advances in micrornas: implications for gene therapists

HUMAN GENE THERAPY 19:27–37 (January 2008)© Mary Ann Liebert, Inc.DOI: 10.1089/hum.2007.147

Review

Advances in MicroRNAs: Implications for Gene Therapists

REBECCA T. MARQUEZ and ANTON P. MCCAFFREY

ABSTRACT

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate the expression of asmany as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diversebiological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Ev-idence shows that numerous viruses interact with the miRNA machinery, and that a number of viruses en-code their own miRNAs. It seems likely that miRNAs will be implicated in many human diseases. Manipula-tion of miRNA levels by gene therapy provides an attractive new approach for therapeutic development. Thisreview focuses on approaches to manipulate miRNA levels in cells and in vivo, and the implications for genetherapy. Furthermore, we discuss the use of endogenous miRNAs as scaffolds for the expression of RNA in-terference (RNAi) as well as competition between exogenous RNAi triggers and endogenous miRNAs. Becauseshort interfering RNAs can also act as miRNAs, seed matches with the 3� untranslated regions of genes shouldbe avoided to prevent off-target effects. Last, we discuss the use of miRNAs to avoid immune responses to vi-ral vectors.

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BACKGROUND

MICRORNAs (miRNAs) ARE SMALL, �22-nucleotide non-coding regulatory RNAs involved in RNA-mediated gene

silencing (Lagos-Quintana et al., 2001). At present, more than500 human miRNAs have been identified. miRNAs are impor-tant gene regulators during processes that include differentia-tion, proliferation, apoptosis, and insulin secretion as well asheart, brain, and skeletal muscle development (see Bartel[2004], Harfe [2005], Kloosterman and Plasterk [2006], andKrutzfeldt and Stoffel [2006] for comprehensive reviews). ThemiRNA pathway involves several processing steps to producethe mature miRNA used for gene silencing (Fig. 1). Long pri-mary miRNA transcripts (pri-miRNAs) transcribed by poly-merase II or III are cleaved by a complex of Drosha and DGCR8(DiGeorge critical region-8) into the pre-miRNA. The pre-miRNA is exported from the nucleus to the cytoplasm by Ex-portin 5, where it undergoes a second cleavage step byDicer/TRBP (HIV TAR RNA-binding protein) to produce ashort, �20-nucleotide duplex RNA with two base 3� overhangs.Usually, a single strand of this duplex RNA is incorporated into

the RNA-induced silencing complex (RISC), although a subsetof miRNAs incorporate both strands individually into distinctRISCs. The mature miRNA strand in RISC is called the guidestrand, and the opposite strand not incorporated is referred toas the passenger strand. The minimal RISC is composed of anArgonaute (Ago) protein, Dicer, and TRBP, although it is likelythat RISC interacts with numerous accessory proteins (Petersand Meister, 2007). Once the mature miRNA guide strand isloaded into the RISC, it binds to the 3� untranslated region(UTR) of its mRNA target and induces silencing. It is currentlythought that the specificity of the miRNA for its target mRNAis primarily (although not exclusively) specified by the “seedsequence” (nucleotides 2–8 of the guide strand; Fig. 2). Oneimplication, evidenced by the short length of the seed sequence,is that miRNAs are remarkably promiscuous. It has been sug-gested that some miRNAs can target hundreds of mRNAs (Farhet al., 2005; Lim et al., 2005; Stark et al., 2005). miRNAs re-press their target mRNAs by a variety of mechanisms. miRNAswith perfect complementarity to their target mRNAs cause di-rect mRNA cleavage, although this is thought to be rare in mam-mals. More often, miRNAs with partial complementarity to

Department of Internal Medicine, University of Iowa, Iowa City, IA 52242.

mRNA 3� UTRs direct translational repression, decapping, ordeadenylation.

The use of RNA interference (RNAi) for gene silencing hasbecome widespread, both for functional studies and for thera-peutic purposes in a gene therapy setting. Commonly, RNAi isinitiated by transfection of cells or animals with synthetic shortinterfering RNAs (siRNAs) or introduction of plasmids or re-combinant viruses expressing short hairpin RNAs (shRNAs). Ithas become apparent that exogenous synthetic siRNAs can si-lence genes because they resemble the endogenous miRNAsgenerated by Dicer/TRBP, and thus can be loaded into the RISC(Fig. 1). Likewise, shRNAs resemble the pre-miRNA. Exoge-nous siRNA and shRNAs are designed to be perfectly comple-mentary to their target mRNA, and therefore they direct cleav-age. However, they can also act as miRNAs. The consequencesfor design of siRNAs/shRNAs are described below. These ex-ogenous siRNAs and shRNAs also have the potential to over-load the miRNA-processing pathway.

MARQUEZ AND MCCAFFREY 28

FIG. 1. The miRNA/RNAi pathway. miRNAs are transcribed from Pol II or Pol III promoters as long transcripts. These pri-miRNAs are processed by Drosha/DGCR8 to form pre-miRNAs. Pre-miRNAs are exported to the cytoplasm by Exportin 5,where they are further processed into a short duplex by Dicer/TRBP. One strand of the duplex (the guide strand) is incorporatedinto RISC and the other strand is destroyed. RISC mediates gene silencing by inhibition of translation, decapping, deadenyla-tion, or direct target cleavage. shRNAs or siRNAs enter the miRNA/RNAi pathway and mediate target cleavage. Ago, Arg-onaute protein; DGCR8, DiGeorge critical region-8; miRNA, microRNA; Pol, RNA polymerase; pri-miRNA, primary miRNAtranscript; RISC, RNA-induced silencing complex; shRNA, short hairpin RNA; siRNA, short interfering RNAs; TRBP, HIV-1TAR RNA-binding protein.

FIG. 2. The miRNA seed sequence determines specificity.The seed sequence (nucleotides 2–8 of the miRNA, boxed andin boldface) is the primary determinant of miRNA specificity.The predicted base pairing of the hepatitis C virus 5� UTR with miR-122 is shown. [Adapted from Jopling et al., Science309:1577 (9/2/2005). Reprinted with permission from AAAS.]

The last few years has witnessed an explosion of publica-tions on miRNAs. This review is not intended as a compre-hensive survey of the miRNA literature. Rather, it highlightskey issues of importance to researchers who seek to integrategene therapy approaches with our emerging understanding ofmiRNAs.

miRNAs and cancer: diagnosis, prognosis, andtherapeutic potential

High-throughput strategies, such as miRNA microarrays (Liuet al., 2004), bead-based flow cytometric miRNA expression pro-filing (Lu et al., 2005), and quantitative polymerase chain reac-tion (qPCR) (Chen et al., 2005; Jiang et al., 2005; Murakami etal., 2005; Raymond et al., 2005), have been developed formiRNA profiling. These high-throughput methods have shownthat miRNA levels were dramatically shifted in various cancers(Liu et al., 2004; He et al., 2005; Iorio et al., 2005; Lu et al.,2005; Murakami et al., 2005) (refer to Calin and Croce [2006]and Esquela-Kerscher and Slack [2006] for a comprehensive re-view of miRNAs and cancer). Interestingly, most differentiallyexpressed miRNAs were downregulated in tumors comparedwith normal tissues. These studies revealed that miRNA profil-ing could be used to distinguish between normal and canceroustissues, developmental lineage, as well as differentiation. Identi-fying miRNA expression profiles during tumorigenesis willlikely provide additional targets for gene therapy, while improv-ing predictions for diagnosis and prognosis.

For most of the miRNAs found to be dysregulated in can-cer, a causative relationship between differential expression andcancer has not been established; however, for a subset there isnow convincing evidence that some miRNAs are directly in-volved in cancer formation. These “onco-miRs” can functionas tumor suppressors (such as miR-15a and miR-16-1 [Calin etal., 2002, 2005; Cimmino et al., 2005] and the let-7 family ofmiRNAs [Calin et al., 2002; Takamizawa et al., 2004; Johnsonet al., 2005, 2007]) and as oncogenes (such as the miR-17–92cluster [He et al., 2005], miR-155 [Costinean et al., 2006], andmiR-21 [Si et al., 2007]). A few examples are provided below.

The first tumor suppressor miRNAs were identified in B cellchronic lymphocytic leukemia (CLL) (Calin et al., 2002). miR-15a and miR-16-1 are located in a cluster on chromosome13q14, which is deleted in more than half of all CLL cases.This correlates with the loss of expression of miR-15a and miR-16-1. It was later shown that miR-15a and miR-16-1 negativelyregulated expression of the antiapoptotic gene Bcl2 (Cimminoet al., 2005). After discovering that miRNAs were locatedwithin a frequently deleted chromosomal region in CLL, re-searchers investigated the presence of miRNAs in “fragile sites”or chromosomal loci that are susceptible to chromosome break-age, amplification, and fusion with other chromosomes (Calinet al., 2004). They found that 50% of miRNAs were locatedwithin human fragile sites. This provided further evidence thatmiRNAs are involved in cancer and that they may be usefultargets for therapy. Interestingly, in one report, Donsante andcoworkers found that hepatocellular carcinoma associated withadeno-associated virus integration resulted from a vector in-sertion near a micro-RNA cluster (Donsante et al., 2007).

One of the most studied oncogenic miRNAs is the miR-17–92 cluster. The miR-17–92 cluster is at a locus that is am-plified in human B cell lymphomas and therefore upregulated

relative to normal tissues (He et al., 2005). Furthermore, en-forced expression of the miR-17–92 cluster in the context of c-myc overexpression accelerated tumor development in amouse B cell lymphoma model.

Another example of an oncogenic miRNA is miR-155. miR-155 is overexpressed in various types of B cell malignancies(Metzler et al., 2004; Eis et al., 2005; Kluiver et al., 2005).Costinean and coworkers produced a transgenic mouse thatoverexpresses miR-155 in B cells (Costinean et al., 2006).These mice exhibited polyclonal preleukemic pre-B cell pro-liferation followed by B cell malignancy, which strongly sug-gests that miR-155 is directly implicated in the initiation and/orprogression of these diseases.

Although the examples discussed above are only a small sub-set of miRNAs implicated in cancer development, they illus-trate the point that targeting aberrantly expressed miRNAs bytherapeutic approaches could have a tremendous impact on fu-ture cancer treatments. Strategies for manipulating miRNA lev-els are discussed below.

Viruses and miRNAs

Increasing evidence suggests that viruses interact with themiRNA machinery (reviewed in Sullivan et al. [2006] andDykxhoorn [2007]). Cloning of miRNAs from virus-infectedcells revealed that many latent DNA viruses encode their ownmiRNAs (Pfeffer et al., 2004). To date, 11 viruses have beenshown to encode their own miRNAs (see the miRBase Se-quence Database [previously the miRNA Registry] at http://mi-crorna.sanger.ac.uk/sequences/index.shtml). These include Ep-stein–Barr virus (EBV) (Pfeffer et al., 2004), herpes simplexvirus type 1 (HSV-1) (Cui et al., 2006), human cytomegalovirus(Pfeffer et al., 2005), human immunodeficiency virus type 1(Omoto and Fujii, 2005), Kaposi sarcoma-associated her-pesvirus (Cai et al., 2005; Pfeffer et al., 2005; Samols et al.,2005), Marek’s disease virus (Burnside et al., 2006), Marek’sdisease virus type 2 (Yao et al., 2007), mouse gammaher-pesvirus 68 (Pfeffer et al., 2005), rhesus lymphocryptovirus(Cai et al., 2006), rhesus monkey rhadinovirus (Schafer et al.,2007), and simian virus 40 (SV40) (Sullivan and Ganem, 2005).These viral miRNAs represent potential antiviral targets andcould also serve as diagnostic markers for viral infection orstage of infection/latency. Below, we discuss several examplesof the interplay of host and viruses via the miRNA pathway.

Viruses use miRNAs in various ways, such as to inhibit vi-ral or host transcripts or to recruit host miRNAs for viral repli-cation. EBV and SV40 are examples of viruses that encodemiRNAs that target their own viral transcripts. EBV encodesat least 18 miRNAs (Pfeffer et al., 2004; Sullivan et al., 2006),one of which targets the transcript encoding the viral poly-merase to negatively regulate it later during infection (Furnariet al., 1993; Pfeffer et al., 2005). Likewise, SV40 encodes twoviral miRNAs that target the viral T-antigen transcript (Sulli-van et al., 2005). This regulatory mechanism may aid the virusin immune evasion.

HSV-1 provides an example of a virus that encodes miRNAsthat target host transcripts. The HSV-1 latency-associated tran-script encodes an miRNA predicted to target two host tran-scripts involved in apoptosis (transforming growth factor-� andmothers against decapentaplegic homolog-3) (Gupta et al.,2006).

ADVANCES IN MicroRNAs 29

Host miRNAs can also be used for viral replication as wellas antiviral defense. The endogenous miRNA, miR-122, is re-quired for replication of hepatitis C virus (Jopling et al., 2005).Figure 2 shows the interaction of miR-122 with the 5� UTR ofhepatitis C virus. As discussed below, two groups have suc-cessfully used antisense approaches to modulate the levels ofmiR-122 in mouse models (Krutzfeldt et al., 2005; Esau et al.,2006). Inhibition of viral replication in vivo using this strategyremains to be demonstrated. In contrast, primate foamy virustype 1 (PFV-1) has an mRNA that is the target of the endoge-nous host miRNA, miR-32 (Lecellier et al., 2005). Interestingly,PFV-1 encodes a protein (Tas) that may interfere with the hostmiRNA pathway (Lecellier et al., 2005). These examples sug-gest that miRNA overexpression or ablation may represent anovel antiviral therapeutic strategy.

Last, at least one commonly used gene transfer vector en-codes an RNA that inhibits the miRNA pathway. The highlyexpressed adenoviral virus-associated type I (VAI) RNA wasshown to be a competitive inhibitor of at least two steps in themiRNA pathway (Lu and Cullen, 2004; Andersson et al., 2005).It is a competitive inhibitor of Exportin 5 as well as Dicer andsmall VAI RNAs generated by Dicer cleavage have been shownto associate with RISC. These findings suggest that saturationof the endogenous miRNA pathway should be assessed in hu-man clinical trials with adenoviral vectors, when possible.

Other miRNA/disease associations

Although the vast majority of associations between miRNAsand human disease are related to tumorigenesis, there is in-creasing evidence that miRNAs could be involved in multiplediseases. For example, 28 miRNAs were found to be dysregu-lated in two mouse models of cardiac hypertrophy (van Rooijet al., 2006). miR-9 and miR-128a were overexpressed in thebrains of Alzheimer’s patients (Lukiw, 2007) and 16 miRNAswere differentially expressed in the brains of schizophrenics(Perkins et al., 2007). Last, a mutation in the 3� UTR of theSLITRK1 gene that is associated with Tourette’s syndrome ledto enhanced repression by miR-189 (Abelson et al., 2005).

THERAPEUTIC MANIPULATION OF miRNAs

As described above, miRNAs are likely to be involved in nu-merous genetic diseases, such as cancer, as well as some viral in-fections. These miRNAs represent potentially novel therapeutictargets. It would clearly be desirable to specifically modulate thelevels of individual miRNAs in vivo. Fortunately, decades of re-search in the fields of antisense, ribozymes, and gene therapy pro-vide a fertile tool chest that serves as a starting point for manip-ulating miRNA levels in vivo. Below, we discuss methods foroverexpressing and ablating miRNAs and provide examples ofinitial efforts to apply these techniques in disease settings. Again,this is not intended to be an exhaustive review, but rather a re-view of important paradigms for gene therapists.

Inhibition of miRNA function through antisense andmiRNA sponges

Numerous groups have now shown that antisense oligonu-cleotides complementary to the guide strand of miRNAs can

inhibit their function in cultured cells (Esau et al., 2004; Meis-ter et al., 2004; Poy et al., 2004; Cheng et al., 2005; Lee et al.,2005; Schratt et al., 2006), flies (Boutla et al., 2003; Hutvagneret al., 2004; Leaman et al., 2005), and mice (Hutvagner et al.,2004; Esau et al., 2006; Krutzfeldt et al., 2007). These inhibi-tors, referred to as “anti-miRs” or “antago-miRs,” could havesignificant clinical potential (Poy et al., 2004; Jopling et al.,2005; Krutzfeldt et al., 2005; Esau et al., 2006; Weiler et al.,2006). Early studies showed that 2�-O-methyl oligoribonu-cleotides were able to block miRNA function in cultured cells(Meister et al., 2004) and in flies (Hutvagner et al., 2004). Incontrast, unmodified 2�-deoxy oligonucleotides (Meister et al.,2004) and 2�-deoxy phosphorothioate oligonucleotides (Daviset al., 2006) were unable to inhibit miRNA activity in culturedcells. Several reports suggest that anti-miRs interfere withmiRNA-mediated silencing by binding to the mature miRNAguide strand in the RISC complex (Hutvagner et al., 2004;Davis et al., 2006; Krutzfeldt et al., 2007). Interestingly, it ap-pears that anti-miR binding leads to degradation of the targetedmiRNA by some previously undescribed mechanism (Esau etal., 2006; Krutzfeldt et al., 2007).

Although 2�-O-methyl oligoribonucleotides appeared to beeffective inhibitors of miRNA function, more potent inhibitorshave been described. Davis and coworkers conducted a limitedcomparison of the anti-miR activities of various oligonu-cleotides with different backbones and 2� modifications (Daviset al., 2006). They found that uniformly 2�-O-methoxyethyloligonucleotides (2�-MOEs) and oligonucleotides in whichevery third nucleotide was substituted with a locked nucleicacid (LNA) residue (Exiqon, Woburn, MA) were the most po-tent. Another report also showed that LNA-substituted oligonu-cleotides were effective at antagonizing miRNAs (Orom et al.,2006). Several vendors sell LNA-modified oligonucleotides (including Integrated DNA Technologies [Coralville, IA] andExiqon [Vedbaek, Denmark]). 2�-O-Methoxyethyl oligonu-cleotides (Isis Pharmaceuticals, Carlsbad, CA) are not com-mercially available. 2�-Fluoro-oligonucleotides with a phos-phorothioate backbone also showed significant activity incultured cells (Davis et al., 2006). Oligonucleotides with cen-tral stretches of 2-deoxy residues can direct RNase H-mediateddegradation of RNA/oligonucleotide duplexes. However, theinability of such oligonucleotides to antagonize miRNA func-tion suggested that RNase H may not have access to miRNA-loaded RISCs bound to anti-miRs (Davis et al., 2006). Last, be-cause transfection of cells with oligonucleotides with somechemistries can lead to a loss of cell viability at high doses(Davis et al., 2006), care should be taken to control for toxicityin anti-miR studies.

A significant barrier to the application of anti-miRs in vivowill concern efficient delivery of these molecules either sys-temically or locally. However, two groups have reported suc-cessful miRNA inhibition in vivo. Krutzfeldt and coworkers tar-geted several different miRNAs in mice, using antago-miRs(Krutzfeldt et al., 2005). These are 2�-O-methyl oligonu-cleotides with two and four phosphorothioate linkages at the 5�and 3� ends, respectively. The antago-miRs were conjugated tocholesterol to reduce renal clearance and enhance uptake. Ad-ministration of three injections of high doses of antago-miR (80mg/kg) resulted in substantial decreases in the levels of en-dogenous miR-16 in all tissues tested except for the brain. The


authors then targeted the abundant liver miRNA, miR-122.They conducted gene expression profiling and identified hun-dreds of genes that were upregulated in the liver on treatmentwith an miR-122 antago-miR. Many of these genes were in-volved in cholesterol biosynthesis and, indeed, miR-122 antago-miR treatment led to a 40% reduction in plasma cholesterol lev-els. These findings provided the proof-of-principle thatanti-miRs have the potential to be therapeutic. Because miR-122 is required for viral replication, miRNA downregulationmay have antiviral activity.

In a separate article, the authors found that longer antago-miRs(25 nucleotides) were more effective in vivo (Krutzfeldt et al.,2007). This was presumably because the phosphorothioate basesat the ends decreased the melting temperature of the miRNA–an-tago-miR duplex. Lengthening the antago-miR resulted in morepairing with the more stable interior 2�-deoxy bases, while main-taining the nuclease stabilization afforded by the terminal phos-phorothioate linkages. The authors also showed that some antago-miRs exhibit mismatch specificity, although this was highlyposition dependent (Krutzfeldt et al., 2007).

In a similar study, Esau and coworkers reported that uncon-jugated 2�-MOE miR-122 anti-miRs reduced the levels of miR-122 in mouse liver after intraperitoneal injection of 12.5–75mg/kg twice weekly for 4 weeks (Esau et al., 2006). As withthe above-described study, the authors observed elevations inthe levels of predicted target mRNAs and decreases in plasmacholesterol. The authors then applied this anti-miR strategy ina disease model of diet-induced obesity in mice. Treatment withmiR-122 anti-miR reduced plasma cholesterol by 35%. Treatedanimals also had lower levels of steatosis.

Ebert and coworkers described another approach to inhibit-ing the function of specific miRNAs in cells (Ebert et al., 2007).They reasoned that by expressing an mRNA containing multi-ple binding sites for an endogenous miRNA, they could bindup the miRNA and prevent its association with its endogenoustargets. They referred to these synthetic mRNAs as “miRNAsponges.” To prevent cleavage of the mRNA containing themiRNA-binding sites, they introduced central mismatches inthe miRNA/mRNA duplex at positions 9–12, which were es-sential for activity. This should prevent release and recyclingof the miRNA. They designed both polymerase (Pol) II- andPol III-driven miRNA sponges, although they observed somenonspecific depression of luciferase expression with Pol III-dri-ven sponges. The use of Pol II promoters should enable the cre-ation of inducible and tissue-specific miRNA sponges. Theyshowed that in cultured cells, their miRNA sponges were atleast as effective as LNA anti-miRs. miRNA sponges were alsoeffective at repressing multiple members of an miRNA family.Stably integrated, miRNA sponge expression cassettes yielded�40% as much inhibition as transiently transfected plasmids.Although it remains to be demonstrated, these results suggestthat the generation of transgenic animals expressing induciblemiRNA sponges may be feasible. Interestingly, one report sug-gests that nature uses miRNA sponges. Franco-Zorrilla andcoworkers showed that in Arabidopsis, the noncoding IPS1RNA serves as a sponge for miR-399 (Franco-Zorrilla et al.,2007). As described above, the IPS1/miR-399 hybrid containsinternal mismatches that are essential for its activity.

The examples described above demonstrate the proof of prin-ciple that miRNA function can be inhibited in vivo and in cul-

tured cells. However, there is clearly a need to refine and op-timize these approaches. Creative methods that mimic naturalbiological processes may provide clues as to how to accomplishthis.

Downregulating expression of miRNAs involved in cancer

Because miRNAs are often dysregulated in cancer, modu-lating their levels may have therapeutic benefit. miR-21 was anearly example of how downregulating an miRNA inhibited can-cer development. miR-21 is overexpressed in many differentcancer types, such as breast cancer (Iorio et al., 2005; Si et al.,2007), ovarian cancer (Iorio et al., 2007), hepatocellular carci-noma (Meng et al., 2007), gliomas (Chan et al., 2005), pan-creatic cancer (Lee et al., 2007), and CLL (Fulci et al., 2007).miR-21 plays a critical role in cell proliferation by targetingand, therefore, inhibiting the tumor suppressor genes TPM1(tropomyosin 1) (Zhu et al., 2007) and PTEN (phosphatase andtensin homolog) (Meng et al., 2007). Using a xenograft carci-noma model, researchers transiently transfected MCF-7 cellswith 2�-O-methyl oligonucleotides complementary to miR-21(or negative control oligonucleotide), and then injected theminto mammary pads of female nude mice (Si et al., 2007). Af-ter 28 days, tumors derived from MCF-7 cells transfected withanti-miR-21 were 50% smaller in size compared with tumorsof mice receiving cells transfected with negative control oligo-nucleotide. The authors showed that suppression of miR-21lasted 2 weeks. Although the model system is somewhat arti-ficial (ex vivo treatment of tumors), this report demonstratesthat knockdown of oncogenic miRNAs can inhibit tumorgrowth. Thus, knockdown of miRNAs by systemic or local de-livery of anti-miRs could represent a novel anticancer therapy.

Overexpression of miRNAs

When miRNA levels are reduced in human disease, it maybe desirable to use gene therapy-based approaches to restoremiRNA expression in certain tissues. The considerable experi-ence with nonviral and viral delivery of transgenes that has ac-cumulated over the past few decades will certainly facilitate thisgoal. As is discussed below, the endogenous miRs miR-26a(McManus et al., 2002) and miR-30 (Zeng et al., 2002; Bodenet al., 2004) have been used as scaffolds for the expression ofRNAi triggers. Clearly, these well-characterized systems couldbe used to produce mature miRNA guide strands in cells. Forexample, overexpression of an miRNA (or miRNAs) whose ex-pression is downregulated in cancer may be beneficial as treat-ment. In some cases, it may be desirable to express endogenousmiRNAs in their native context with their own promoters, thusmaintaining proper regulation. As more complete miRNA tran-scription units are characterized, this may become increasinglypossible.

Reexpression of miRNAs downregulated during cancer

Loss of tumor suppressor genes is a common theme duringtumorigenesis. As previously discussed, most differentially ex-pressed miRNAs in tumors were downregulated compared withnormal tissues. Exogenous expression of miRNAs downregu-lated during tumorigenesis may have potential as a therapeutic


strategy. Researchers have demonstrated that miR-34a wasdownregulated in 36% of human colon cancers compared withcounterpart normal tissues (Tazawa et al., 2007). In vivo re-expression of miR-34a suppressed growth of HCT 116 andRKO cells in mice. Furthermore, miR-34a functions as a po-tent suppressor of cell proliferation through modulation of the E2F signaling pathway. Loss of miR-34a expression couldcontribute to aberrant cell proliferation, leading to colon can-cer development (Tazawa et al., 2007). Thus, exogenous ex-pression of miRNAs may have application as an anticancerstrategy.

Synthetic miRNAs targeting genes associated with metastasis

An alternative approach for cancer therapeutics involves cre-ating synthetic miRNAs that target genes responsible for onco-genesis or metastasis. Loss of certain miRNAs in breast tumorcells can lead to increased invasion and metastasis of breastcancer. Inhibition of chemokine (C-X-C motif) receptor 4(CXCR4) with siRNA inhibited metastasis of breast cells invivo (Liang et al., 2004, 2005). Therefore, researchers designedand synthesized a pre-miRNA with an miR-155 backbone thattargeted CXCR4 in order to determine whether the CXCR4/SDF-1 pathway was regulated by expression of miRNAs (Liang et al., 2007). After transfecting breast tumor cells withthe synthesized miRNA, they observed a significant decreasein CXCR4 in breast tumor cells and reduced migration and in-vasion. Furthermore, they formed fewer lung metastases in vivocompared with control miRNA-transfected cells.

Another example of inhibiting metastatic-related genes in-volves PRL-3 (protein tyrosine phosphatase of regeneratingliver-3), whose high expression is associated with lymph nodemetastasis in gastric carcinoma. This study used an artificialmiRNA with an miR-155 backbone that targeted PRL-3. PRL-3 knockdown effectively suppressed the growth of peritonealmetastases and improved the prognosis for nude mice (Li et al.,2006). Therefore, synthetic miRNAs that target genes involvedin metastasis serve as an alternative means for treating cancer.

Epigenetic gene therapy in cancer

Manipulation of miRNA levels may also be a means to al-ter DNA methylation. DNA methylation is a crucial mechanismassociated with epigenetic regulation. Changes in the pattern ofDNA methylation, either increased (hypermethylation) or de-creased (hypomethylation), have been identified in all types ofcancer cells examined so far. DNA methyltransferase inhibi-tors, both nucleoside and nonnucleoside analogs, are currentlybeing tested for cancer therapy (reviewed in Brueckner et al.,2007). However, similar to most cancer treatments, there arerestrictions associated with efficacy and cytotoxicity. Thereforedeveloping a new means of targeting DNA hypermethylationis of great interest. The miR-29 family is downregulated inmany cancers that contain aberrant DNA hypermethylation pat-terns, including lung cancer. It was shown that miR-29 targetsDNA methyltransferase-3A (DNMT3A) and -3B (Fabbri et al.,2007). On enforced expression of miR-29, normal methylationpatterns in lung cells were reestablished and tumor suppressorspreviously silenced by promoter hypermethylation were reacti-vated. Furthermore, expression of miR-29 inhibited tumori-

genicity both in vitro and in vivo. Epigenetic gene therapy approaches can be useful across many tumor types. miRNA-mediated therapy may be useful in combination with DNAmethyltransferase inhibitors that are nontoxic, but have limitedefficacy. One study showed that treatment of malignant cholan-giocytes with gemcitabine, a nucleoside inhibitor, alters themiRNA expression profiles (Meng et al., 2006). Furthermore,inhibiting miRNAs highly overexpressed in malignant cholan-giocytes, such as miR-21, increased gemcitabine cytotoxicity.Therefore miRNAs may contribute to chemoresistance and in-hibiting miRNAs overexpressed in cancer may increase the ef-ficacy of chemotherapy.

IMPLICATIONS OF miRNAs FOR RNAi GENE THERAPY

miRNAs as scaffolds for RNAi triggers

Since the discovery of RNAi, researchers have been inter-ested in increasing the efficacy of gene-silencing tools. One ap-proach involves expressing RNAi triggers in the context of naturally occurring miRNAs. This can be accomplished by re-placing sequences in the central stem of endogenous pre-miRNAs, such as miR-26a (McManus et al., 2002) and miR-30 (Zeng et al., 2002; Boden et al., 2004), with sequences thatwill direct RNAi against a target of interest (RNAi triggers).The premise for this is that endogenous miRNA scaffolds willbe recognized and efficiently processed by the miRNA/RNAimachinery. When designing miRNA-based RNAi triggers, careshould be taken to maintain the native sequence around theDrosha cleavage site, as that can significantly influence silenc-ing efficiency (Zhou et al., 2005). By increasing our under-standing of the processing and targeting of natural miRNAs wewill be able to design a more robust silencing tool. Boden andcoworkers reported that pri-miRNAs may be processed moreefficiently than shRNAs, leading to 80% improvement in si-lencing efficiency (Boden et al., 2004). Hairpins containingloop sequences derived from pri-miRNAs were more efficientlytransported to the cytoplasm than were hairpins containing ar-tificial loops (Kawasaki and Taira, 2003). Numerous groups,including our own, have now used this approach. Silencing ef-ficiencies of �95% can be achieved with miRNA-derivedRNAi triggers (A. McCaffrey, unpublished results). Impor-tantly, pri-miRNAs can be expressed from polymerase II pro-moters (Boden et al., 2004), which enables the use of tissue-specific promoters, as well as inducible expression.Alternatively, miRNA-based RNAi triggers can be expressedfrom polymerase III promoters for high-level expression. Thus,expression of RNAi triggers in the context of endogenous miRNAs may offer significant advantages.

Competition between exogenous RNAi triggers andendogenous miRNAs

All drugs have a “therapeutic window”; insufficient dosesare not efficacious and excessive doses cause toxicity. In asobering report, Grimm and coworkers showed that, whenshRNAs are highly overexpressed in mice, they can cause acutetoxicity (Grimm et al., 2006). The authors speculate that


shRNAs were saturating the essential miRNA nuclear exportfactor Exportin 5, and thus competing for export with endoge-nous miRNAs. Long-term expression of the hairpins led to adepletion of endogenous liver miRNAs such as miR-122. Itshould be noted that high doses of adeno-associated virus(AAV) serotype 8 were used in these studies in order to achieve�100% transduction of hepatocytes. These results indicate thatit would be desirable to design the most potent RNAi triggerspossible such that subsaturating doses are efficacious. Regu-lated and tissue-specific expression also seems prudent. It willbe essential to monitor for saturation of the miRNA machineryin preclinical and human clinical trials with RNAi, when pos-sible.

If Exportin 5 is the only bottleneck at which RNAi triggerscompete with endogenous miRNAs for the RNAi/miRNA ma-chinery, one would predict that siRNAs (which do not requireExportin 5) would not compete with endogenous miRNAs.However, the Rossi group showed that both exogenous shRNAsand siRNAs competed with miR-21 in cultured cells, suggest-ing that factors downstream of Exportin 5 can be saturated (Cas-tanotto et al., 2007). Intriguingly, an exogenous miRNA-basedRNAi trigger with the same guide strand did not compete withmiR-21 (Castanotto et al., 2007). Although this suggests thatexpression of RNAi triggers in the context of endogenousmiRNAs may increase safety, further studies are required toconfirm this result.

John and coworkers reported that high doses of siRNAs for-mulated with lipid nanoparticles transiently mediated efficientsilencing of two liver transcripts in mice without interferingwith the levels of three liver miRNAs (John et al., 2007). It isimportant to note that only two time points were examined inthis experiment (2 days and 30 days). Silencing was observedonly on day 2. In contrast, Grimm and coworkers observed tox-icity only at late time points after chronic expression of theshRNA from viral vectors (greater than �25 days) (Grimm etal., 2006). John and coworkers conducted further long-term si-lencing studies with lipid nanoparticle-formulated siRNAs inhamsters (21 days) and observed efficient silencing of the he-patocyte-expressed sterol regulatory element-binding protein(SREBP) transcript without decreases in expression of miR-122. miR-122 is by far the most abundant liver miRNA. Itwould be interesting to determine whether longer term silenc-ing (�25 days) would affect the levels of miR-122 or the lev-els of a less abundant liver miRNA. Competition between pro-cessing of siRNAs and endogenous miRNAs requires furtherinvestigation. Identifying the proper balance between efficacyand toxicity may be required for each individual exogenousRNAi trigger.

Seed sequence of siRNAs is critical for predicting off-target silencing

When siRNAs were first applied as gene-silencing tools, itwas initially thought that they were exquisitely specific. Later,more comprehensive studies using microarray analysis indi-cated that some siRNAs (and presumably some shRNAs) cancause off-target cleavage of mRNAs containing closely relatedsequences (Jackson et al., 2003; Scacheri et al., 2004). Morerecently it has been appreciated that siRNAs can cause off-tar-get cleavage of mRNAs with as few as seven nucleotide

matches in the 3� UTR (Lin et al., 2005). A bioinformatics andmicroarray analysis of off-target cleavage by 12 different si-RNAs suggested that siRNAs can function as miRNAs duringoff-target silencing (Birmingham et al., 2006). Complemen-tarity between the nucleotides 2–7 or 2–8 (of either strand ofthe siRNA) and sequences in the 3� UTRs of mRNAs was as-sociated with off-target cleavage. Nucleotides 2–8 correspondto the equivalent of the miRNA seed sequence in the siRNA.Multiple seed matches within the 3� UTR were even morehighly correlated with off-target cleavage. Homology with the5� UTR or coding region was not associated with off-targetcleavage. Thus, when selecting siRNA sequences, special careshould be taken to avoid guide or passenger strand sequenceswhose “seed sequence” has homology to the 3� UTR of mRNAs. It seems quite likely that this same paradigm appliesto the siRNAs produced by Dicer cleavage of shRNAs. Clearlythese results have implications for the selection of therapeuticsiRNA or shRNA sequences. Dharmacon/Thermo Fisher Sci-entific (Lafayette, CO) provides a Web tool with which tosearch for seed matches in siRNA sequences (see http://www.dharmacon.com/seedlocator/default.aspx). These resultsalso underscore the importance of proper controls in siRNAscreens. If a particular phenotype is indeed associated with theknockdown of a particular mRNA, then multiple distinct si-RNAs targeting that mRNA should produce the same pheno-type. A more rigorous approach is to demonstrate rescue withan “immunized” transgene that contains silent mutations in thesiRNA-binding site, thus preventing RNAi silencing. Last, off-target effects are dose dependent (Birmingham et al., 2007),and therefore the minimal dose required to obtain sufficientknockdown should be used.

Thus, because RNAi and miRNA use overlapping machin-ery, care must be taken when designing RNAi triggers. Maxi-mal potency will reduce the dose required and reduce the pos-sibility of competition with the endogenous miRNA machinery.Care must also be taken to avoid off-target effects due to RNAitriggers acting as miRNAs.

USING miRNAs TO AVOID IMMUNERESPONSES TO VIRAL VECTORS

Naldini and coworkers described a clever use of miRNAsfor gene therapy (Brown et al., 2006). A significant hurdle tothe application of gene replacement therapy is the developmentof transgene-specific immunity (Thomas et al., 2003). For ex-ample, clinical trials for the treatment of hemophilia have beenhampered by the development of immune responses against vi-ral vectors and transgenes (High, 2005). A major cause of trans-gene-specific immunity is the expression of transgenes in pro-fessional antigen-presenting cells (APCs) (De Geest et al.,2003). Tissue-specific promoters are not sufficient to preventexpression in APCs because they are leaky. In an elegant proof-of-principle article, Brown and coworkers (2006) exploited thefact that the expression of miR-142-3p is enriched in hemato-poietic cells (Chen et al., 2004; Baskerville and Bartel, 2005).They placed four sites with perfect complementarity to miR-142-3p in the 3� UTR of the lentivirus carrying the gene en-coding green fluorescent protein (GFP), which under normalcircumstances is a potent neoantigen (Stripecke et al., 1999).


When they injected mice with a control lentiviral vector lack-ing the miR-142-3p sites, they observed GFP expression in he-patocytes, endothelial cells, Kupffer cells (liver macrophages),and splenocytes on day 5. However, by day 14, little or no GFPexpression was observed, presumably because of immune clear-ance. In contrast, when they injected mice with a lentivirus car-rying the GFP gene and with four miR-142-3p-binding sites inthe 3� UTR, they observed GFP expression in �1% of spleno-cytes, and then only in the marginal zone, which is not of he-matopoietic lineage. Presumably because they avoided host im-mune responses, with this vector they observed long-term GFPexpression (�120 days). Given the above-described report dis-cussing miRNA sponges, one concern is that high-level ex-pression of a transgene containing multiple binding sites for aparticular miRNA could compete with endogenous miRNA tar-gets for their cognate miRNA. Interestingly, when the authorsperformed the transfection with a second reporter containingfour miR-142-3p-binding sites, they did not see an increase inGFP expression. This suggests that miR-142-3p was not limit-ing in this system. Although speculative, this may be becausethe complementarity of the miRNA to the target was perfect.Thus, one would predict that the miRNA would cleave the tar-get and recycle rather than remain bound to it as is the case formiRNA sponges. In a separate report, Brown and coworkersapplied the same strategy to achieve long-term expression ofhuman clotting factor IX in hemophilia B mice (Brown et al.,2007). It seems likely that this strategy could be applied to thelong-term expression of other transgenes in vivo.

CONCLUDING REMARKS

There has been an explosive increase in our understandingof the role of miRNAs in normal gene regulation and in humandisease. Gene therapists are in a unique position to rapidly ap-ply approaches developed for antisense, ribozymes, and genetransfer to miRNAs. It seems likely that miRNA expression lev-els can be used as novel diagnostic markers. Manipulation ofmiRNA levels could also have therapeutic benefit. Last, by ex-ploiting our emerging understanding of miRNA biology, genetherapists may be able to design safer and more effective RNAitherapeutics as well as safer gene transfer vectors.

ACKNOWLEDGMENTS

The authors thank Ramona McCaffrey for editorial assis-tance. A.P.M. is supported by NIH RO1 AI068885. R.T.M. issupported by a Training Grant in Molecular Virology andPathogenesis (NIH RO1 AI007533) and the University of IowaDean’s Graduate Fellowship Award.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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Address reprint requests to:Dr. Anton McCaffrey

Department of Internal MedicineUniversity of Iowa

Iowa City, IA 52242

E-mail: [email protected]

Received for publication November 6, 2007; accepted for pub-lication November 22, 2007.

Published online: December 19, 2007.


advances in micrornas: implications for gene therapists

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