The Journal of Allergy and Clinical Immunology
Volume 121, Issue 2 , Pages 309-319, February 2008

Not miR-ly small RNAs: Big potential for microRNAs in therapy

  • Tara M. Love, PhD

      Affiliations

    • Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Mass
    • Department of Pathology, Harvard Medical School, Boston, Mass
    • ,
  • Howell F. Moffett, BA

      Affiliations

    • Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Mass
    • Department of Pathology, Harvard Medical School, Boston, Mass
    • ,
  • Carl D. Novina, MD, PhD

      Affiliations

    • Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Mass
    • Department of Pathology, Harvard Medical School, Boston, Mass
    • Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Mass
    • Corresponding Author InformationReprint requests: Carl D. Novina, MD, PhD, Department of Pathology, Harvard Medical School, Boston, MA 02115.

Received 30 October 2007; received in revised form 21 December 2007; accepted 26 December 2007.

Article Outline

RNA interference (RNAi) describes a set of natural processes in which genes are silenced by small RNAs. RNAi has been widely used as an experimental tool that has recently become the focus of drug development efforts to treat a variety of diseases and disorders. Like all molecular therapies, in vivo delivery is the major hurdle to realizing therapeutic RNAi. Several strategies have been developed that increase small RNA half-life in the blood, facilitate transduction across biological membranes, and mediate cell-specific delivery. Importantly, these strategies permit targeting of mRNAs as well as microRNAs (miRNAs), a class of small RNAs encoded in the genome. miRNAs are required for multiple developmental and cellular processes. Dysfunction of miRNAs can result in a host of pathologies, suggesting that miRNAs are potential targets of therapy. Recent studies of miRNA function in immune-specific pathways indicate that specific miRNAs might be exploited as therapeutic targets to treat immune disorders, including autoimmunity, allergy, and hematopoietic cancers.

Key words: RNA interference, small interfering RNA, microRNA, antagomiR

Abbreviations used: AMD, Age-related macular degeneration, CLL, Chronic lymphocytic leukemia, CPP, Cell-penetrating peptide, dsRNA, Double-strand RNA, HBV, Hepatitis B virus, miRNA, MicroRNA, RISC, RNA-induced silencing complex, RNAi, RNA interference, RSV, Respiratory syncytial virus, shRNA, Short hairpin RNA, siRNA, Small interfering RNA, SNALP, Stable nucleic acid lipid particles, TP, Target protector, UTR, Untranslated region

 

RNA interference (RNAi) is a collection of processes that use small RNAs to silence genes. As a naturally occurring pathway, one class of endogenous small RNAs, called microRNAs (miRNAs), is transcribed from cellular genes estimated to represent about 1% of all human genes.1 More than 1/3 of the human genome is predicted to be targeted by miRNAs,2 suggesting that numerous genetic networks may be affected by miRNAs. Indeed, miRNA functions are critical for several cellular processes including differentiation, apoptosis, proliferation, organogenesis, limb development, antiviral defense, and insulin secretion (see review3). It is therefore not surprising that miRNA dysfunction participates in a host of pathologies, most notably cancers. In fact, the term oncomiR has been coined to describe miRNAs that play a role in cancer formation, because particular miRNAs are now being considered oncogenes and tumor suppressors.4

As a tool, RNAi has transformed the way biomedical research is conducted and has already gained success in clinical trials. As an experimental method for discovery of gene function, RNAi uses exogenously introduced small interfering RNAs (siRNAs), which target particular mRNAs for destruction. Currently, siRNAs are the focus of RNAi-based clinical trials, although a large portion of academic, biotechnology, and big pharmaceutical efforts are now centered on the use of chemically synthesized small RNAs designed to manipulate miRNA expression. Each of these RNAi-based tools holds tremendous promise in molecular therapy. Importantly, the use of RNAi as a tool has grown up in parallel with the basic biology of RNAi. Therefore, the users of RNAi inform the scientists who investigate the biological mechanisms of RNAi in a previously unheralded manner. These and other properties of RNAi have led to the notion that the small RNA mediators of RNAi are especially good effectors and targets for therapeutic gene silencing.

Although miRNAs share many physical and biological properties with siRNAs, they produce very different functional outcomes in mammals. siRNAs are double-strand RNAs of 21 to 23 nucleotides that bind to mRNAs with perfect sequence complementarity and cause cleavage of the mRNAs (Table I; Fig 1, A). miRNAs are single-strand RNAs of 21 to 23 nucleotides and generally do not cleave mRNA targets. Instead, they bind to 3′ untranslated regions (UTRs) of mRNAs with imperfect sequence complementarity and repress their translation (Table I). Notably, one siRNA cleaving its mRNA target may provide robust gene silencing (sometimes more than 90% reduction) of target mRNA expression. Conversely, a single miRNA leading to translational repression generally does not lead to robust gene silencing of its target. In fact, the subtlety and power of miRNA-based gene silencing may derive from the fact that one miRNA targets multiple genes,5 many of which participate in common signaling pathways. Furthermore, several different miRNAs may target a single mRNA.5 Collectively, multiple miRNAs provide robust gene silencing of multiple target mRNAs. Indeed, the promiscuity of miRNAs make them attractive candidates for therapeutic gene silencing, especially because most, if not all, diseases are heterogeneous in nature and may be the product of dysregulation of more than 1 gene.

Table I. Comparison of siRNA and miRNA properties
siRNAmiRNA
ArchitectureDouble-strandSingle-strand
OriginExogenousEndogenous
No. of intended target genes per small RNA1Multiple
Degree of gene silencing per small RNAHighLow
Complementarity to mRNA targetPerfectImperfect
Effect on mRNAEndonucleolytic cleavageRepressed translation
  • View full-size image.
  • Fig 1. 

    Schematic of RNAi-based technologies. A, siRNAs cleave mRNAs and reduce mRNA levels. siRNAs are 21-nucleotide to 23-nucleotide RNA duplexes with 5′ recessed ends and 3′ overhanging ends that bind with perfect sequence complementarity to mRNAs and destroy them by endonucleolytic cleavage. B, Target protectors (TPs) block miRNA binding sites and increase mRNA levels. TPs are 25-nucleotide single-strand RNAs that bind specific mRNAs at miRNA-binding sites. TPs exhibit specificity in their target recognition by exhibiting perfect sequence complementarity to the seed region of the mRNA and 16 nucleotides outside the seed region that are not conserved between different target mRNAs. C, miRNA-mimetics reconstitute reduced miRNAs. miRNA-mimetics are duplexed RNAs with siRNA architecture whose gene-targeting strands are composed of exact sequences of mature miRNAs. Delivery of miRNA-mimetics can result in translational inhibition of multiple mRNAs (shown in different colors). D, AntagomiRs inhibit increased miRNAs. AntagomiRs are single-strand antisense RNAs with perfect sequence complementarity to mature miRNAs They permit effective inhibition of miRNA functions. Delivery of antagomiRs can result in de-repression of translation of multiple genes (shown in different colors). Addition of a cholesterol moiety (chol) to the 5′ end facilitates their uptake in vivo.61

Much of our knowledge on the use of siRNAs in therapy may be directly applied to the use of small RNAs that modulate miRNA pathways. The advent of RNAi has not only altered the way biomedical research is conducted but also it has progressed to the bedside at an astounding rate from the time of its discovery less than 10 years ago! In this review, we discuss the current state of the use of siRNAs in therapy and introduce current ideas for targeting miRNAs for therapy, which are not far behind siRNAs in the race for clinical trials. We also speculate on potential therapies for immune-specific diseases by targeting specific miRNA-regulated pathways implicated in normal immune function.

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siRNAs as a new class of drugs 

The use of RNAi as a therapeutic modality is making considerable progress, and siRNAs are now considered by many to be a new category of drugs. Currently there are 7 clinical trials underway that use siRNAs for treatment of a variety of diseases and disorders (Table II), and many more are in the pipeline to commence phase I clinical trials in 2008. The success of siRNAs in therapy derives in part from their powerful and efficient mechanism of gene knockdown, sequence-specific cleavage of mRNAs (Table I; Fig 1, A). Cleavage of targeted mRNAs is mediated by the RNA-induced silencing complex (RISC), an efficient, multiple turnover enzyme.6 That is, 1 RISC is recycled multiple times to cleave multiple copies of the same target mRNA. Consequently, very small amounts of siRNAs (nmol/L) are necessary to elicit a potent gene silencing response in vivo. Although a systematic and comparative analysis on multiple target mRNAs using siRNAs, antisense oligonucleotides, and ribozymes has not been performed, it is likely that siRNAs are more sensitive and potent than these other RNA-based forms of therapy. However, modifications to the newer generation of antisense molecules and ribozymes have significantly improved their performance (see reviews7, 8).

Table II. Current siRNA-based clinical trials
DrugDisease/conditionDrug targetTarget tissueNature and method of RNAi deliveryCompanyClinical trial status
BevasiranibWet AMDVEGFEyeNaked siRNA, intravitreal injectionOpko (Miami, Fla)August 2007, phase III
Sirna-027/AGN211745Wet AMDVEGF receptorEyeNaked siRNA, intravitreal injectionMerck/Sirna (San Francisco, Calif)/Allergan (Irvine, Calif)August 2007, phase II
ALN-RSV-01RSV infectionRSV nucleocapsid (N)LungNaked siRNA, intranasal deliveryAlnylam (Cambridge, Mass)June 2007, phase II
RTP801i-14Wet AMDHypoxia-inducible gene (RTP801)EyeStabilized chemically modified siRNA, intravitreal injectionQuark (Fremont, Calif)/Silence (London, United Kingdom [UK])/Pfizer (New York, NY)November 2007, phase I/IIa
AKIi-5Acute renal failurep53 geneKidneyStabilized chemically modified siRNA, intravenous deliveryQuark/SilenceNovember 2007, phase I
CALAA-01Solid and metastatic tumorsM2 subunit of ribonucleotide reductaseTumorNanoparticle-packaged siRNA with transferrin to target tumors, intravenous deliveryCalando (Pasadena, Calif)End of 2007, preclinical/phase I
NUCB1000HBV infection4 HBV genesLiverCationic lipid-packaged DNA-based plasmid expressing 4 shRNAs, intravenous deliveryNucleonics (Horsham, Pa)/Novosom (Halle, Germany)May 2007, phase I
rHIV7-shl-TAR-CCR5RZAIDS-related lymphomaHIV tat/rev gene, TAT responsive elements, CCR5 receptorStem cellsDNA-based plasmid expressing anti-HIV RNA, including shRNA, lentiviral infection of stem cells with plasmid followed by stem cell transplantBenitec (Melbourne, Australia)June 2007, phase I

VEGF, Vascular endothelial growth factor.

Mammals tolerate very large doses of siRNAs. Mice receiving 2.0 to 2.5 mg siRNAs injected directly into their bloodstreams did not demonstrate signs of toxicity.9 In contrast, mice receiving high titers of adeno-associated viruses expressing multiple different short hairpin RNAs (shRNAs) to 6 target genes died from toxicity.10 This latter strategy requires expression of shRNAs, which are double-strand RNAs that form a hairpin structure containing a stem of perfectly complementary sequences that get processed into siRNAs by endogenous endonucleases. Thus, shRNAs enter the natural RNAi pathway at an earlier point than siRNAs. The lethality in mice receiving shRNAs was attributed to saturation of the stabilizer and transporter of siRNA/miRNA precursors, Exportin 5 (limiting in cells), which, in turn, caused a global decrease in endogenous miRNAs, leading to liver toxicity and morbidity.10 Together, these observations support the notion that, if necessary, siRNAs may be administered at high doses because they enter the natural RNAi pathway at a step later than Exportin 5 function and therefore do not induce toxicity. This property of siRNAs is especially useful in cases in which higher doses manifest stronger therapeutic effects. For example, Merck's drug AGN211745 (Merck, Whitehouse Station, NJ) showed varied dose-dependent effects during phase I clinical trials to treat age-related macular degeneration (AMD; Table II). A single intravitreal injection resulted in maintenance of visual acuity at lower doses (100 μg), but at higher doses (as much as 800 μg), visual acuity was greatly improved without toxic side effects.11

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Unintended effects of siRNAs 

One major concern with the use of siRNAs in therapy is the potential for off-target effects, gene silencing by siRNAs on unintended mRNA targets. In one study, introduction of an individual siRNA into cells had small (∼2-fold) effects on hundreds of genes.12 It is likely that the 2 strands of the introduced siRNA had imperfectly complementary base-pairing with certain mRNAs; that is, the siRNA strands may have mimicked miRNA targeting of mRNAs. Efforts to reduce off-target effects of siRNAs include choosing siRNA design algorithms that result in more functional siRNA targeting and avoid sequence similarities with off-target mRNAs. These algorithms aim to reduce the amount of siRNAs required for on-target silencing. Additional efforts to reduce off-target effects have focused on modifying the sense and antisense strands of siRNAs (see reviews13, 14). For example, 2′ O-methyl modification at position 2 of the antisense strand of an siRNA duplex significantly reduces off-target effects without compromising silencing of the intended mRNA target.15 Still, some effects on unintended targets cannot be avoided; however, in gene therapy and gene discovery, small effects on unintended targets may be acceptable to achieve very large effects on intended targets.

Another concern with using siRNAs in therapy is their potential to be immunogenic. Generally, long double-strand RNAs (dsRNAs) introduced into mammalian cells activate protein kinase R, a dsRNA-dependent protein kinase and key mediator of the IFN pathway. Shorter dsRNAs, including siRNAs (<30 bp), have previously been reported to avoid induction of the IFN pathways16, 17, 18, 19, 20; however, multiple reports have also demonstrated cases in which siRNAs do trigger an IFN response.20, 21, 22 This property may depend on the cell context20 and the sequences of the siRNAs.23, 24, 25 Therefore, any indication of IFN activation by particular siRNAs should be followed up by using siRNAs with different sequences devoid of certain immunogenic motifs (G-U-rich23, 26). In addition, one or two 2′ O-methyl base modifications in 1 strand of an siRNA duplex can significantly decrease the immunostimulatory characteristics of that siRNA.26

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Delivery of siRNAs 

The delivery of siRNAs has been the biggest hurdle to overcome in RNAi-based therapies. Indeed, the success of siRNAs in therapy depends critically on efficient delivery. siRNA delivery schemes benefit from lessons learned through use of other nucleic acid–based delivery schemes. However, siRNAs have properties that might be exploited for their efficient delivery, which may reduce the height of this hurdle. Compared with antisense oligonucleotides, siRNAs work via a more potent and efficient catalytic mechanism, suggesting that lower amounts may be required for effective gene silencing. Compared with ribozymes and plasmid-based therapies, the small size of siRNAs may better facilitate their uptake by cells. Moreover, the duplex nature of siRNAs makes them fairly resistant to exonuclease-mediated degradation, although their half-life in serum is still relatively short. Several tools exist or are being developed to facilitate delivery of small RNAs. Some of these strategies are discussed here.

Most mammalian tissues do not readily take up naked siRNAs partly due to their dilution in the blood stream. However, certain tissues, such as the eye and respiratory tract, are easily accessible by clinicians and are relatively isolated compartments composed of mostly nondividing cells. Therefore, successfully delivered siRNAs will not be rapidly cleared or diluted and can generally support a sustained gene silencing response from a single dose. In addition, these sites are not exposed to the blood supply, which contains degrading enzymes detrimental to naked siRNAs. Because of all these qualities, relatively lower doses of siRNAs are necessary to elicit potent gene silencing responses in the eyes and lungs. To this end, it is not surprising that the most progress has been made in the eye-based and lung-based clinical trials using siRNAs (Table II). Naked siRNAs are successfully being delivered to the eyes by direct injection into the vitreous fluid to treat AMD, and to the lungs by inhalation to treat respiratory syncytial virus (RSV) infection.

Systemic delivery of siRNAs to tissues is certainly not as trivial as local delivery schemes for siRNAs described. The systemic delivery challenge may be conceptually broken down into 3 separate issues with different strategies under investigation to cross the delivery hurdle: increasing the half-life of siRNAs in the blood, transducing siRNAs across biological membranes, and mediating cell-specific delivery. In our discussion, these issues are artificially separated for purposes of illustration to provide a more complete understanding of specific problems and some common solutions to these problems. It is important to note that many of the siRNA delivery strategies discussed are multifunctional and can be used to overcome multiple aspects of the delivery hurdle. In addition, the most successful siRNA delivery schemes generally make use of more than 1 modification to increase the half-life of siRNAs in the blood, to transduce siRNAs across biological membranes, and to mediate cell-specific delivery. For purposes of clarity, certain strategic modifications to siRNAs are highlighted for their applicability in 1 of the 3 sections.

Virus-mediated delivery of siRNAs has 1 major advantage over the delivery of chemically synthesized siRNAs: strong, constitutive expression of siRNAs in target tissue that will not be diluted as cells divide. However, virus-mediated delivery of any gene or small RNA presents many limitations, including immunogenicity, toxic side effects from viral infection, and in the case of retroviruses, a strong potential for insertional mutagenesis (see review27). In addition, high expression of viral shRNAs may saturate the limiting RNAi machinery, causing problems with endogenous miRNA production.10 Therefore, most efforts for delivery of small RNAs have focused on delivery of chemically synthesized siRNAs rather than on virus-delivered siRNAs. The issues concerning virus-mediated delivery of transgenes have been addressed previously27 and are not further addressed here. Instead, we focus our attention on strategies to deliver chemically synthesized siRNAs, which inform strategies to deliver small RNAs that can be used to modulate miRNA pathways.

Increasing the half-life of siRNAs in the blood 

Naked siRNAs in the bloodstream are easily degraded because the serum contains high amounts of nuclease activities. One way to reduce nuclease sensitivity is through chemical modification of siRNAs. Chemical modifications of siRNAs that increase their stability include phosphorothioate addition to 3′ ends and 2′ O-methyl, 2′-fluoro, and locked nucleic acid–type substitutions to the ribose backbone (see review14). Indeed, some of these modifications are incompatible with RNAi functioning but possess characteristics that make them extremely useful in the modulation of miRNA pathways. Some of these modifications are discussed in more detail in the section on miRNA pathway modulators.

Another means to the reduce nuclease sensitivity is to enclose siRNAs in a synthetic particle, such as a liposome or polymer nanoparticle. These delivery vehicles keep siRNAs in a protected milieu until they are released into target cells. In fact, a combination of chemically modified siRNAs packaged into nonviral carriers (liposomes or polymer nanoparticles) is becoming the standard for stabilization and delivery of siRNAs in vivo. Liposomes are synthetic phospho-lipid bilayers containing a core of aqueous solution. They have been successfully used for in vivo delivery of chemical drugs,28, 29, 30 plasmid DNA,31 and, more recently, siRNAs.32 Typically, cationic lipids are used to create siRNA liposomes. The cationic lipids bind to the net negatively charged siRNAs and form a lipid bilayer around the siRNAs, thereby protecting them from degradation and increasing their half-life and potency.32 When administered, siRNA-liposomes fuse to target cell membranes, and siRNAs are released into the cytoplasm through endocytosis. siRNA-encapsulated liposomes are very easily absorbed by some tissues, such as the epithelium and lamina propria of the mouse vagina. In 2006, Palliser et al33 reported topical vaginal application of siRNA-encapsulated cationic liposomes directed toward herpes simplex virus–specific genes. The result was sustained gene silencing and protection from lethal herpes simplex virus infection, suggesting that topical lipid-based siRNA creams might be an option for treating other epithelial disorders.

In some cases, cationic lipid-mediated delivery of siRNAs has been associated with toxicity34, 35 and induction of the IFN response.25, 36, 37, 38 These properties can be greatly improved by packaging chemically modified siRNAs into specialized cationic liposomes, as was done by Morrissey et al38 in 2005 to suppress hepatitis B virus (HBV) replication in mice. Referred to as stable nucleic acid lipid particles (SNALP), these were refractory to immunostimulation and worked potently at dosage levels 10 times less than cationic liposomes containing unmodified siRNAs.38 Another way to reduce toxicity and immunogenicity of siRNA-liposomes is to adjust lipid composition, for example, by using neutral lipids. In mouse models, the use of neutral lipids to create siRNA liposomes greatly enhanced efficiency of delivery and potency of silencing by using very low amounts of siRNAs.39, 40, 41

Cationic polymer-based nanoparticles are ideal carriers of siRNAs because of their ability to stabilize siRNAs and efficiently deliver them in a targeted fashion. Nanoparticle formulations allow the addition of targeting ligands to surfaces of nanoparticles. Furthermore, large-scale manufacturing of such particles is feasible, and in general, nanoparticles are less toxic than their liposome counterparts, offering the advantage of being biodegradable. A widely used method of nanoparticle-based siRNA protection and delivery is polyethyleneimine complexation. Polyethyleneimines are highly positively charged, bind with high affinity to siRNAs, and have demonstrated efficient endocytotic uptake of siRNAs in vivo (see review42).

Transducing siRNAs across biological membranes 

In addition to making sure that delivered siRNAs are protected from degradation, it is important that they enter their target cells. As mentioned, most mammalian cells do not efficiently take up naked siRNAs. Instead, the siRNAs need to be packaged in some way that allows them to traverse the lipid bilayer of cell membranes. This has been achieved by using synthetic packaging particles, such as the liposomes and polymer nanoparticles described above, which can facilitate the endocytosis of packaged siRNAs. An alternative strategy for getting siRNAs into cells is to add a cholesterol moiety to the siRNAs. In 2004, Soutschek et al43 showed that the addition of a cholesterol moiety to siRNAs greatly enhanced their uptake into liver cells and caused potent knockdown of their target gene, apolipoprotein B. The mechanism of uptake of cholesterol-conjugated siRNAs is thought to be facilitated by the low-density lipoprotein (LDL) receptors present on many cell types. Interestingly, this was the first demonstration of systemic delivery of siRNAs by intravenous injection into mice, which was also the first feasible and clinically relevant mode of delivery for siRNAs that had been reported.

Another method for packaging and cellular delivery of siRNAs is the use of cationic peptides, called cell-penetrating peptides (CPPs). The strong positive charge on these peptides promotes binding and condensation of negatively charged biomolecules, including siRNAs. Discovered more than a decade ago, CPPs have been shown to transduce oligonucleotides and other biologically active molecules efficiently across cellular membranes in a receptor protein–independent manner (see review44). siRNAs can be packaged with CPPs either covalently or through electrostatic interactions, and treatment of cells with siRNA/CPP complexes has been successful in vitro, with minimal toxicity.45, 46 However, in vivo applications for siRNA/CPP complexes have yet to be successfully executed. One lesson learned from in vitro studies is that siRNA/CPP complexes may not always escape from endosomes once they are taken up by cells. Thus, future formulations of siRNA/CPP complexes are focused on making the CPP portion of the complexes more endosomolytic.46

Mediating cell-specific delivery 

In addition to identifying molecular targets for therapy, defining cellular targets for therapy is critical for increasing intended effects while minimizing unintended effects of siRNAs. Without targeting modules associated with siRNAs, bulk siRNAs injected into the bloodstream will eventually lead to the liver, because many drugs are metabolized by the liver. Efficient systemic delivery of siRNAs to a particular tissue other than the liver requires a specific targeting component to be added to siRNAs. To make this work effectively, it is imperative that the target tissue expresses a cell surface protein that will recognize the targeting ligand. This bodes well for tumor cells, which tend to express cell surface receptors not found in normal cells aberrantly. For example, the transferrin receptor is highly expressed on the surfaces of a broad range of tumor cell types. Capitalizing on this characteristic of cancer cells, Calando Pharmaceuticals (Pasadena, Calif) uses nonchemically modified siRNAs coupled to transferrin receptor targeting agents, composed of specialized cyclodextrin-based nanoparticles (Table II). The siRNAs encapsulated in the nanoparticles are complimentary to the M2 subunit of the ribonucleotide reductase gene, which is a well established cancer target. Thus, the idea is to target a broad range of tumor cells via the transferrin receptor and knock down an aberrantly expressed gene important for the tumor cell's proliferative capacity.

Another method used to deliver siRNAs to specific cell addresses is coupling of siRNAs to antibody-protamine fusion proteins. Protamine is a positively charged protein that naturally packages DNA in sperm cells and thus efficiently packages siRNAs, protecting them from degradation. Because the siRNAs are not covalently bound to the protamine, cocktails of siRNAs can be used in the same delivery module. The antibody component of the fusion is responsible for targeting specific cell surface antigens and thus offers the advantage of targeting very specific subsets of cells. The siRNA payload is then delivered by receptor-mediated endocytosis. By using siRNA-protamine-antibody fusions, Song et al47 demonstrated potent silencing of target genes in HIV-infected cells and HER2-positive cells in mice. Similarly, Wen et al48 used siRNA-protamine-antibody fusions to inhibit HBV gene expression in mice. Next, it will be interesting to see what other cell types are targeted in vivo using this technology. For example, this technology might be used to deliver siRNAs specifically to acute myelogenous leukemia cells in the peripheral blood via the acute myelogenous leukemia–stem cell—specific marker, CD96.49

Viral peptides have been exploited for their use in targeted siRNA-based therapies. Recently, it was demonstrated that siRNAs could cross the blood-brain barrier.50 Preventing the passage of therapeutic agents to the brain, the blood-brain barrier has been a major obstacle in treating brain pathologies. To get around this obstacle, researchers used a 29–amino acid peptide from the rabies virus (which binds the acetylcholine receptor of neuronal cells) as a carrier for targeted delivery of siRNAs into the brain, thus successfully protecting mice from Japanese encephalitis virus infection.50 This was a major breakthrough, not only from the standpoint of targeted RNAi therapeutics. It was the first successful demonstration of intravenous delivery of a therapeutic agent to the brain.

Another avenue for targeting siRNAs to specific cell types has been derived from evolutionary tools used to define novel binding motifs on surfaces of specific cells. An example of one such tool is the tryptophan cage, an independently folding 20–amino acid protein domain derived from a longer peptide isolated from the Gila monster (Heloderma suspectum). The tryptophan cage is a hydrophobic cluster of amino acids with tryptophan buried in a central location and has been optimized for efficient folding. Also, tryptophan cages are highly amenable to modifications at several amino acid positions. Herman et al51 exploited these properties of tryptophan cages by expressing them in a phage-display library such that different combinations of amino acids composing the tryptophan cage form motifs that exhibit cell type–specific binding in vivo. Therefore, these tryptophan cage variants recognize different molecular zip codes by binding to specific cell surface proteins. Tryptophan cages may provide another avenue for siRNA conjugation and targeting to specific cells and offer the advantage of being much smaller than antibodies, making them easier to manufacture on a large scale.

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miRNA pathway mediators as the next new class of drugs 

Pharmaceutical companies have recently begun to move toward the use of miRNAs as a therapeutic modality. If siRNAs are efficient at targeting mRNAs by co-opting a natural endogenous pathway that is already in place for miRNA-mediated silencing of genes, one idea is to eliminate the middle man and target miRNAs directly. Several new companies are predicated on using miRNAs as diagnostic and therapeutic tools for a range of diseases. Interestingly, companies that have already developed and marketed other RNA-based therapies, such as antisense RNA drugs, are helping to accelerate the progress of therapeutic miRNA technologies. Possessing long-standing experience and expertise in RNA chemistry, these companies have jumped into the RNAi therapeutic race in full force. Lessons learned about delivery and use of antisense RNAs can be applied to siRNAs and miRNAs (see review52). Then again, there has been a renaissance in antisense technologies since the discovery of RNAi. RNAi not only has benefited from antisense technologies but also may inform further development of antisense technologies as more people effectively use RNAi. Thus, collaborative efforts of old and new RNA companies pose a powerful force in small RNA therapeutics, including miRNA therapeutics.

Every cell has a unique miRNA expression signature that reflects the identity and functions of that cell. Profiling of miRNA expression patterns in numerous cell types and numerous pathways has identified differential expression of miRNAs characteristic of certain tissues under specific physiological stimuli. miRNA expression profiling can identify cell types in normal and diseased states, such as in cancer (see review53). Therefore, it is possible to target miRNAs that mediate a particular cellular process without knowing all of the genes involved in that process. This presents a major advantage to using RNAi to target miRNAs rather than to target mRNAs. Globally, miRNAs are predicted to target more than 1/3 of the genome, which is roughly more than 6600 genes.2 Whereas an siRNA targets 1 gene, a single miRNA is predicted to target between 100 and 200 genes.5

One emerging paradigm for miRNA function in mammals is that the effect of a single miRNA on translational repression of a particular mRNA containing 1 miRNA binding site is very small in degree. To obtain robust gene silencing by a miRNA, multiple miRNA binding sites must be present in a particular mRNA that act cooperatively.54 As stated, the emerging paradigm for miRNA function in mammals is that a single miRNA may target multiple genes of a particular signaling pathway. Because some genes are involved in multiple pathways, altering expression of any 1 miRNA may lead to effects on unintended pathways. However, the effect of altering multiple gene targets of that miRNA in the intended pathway will be amplified by having small-fold intended effects become multiplicative. Because certain miRNAs and mRNAs (and subsequently the pathways they mediate) will be selectively activated, it is likely that altering a particular miRNA will have small effects on unintended pathways and large effects on intended pathways. Thus, the power of targeting a single miRNA far exceeds that of targeting a single gene, as with siRNAs.

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Tools for altering cellular miRNA pathways 

Several tools are available that enable selective targeting of miRNA pathways, including tools that reconstitute reduced miRNAs (miRNA-mimetics; Fig 1, C) and reduce overexpressed miRNAs (antagomiRs; Fig 1, D). Recently, technologies have been described that permit selective protection from miRNAs through accessibility to cognate miRNA binding sites (target protectors; Fig 1, B).

miRNA-mimetics 

For cases in which miRNAs are consistently reduced in disease processes or overexpression of miRNAs would be a therapeutic benefit, miRNA-mimetics can be used (Fig 1, C). miRNA-mimetics are synthetic small RNAs usually containing the exact sequence of the endogenous miRNA. Instead of delivering a single strand, miRNA-mimetics are delivered as perfectly complementary duplexes, like the architecture of siRNAs. This is because RISC is much more efficient in loading small RNAs originating from a duplex rather than a single strand.55 A powerful advantage of using miRNA-mimetics is that these synthetic miRNAs can be modified to be more potent than their naturally occurring forms. For example, miRNA-mimetics have been designed to target glioma-associated antigen 1 (Gli-1) with a much higher binding affinity than naturally occurring miRNAs that target Gli-1.56 These modified miRNA-mimetics more strongly inhibited Gli-1–mediated proliferation of pancreatic tumor cell lines than their natural counterparts.56 Currently, the delivery of miRNA-mimetics has yet to be demonstrated in mouse models with the ultimate goal of replacing lost miRNA functions in human clinical models.

AntagomiRs 

For cases in which miRNAs are consistently upregulated in disease processes or reduction of miRNAs would be a therapeutic benefit, antagomiRs can be used to block those miRNAs directly and specifically. AntagomiR is used here as a generic term to describe any antisense oligonucleotide that directly binds to miRNAs and blocks their activities. AntagomiRs may work by stoichiometric interaction with mature miRNAs by titrating them from biologically active pools of mature miRNAs (Fig 1, D). AntagomiRs may also work by binding to miRNA precursors and inhibiting the biogenesis of mature miRNAs. Although it is not exactly clear how antagomiRs inhibit miRNA functions, their effect is to prevent incorporation of mature miRNAs into RISC. An advantage of using antagomiRs in therapy is that they potently and specifically inhibit miRNAs and therefore affect hundreds of genes. This is also a disadvantage if only a subset of those genes is the intended point of therapy.

Early reports on antagomiR use described 2′ O-methyl RNA oligonucleotides that were perfectly complementary to the antisense strand of siRNAs and blocked their function in vitro.57, 58 The first in vivo demonstration of the use of antagomiRs to block miRNA activities was in worms, in which 2′ O-methyl oligonucleotides perfectly complementary to let-7 induced a let-7 loss-of-function phenotype.58 Since the first reports, different types of antagomiRs have been widely used as tools to probe the functions of miRNAs in mammalian cells. For example, miR-21 was potently inhibited in glioblastoma cells by using either 2′ O-methyl or locked nucleic acid–based antagomiRs.59 In addition, antagomiRs not only have been used to target mature miRNAs but also have been designed to block miRNA biogenesis by targeting miRNA precursors.60

A major step toward the goal of probing miRNA functions in vivo was successful systemic delivery of antagomiRs in mice to knock down miR-122, a liver-specific miRNA.61 The antagomiR used in these studies was 2′ O-methylated and contained a cholesterol moiety on the 5′ end, which facilitated its uptake into hepatocytes, causing specific miR-122 silencing for more than 1 week after a single intravenous injection.61 In these studies, several genes involved in cholesterol biosynthesis were upregulated on miR-122 inhibition, and serum cholesterol levels went down by 44%.61 Further studies using miR-122–specific antagomiRs in vivo revealed specific roles for miR-122 in lipid metabolism and further supported the notion that miRNA-specific inhibition by antagomiRs holds great therapeutic promise.62

Target protectors 

A possible option to affect only certain miRNA target mRNAs is to use RNA-binding antisense oligonucleotides. Target protectors (TPs; Fig 1, B) are complementary to miRNA binding sites in the 3′ UTR of specific genes, thus preventing miRNA access to those sites. These specialized oligonucleotides have recently been reported to interfere with miR-430–mediated repression of specific 3′ UTRs in zebrafish.63 The specificity of TPs for select miRNA binding sites within 3′ UTRs relies on certain miRNA:mRNA binding properties. A miRNA:mRNA pair usually possesses perfect complementarity between nucleotides 2 and 7 from the 5′ end of the miRNA and its binding site in the target mRNA. This is called the seed region of the mRNA and is highly conserved among the mRNA targets of any 1 specific miRNA. TPs take advantage of the fact that flanking sequences 5′ and 3′ of the seed region are much less conserved among miRNA binding sites within specific 3′ UTRs. Thus, these flanking regions provide a fingerprint unique to each miRNA target. The TPs used in the zebrafish studies were 25 nucleotides long and were complementary to 9 nucleotides encompassing the seed region of miR-430 and 16 more nucleotides 5′ or 3′ of the seed region of the mRNA62 (Fig 1, B). These TPs were effective at selectively blocking miR-430–mediated inhibition of 2 different targets.63 Therefore, TPs are advantageous in cases in which the mRNA targets of miRNAs are known. In the years to come, as more and more targets of miRNAs get validated, this technology will become immensely useful.

Delivery of miRNA pathway modulators 

Delivery issues for miRNA-modulating small RNAs are similar to issues concerning siRNAs. For example, once the small RNA gets to its location, how can persistence of therapeutic activity be ensured? For nondividing cells, this is less of an issue. For rapidly dividing cells, like cancer cells, small RNAs will be diluted with each cell division. In these cases, dosage may be important such that high quantities of miRNA-modulating small RNAs may be needed to ensure a long enough duration of the biological effect. However, the problem with using large amounts of miRNA-modulating small RNAs might be that cellular pathways are affected that are not germane to therapy. Constitutive expression of miRNA-modulating small RNAs using viral vectors would likely support persistent therapy in rapidly dividing cells. In these cases, it may be important to consider side effects of viral vectors. It may also be that temporary modulation of miRNAs by transient delivery of miRNA pathway–modulating small RNAs will be beneficial in some cases. For transient biological processes, like the inflammatory response in which temporary miRNA modulation is logical, delivery of miRNA-modulating small RNAs might be an effective means of changing immune responses temporarily. For these transient biological processes, the dilution of small RNAs as cells divide might make the strategy more reasonable. One might even envision a quick fix to alter miRNA levels in the treatment of disorders with acute exacerbations, such as asthma.

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Potential applications of miRNA pathway modulators in immune diseases 

During allergy and autoimmunity, the immune system retains a relatively normal response to pathogens. At the same time, the immune system demonstrates pathological responses to self or environmental antigens. Recent research suggests that miRNAs play important roles in regulation of innate and adaptive immune responses, as well as in the interplay between immune and nonimmune tissues. In addition, dysregulated miRNA expression has been linked to numerous hematopoietic cancers in human beings and leukemogenesis and lymphomagenesis in animal models. Together, these reports suggest that autoimmunity, allergy, and hematopoietic cancers are cases in which the subtle yet pervasive effects of miRNAs could be harnessed to affect positive clinical outcomes. We present examples of potential applications for immune-specific miRNA pathway modulators.

Autoimmunity and allergy 

Much of current drug therapy for T-cell–dependent autoimmune conditions depends on nonspecific immune suppression by immunomodulating agents such as prednisone, cyclosporine, or azathioprine. Use of these agents is hampered by significant off-target side effects, hematopoietic toxicity, and immune suppression, with an attendant risk of opportunistic infection. Manipulating miRNA expression has the potential to inhibit autoimmune reactions significantly without side effects of current chemotherapeutic regimens.

miR-181a plays an important role in lymphocyte development.64, 65 In addition, increases in miR-181a expression in T cells have been shown to lower the threshold for T-cell responses.66 Thus, a potentially interesting target for therapy is miR-181a. Modulating miR-181a may affect regulation of T-cell responses to stimuli. The majority of autoimmune T cells have very low affinity for their cognate antigens, which allows them to escape multiple rounds of selection against autoreactive T-cell receptors during T-cell development. These autoimmune cells will thus be highly sensitive to slight changes in the threshold of T-cell activation, which is controlled by miR-181a levels. Targeted delivery of miR-181a–specific antagomiRs to peripheral T cells might preferentially inhibit low-affinity autoimmune responses while preserving high-affinity T-cell responses to pathogens.67

Two other miRNAs that play important roles in both adaptive and innate immunity are miR-146 and miR-155. Expression of these 2 miRNAs is induced in response to activating signals, either by T-cell and B-cell receptor ligation, or by stimulation of innate immune cells through Toll-like receptors. Recently, 2 mouse knockouts of miR-155 were described (see review68). Mice lacking miR-155 had severe defects in adaptive immune responses to bacterial pathogen challenge, which was associated with TH2-biased cytokine secretion from CD4+ T cells and dendritic cells as well as impaired B-cell differentiation. In addition, these mice demonstrated autoimmune phenotypes in the lungs, with increased airway remodeling and leukocyte invasion.69, 70 These observations indicate that modulation of miR-155 may allow therapeutic manipulation of the immune response.

In B cells, miR-155 is required for differentiation in response to antigen and formation of a high-avidity antibody response. Consequently, inhibition of miR-155 would be predicted to significantly repress formation of a new antibody or autoantibody response. In T cells, modulating miR-155 expression may allow for shifting the focus of an ongoing immune response. In peripheral T cells, increasing miR-155 levels with miR-155 mimetics would lead to a TH1-like cytokine profile and enhancement of the T-cell response. Conversely, decreasing miR-155 levels with specific antagomiRs would lead to a TH2-like cytokine profile and inhibition of the T-cell response. The role of miR-155 in innate immunity is less well defined but may also serve as a therapeutic target in autoimmune diseases involving local or systemic inflammation.

The gene encoding miR-146 has not been knocked out. However, the expression and target specificity of miR-146 suggest that this miRNA plays an important role in the regulation of cytokine responses. miR-146 is preferentially expressed in TH1-type cells and has been shown to regulate the key cytokine signaling adapter molecules, IL-1 receptor–associated kinase (IRAK1) and TNF receptor–associated factor (TRAF) 6, in innate immune cells.71, 72 Although additional research is needed, these results suggest that manipulation of miR-146 expression could significantly affect the TH1/TH2 cytokine balance.

The ability to modify cytokine secretion therapeutically should have significant implications for treatment of TH1-biased autoimmune conditions, such as type 1 diabetes, as well as allergic diseases, including asthma, which are highly dependent on TH2 cytokine secretion. TH2 cytokines drive allergic responses such as asthma, whereas TH1 cytokines can actually inhibit allergic responses. Consistent with this notion, the miR-155 knockout mice demonstrated phenotypes similar to those seen in asthma, with increased airway thickness and leukocyte invasion.69, 70 Interestingly, miR-155 lies within a chromosomal locus associated with asthma in human populations,73 supporting a role for miR-155 in human asthma.

miRNAs also participate in regulation of immune responses by nonhematopoietic cells. miR-203 is upregulated in keratinocytes during autoimmune psoriasis and has been associated with maintenance of local inflammation in this condition.74 One regulatory target of miR-203 is a gene known as suppressor of cytokine signaling 3 (SOCS-3). Transdermal delivery of siRNAs to disturbed epithelium has been reported.75 Inhibition of miR-203 with antagomiRs or blockade of the miR-203 binding site on SOCS-3 using TPs in keratinocytes could break the cycle of inflammation that leads to psoriatic lesions.

Hematopoietic cancers 

Efforts to understand biological roles of miRNAs have been bolstered by findings that miRNAs are linked to cancer. The first report implicating miRNAs in human cancer showed that 2 miRNA genes, coding for miR-15a and miR-16-1, were located at a chromosomal region frequently deleted or downregulated in chronic lymphocytic leukemia (CLL).76 In fact, a genome-wide analysis of 186 miRNA loci revealed that miRNA genes are often located in genomic regions that are deleted or amplified in several cancers, including follicular lymphoma and prolymphocytic leukemia.77 In addition to loss of miR-15a and miR16-1 in CLL, other hematopoietic malignancies, such as B-cell lymphomas, have been characterized by overexpression of particular miRNAs, including miR-15578, 79 and the miR-17-92 cluster.80 Thus, the emerging view from genomic and miRNA expression data has been that miRNAs lost in cancers might be tumor suppressors and those amplified in cancers might be oncogenes. In support of this notion, global profiling of miRNA expression levels in hundreds of different cancers revealed that miRNAs are most often reduced in cancers, with cases (albeit many fewer) in which miRNAs are increased.81 In addition, these profiling studies introduced the idea of classification of cancers on the basis of miRNA profiles, such that each profile was a fingerprint for cellular origin and differentiation state of a particular cancer.81 These studies suggested that miRNA expression profiles can be used as a diagnostic tool and to predict prognosis or response to therapy. In fact, multiple groups have reported a strong correlation between specific miRNA expression signatures and the progression and prognosis of various cancers, including CLL (see review53).

Although miRNA expression profiles can serve as markers of cancer, they do not directly address the causality of miRNAs to cancer. The first direct demonstration that miRNAs could indeed cause cancer came from work showing that overexpression of RNA from the BIC locus, later found to encode miR-155, cooperated with Myc to drive erythroleukemogenesis and lymphomagenesis in chickens.82 Similarly, the miR-17-92 polycistron was shown to cooperate with Myc to drive B-cell lymphoma formation in mice.80 These data immediately suggest that miR-155 and miR-17-92 may be reasonable targets of antagomiR-based therapy for certain leukemias and lymphomas.

Other miRNAs may also be therapeutic targets for hematopoietic malignancies on the basis of intriguing cell culture data. For example, overexpression of miR-223 in an acute promyelocytic leukemia cell line enhanced differentiation of granulocytes.83 This suggests that administering a miR-223 mimetic might be a treatment for acute promyelocytic leukemia, which is characterized by a loss of differentiation and proliferation of myeloid precursor cells, including pregranulocytes. Similarly, the administration of a miR-221/222 mimetic might be a treatment for erythroid leukemias because the overexpression of miR-221 and miR-222 reduced proliferation of a erythroleukemia cell line.84 A recent report revealed that overexpression of let-7a in a Burkitt lymphoma cell line had a strong antiproliferative effect because of specific targeting of Myc by let-7a.85 Thus, perhaps either let-7a–targeted antagomiRs or Myc-targeted TPs would be options for treatment of Burkitt lymphoma and possibly other MYC-dysregulated hematopoietic malignancies.

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Future directions 

miRNA expression profiling efforts have provided a molecular taxonomy of cancers and, by extension, of normal cells. miRNA expression patterns may be used to classify cells and to identify the origin of cells from a variety of pathologies. Although it is clear that miRNAs that cause disease may be targets for therapy, it is also possible that miRNAs that are not involved in the etiology of pathological processes may also be targets for therapy. That is, a characteristic miRNA expression pattern may be linked to a particular disease prognosis or susceptibility to treatment. Therefore, modulating expression of these signature miRNAs could possibly alter prognosis or susceptibility to treatment.

To illustrate this point, consider the following scenario. let-7 has been found to be globally downregulated in miRNA expression profiling analyses of hundreds of cancers81 and is known to target the RAS pathway.86 RAS may not be directly involved in causing all of these cancers; however, there may be therapeutic benefit to targeting RAS signaling pathways in these cancers. In addition to RAS, it is possible that other genes that are regulated by let-7 may affect prognosis or susceptibility to treatment of particular cancers. Therefore, one might imagine that reconstituting let-7 with let-7 mimetics might confer a better prognosis or susceptibility to treatment in those cancers by affecting RAS and non-RAS pathways simultaneously.

In cancers in which RAS participates in pathogenesis directly, another therapeutic strategy might be to use siRNAs to target the RAS gene to confer antiproliferative properties to those cells. Using RAS-directed siRNAs will likely result in very robust silencing of RAS in cancerous cells. However, a disadvantage of using siRNAs against RAS might be robust silencing of RAS-dependent pathways in noncancerous cells. As an alternative, miRNA pathway modulators may be more effective therapeutic agents than siRNAs because of the breadth and tissue specificity of miRNA-based gene regulation. miRNAs are selectively expressed in certain tissues, and their targets are selectively expressed. Therefore, the possibilities are much lower for off-target and unintended misregulation of specific miRNA pathways compared with siRNA-based therapies. In this case, reconstituting let-7 with let-7 mimetics would selectively affect RAS signaling only in cells in which the RAS pathway is upregulated. This effect may be amplified by modulating other miRNAs affecting the RAS pathway. The latter example will require a more careful and accurate connectivity map that connects certain pathways by miRNA silencing. The subtlety of miRNA-mediated silencing in comparison to siRNA-mediated silencing might suggest a limitation to the use of miRNAs in therapy; however, this quality is what might make them so efficient! That is, some level of gene expression for physiological processes will be intact, but excesses will be reduced.

There may be some cases in which miRNAs work at the point of therapy in the future, but almost certainly RNAi will have multiple roles in adjunctive therapy for diseases. This could be in combination with chemical drugs, or different types of RNAi could be combined. An example of adjunctive RNAi combination therapy could be addition of an siRNA-based therapy to a miRNA pathway–modulated therapy if, for instance, stronger silencing is the goal. This might be needed in a case in which a patient is not responding to or is becoming resistant to a single type of RNAi-based therapy. In these cases, a careful risk-benefit analysis should be considered. In addition, antisense technologies might even be used in combination with siRNA and miRNA pathway modulator technologies to enhance therapeutic outcomes.

We still know very little about the basic biology of miRNA pathways. As small RNA-based tools become more widely used, we will gain more insights into basic mechanisms of miRNA pathways. As we gain more mechanistic insights into miRNA pathways, these small RNA-based tools will improve. There is no need to reinvent the wheel. Nature has already provided us with small RNAs, which may be drugs themselves or druggable targets.

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 Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

PII: S0091-6749(07)03617-2

doi:10.1016/j.jaci.2007.12.1167

The Journal of Allergy and Clinical Immunology
Volume 121, Issue 2 , Pages 309-319, February 2008

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