (98). miR-29 has been shown to inhibit extracellular matrix production and is downregulated in numerous fibrotic diseases (113, 114). Remlarsen restores miR-29 levels and has the potential to inhibit fibrosis. Indeed, it has shown remarkable efficacy in pre-clinical models of ocular, lung, and liver fibrosis.

Even though the miRNA therapeutics field is still in its nascency, there already are two anti-miRNA drugs under clinical development for kidney diseases. A big reason is a general realization that ASOs readily deliver to kidney tubules even without any special formulations. The first drug is an anti-miR-21 ASO that is currently being tested in phase 2 clinical trial for safety and therapeutic efficacy for patients with Alport syndrome (62, 95) and see the section on miR-21). The second drug is RGLS4326, an anti-miR-17 ASO, being developed for ADPKD treatment (18).

RGLS4326 is a single-stranded, short oligonucleotide of only 9 nucleotides in length that bears fall complementarity to the miR-17 seed sequence. It was discovered by screening a custom-made library, which contained 199 anti-miR-17 ASOs that varied in length, biochemical modifications, and base sequences. From this screen, RGLS4326 emerged as the leading drug candidate, in part, because of its preferential distribution to kidney tubules, including collecting duct-derived cysts. As expected, RGLS4326 displaces miR-17 from translationally active polysomes and thereby de-represses multiple miR-17 mRNA targets. Several other characteristics make RGLS4326 uniquely suited for ADPKD. First and most importantly, RGLS4326 treatment attenuates cyst growth in multiple PKD mouse models and human in vitro ADPKD models. It demonstrates therapeutic efficacy that is comparable to tolvaptan. Second, this drug is administered subcutaneously and retains pharmaceutical activity for up to 2 weeks, and daily administration is not needed. This is a favorable property considering that ADPKD is a chronic condition that requires life-long therapy. Finally, RGLS4326 has a unique mechanism of action. miRNAs simultaneously repress many mRNA targets, thereby affecting multiple signaling pathways. Accordingly, RGLS4326 simultaneously modulates several cyst pathogenic pathways such as Ppara, Wnt signaling, mTOR signaling etc. The most intriguing observation is that RGLS4326 directly upregulates the ADPKD genes, PKD1 and PKD2. Thus, some of its efficacy may be because of increasing PKD1/2 gene dosage above a critical threshold. A big question moving forward is conclusively establishing the safety profile of RGLS4326 in humans.

Conclusion

ADPKD is the most common genetic cause of renal failure. Current treatment options are limited. miRNAs have emerged as promising drug targets in ADPKD. The biological effects of three epithelial miRNAs (miR-17~92, miR-21 and miR-193b-3p) and one stromal miRNA (miR-214) have been evaluated in ADPKD. miR-17~92 and miR-21 promote disease progression in ADPKD. Conversely, miR-193b-3p, and miR-214 attenuate cyst growth. These studies have led to the development of miRNA-based drugs for the treatment of ADPKD.