Developing therapeutic microRNAs for cancer

Abstract

Despite substantial progress in understanding the cancer-signaling network, effective therapies remain scarce due to insufficient disruption of oncogenic pathways, drug resistance and drug-induced toxicity. This complexity of cancer defines an urgent goal for researchers and clinicians to develop novel therapeutic strategies. The discovery of microRNAs (miRNAs) provides new hope for accomplishing this task. Supported by solid evidence for a critical role in cancer and bolstered by a unique mechanism of action, miRNAs are likely to yield a new class of targeted therapeutics. In contrast to current cancer medicines, miRNA-based therapies function by subtle repression of gene expression on a yet large number of oncogenic factors and are, therefore, anticipated to be highly efficacious. After the completion of target validation for several candidates, the development of therapeutic miRNAs is now moving to a new stage that involves pharmacological drug delivery, preclinical toxicology and regulatory guidelines.

INTRODUCTION

MicroRNAs (miRNAs) are a recently discovered class of endogenous non-coding RNAs that function as master regulators of the human genome.¹ The frequent aberrant expression and functional implication of miRNAs in many human diseases have lifted these small cellular components to the ranks of preferred drug targets.^2,3 Notably in cancer, certain miRNAs meet the stringent criteria for becoming ideal therapeutic intervention points by functioning as bona fide oncogenes and tumor suppressors. ‘Oncogene addiction’, a term previously reserved for protein-encoding oncogenes, has recently been extended to miRNAs.⁴ Thus, the discovery of the ~1400 human miRNAs known to date not only adds significantly to the pool of novel drug targets but also supplies us with the prospect of creating a new class of therapeutics that operates by a new mechanism of action. The functionality of a therapeutic miRNA is based upon the catalytic process of the natural miRNA, which comprises a 15–22 nt single-stranded RNA that enters the cytoplasmic multiprotein complex RNA-induced silencing complex (RISC) to pair with mRNAs carrying complementary sequences and, consequently, repress gene expression.¹ Imperfect base pairing between miRNAs and mRNAs is common and enables miRNAs to regulate a broad, but nevertheless specific set of genes. Accordingly, a given miRNA can control multiple oncogenes and oncogenic pathways deregulated in cancer. In view of cancer as a heterogenic disease that cannot be successfully treated by targeting a single gene of interest,^5–7 it is this ability of miRNAs that may hold the key to therapeutic success.

THERAPEUTIC APPROACHES

Depending on miRNA function and its status in the diseased tissue, there are two approaches to developing miRNA-based therapies: antagonists and mimics. Each approach shares similarities with each other as well as with other therapies; however, they are sufficiently distinct to suggest that miRNA mimics and antagonists should be viewed as separate therapeutic modalities. A summary of their mechanistic and structural characteristics in comparison with gene therapy, small interfering RNAs (siRNAs) and small-molecule inhibitors is presented in Table 1.

Table 1.

Mechanistic and structural characteristics of miRNA-based therapeutics

	Gene therapy	miRNA mimics	siRNAs	miRNA antagonists	Small-molecule inhibitor
Therapeutic
Type of therapeutic	Protein	Short ss RNA	Short ss RNA	Short ss RNA	Small organic compound
Application	Replacement	Replacement	Inhibition	Inhibition	Inhibition
Status of drug target	Loss of function	Loss of function	Gain of function	Gain of function	Gain of function
Recognition by body as	Native	Native	Foreign	Foreign	Foreign
Delivery
Structure of API	Large DNA vector	Short ds RNA	Short ds RNA	Short ss RNA	Small organic compound
Molecular weighta	~22 000 kDa	~15 kDa	~15 kDa	~7 kDa	~0.5 kDa
Delivery route	Local	Local/systemic	Local/systemic	Local/systemic	Systemic
Endogenous processing into final therapeutic	Transcription translation	RISC	RISC	None	None

Abbreviations: API, active pharmaceutical ingredient; ds, double-stranded; miRNA, microRNA; RISC, RNA-induced silencing complex; siRNA, small interfering RNA; ss, single-stranded.

^aMolecular weight values are based on examples for each category; gene therapy: genome of human adenovirus 5; miRNA mimic: double-stranded let-7a precursor; siRNA: PLK1 siRNA; miRNA antagonist: single-stranded let-7a; small-molecule inhibitor: erlotinib, gefitinib, imatinib.

MicroRNA antagonists are generated to inhibit miRNAs that acquire a gain of function in human disease. Thus, miRNA antagonists are conceptually similar to other inhibitory approaches, such as siRNAs and small-molecule inhibitors, and logistically follow a similar path during drug development that starts with the identification of a disease-related miRNA and continues with the making of a novel molecule that shows adequate specificity and inhibitory activity. During that process, the endogenous miRNA in question can serve as a biomarker for optimizing the pharmacokinetic and pharmaco-dynamic properties of the antagonist. The most common strategy to ablate miRNA function is achieved by single-stranded oligonucleotides with miRNA complementary sequences. The backbones of these are chemically modified to increase the affinity toward the endogenous miRNA and to sequester it in a configuration that is unable to be processed by RISC. Therefore, miRNA inhibition occurs upstream of RISC and presumably independently of cellular cofactors. Examples of miRNA antagonists are anti-miRs, antagomiRs and locked nucleic acids.^8–11

In contrast, miRNA mimics are used to restore miRNAs that show a loss of function. This approach, also known as miRNA replacement therapy, has attracted much interest as it provides a new opportunity to therapeutically exploit tumor suppressors.¹² In this regard, miRNA replacement is reminiscent of traditional gene therapy; however, the low molecular weight of miRNAs permits the delivery of therapeutic miRNAs as short double-stranded oligonucleotides rather than in form of viral vectors used for gene therapy.^13,14 Thus, the technical implementation of miRNA replacement is more closely related to those of siRNAs–both, miRNA mimics and siRNAs, act downstream of RISC and require enzymatic functions of cellular RISC to be catalytically active (Table 1). While siRNAs are designed to repress a single mRNA of interest, miRNA mimics inherently target multiple transcripts, now providing a unique mechanism of action that guides the process for drug development and sets forth the context of its therapeutic application. For instance, as the miRNA itself becomes the therapeutic entity, the primary rationale for its application should be based on its expression levels in diseased tissues rather than on the identity and status of selected target genes. Using this approach, the quest for target genes responsible for the miRNA-induced phenotype becomes theoretically irrelevant, because the mimic carries the same sequence as the endogenous miRNA and is expected to regulate the same, all-inclusive set of genes. This is of particular interest, as a given miRNA can regulate several hundreds of genes, and identifying all genes critical to miRNA function could become a tedious, if not impossible, task. Nonetheless, information about genes and pathways regulated by the miRNA may prove useful in understanding its mechanism of action, monitoring miRNA activity and designing clinical trials. An example for applying a miRNA mimic in the appropriate context is provided by let-7, a miRNA expressed at reduced levels in self-renewing tumor-initiating breast cancer cells.¹⁵ Short hairpin RNA-induced repression of KRAS, a proto-oncogene repressed by let-7,¹⁶ slightly reduced tumorigenesis of these cancer cells. However, restoring the levels of let-7 had superior anti-oncogenic effects.¹⁵ This suggests that the repression of other, presumably unknown target genes is necessary to recapitulate the let-7 phenotype and to harness the full therapeutic benefit of the miRNA.

Interestingly, the therapeutic application of miRNA mimics is not limited to replacement and can also provide the desired response in disease cells with normal miRNA expression levels. For instance, adding miR-34a, a tumor suppressor frequently expressed at reduced levels in various cancer types, not only inhibited the growth of cancer cells devoid of miR-34a but also of those that show normal levels of endogenous miR-34a.¹⁴ Although an experimental demonstration remains absent, it is possible that the delivered miR-34a impinges on pathways that these cancer cells have become addicted to, and modulates these sufficiently to interfere with oncogenicity. Thus, there appears to be an added value for miRNA mimics; however, it remains to be determined what patient will benefit from it.

As miRNA mimics behave like endogenous miRNAs, another aspect to consider is the selection of appropriate assays and measures during drug development. Although the therapeutic activity of miRNA antagonists can be assessed by monitoring the levels of the endogenous miRNA, a phenotypic readout, such as efficacy in a cell or animal model, is usually the preferred method to monitor the activities of miRNA mimics. This approach applies to situations where the chemistry of the mimic has not been finalized and chemical modifications may alter miRNA specificity and functionality. The preference for a phenotypic assay over monitoring one or few target genes is based on the fact that the repression of few mRNAs may not be representative for its overall activity but, rather, may direct development programs into generating compounds with altered, and thus, incorrect target specificity. An alternative to phenotypic assays are genome-wide mRNA expression analyses that can capture all miRNA-induced changes to the transcriptome. Once the chemistry of the mimic has been finalized, single target genes that provide satisfactory sensitivity can be used as biomarkers to monitor therapeutic activity.

microRNAs AS ACTIVE PHARMACEUTICAL INGREDIENTS

Developing active pharmaceutical ingredients that either target or mimic specific miRNAs involves creating oligonucleotides with favorable pharmacological and pharmacokinetic properties. The generation of miRNA antagonists has been achieved by antisense oligonucleotides that are partially or completely complementary to specific miRNA sequences.^9,17,18 Nucleotide analogs, such as 2′-O-methyl, 2′O-methoxy ethyl and locked nucleic acids, must be included in the antisense molecules to ensure selective hybridization with the endogenous miRNAs and prevent the miRNA from interacting with mRNAs. The length and chemical composition of antisense oligonucleotides can be optimized to improve circulation time, cellular internalization and activity, and to enable systemic delivery without a delivery agent.¹⁹ The most advanced miRNA antagonist is a miR-122-specific locked nucleic acid that is currently in Phase II clinical testing for patients infected with hepatitis C virus.²⁰

Developing a miRNA mimic involves creating an RNA molecule with the ability to enter the silencing complex and affect the same target genes as the endogenous miRNA. The active endogenous miRNA comprises an ~22-nucleotide, single-stranded RNA that functions as a guide sequence within RISC (Table 1). This activity can be mimicked using a synthetic, single-stranded RNA molecule that contains the same sequence and chemistry as the mature endogenous miRNA. Consistent with results published for siRNAs,²¹ however, the potency of singled-stranded miRNA mimics tend to be 100–1000-fold less compared with mimics that feature a second, complementary strand. Therefore, double-stranded miRNA mimics are greatly preferred to single-stranded mimics. The passenger strand can either be perfectly complementary to the mature miRNA or it can incorporate mismatches that are consistent with the precursor form that is produced naturally in cells.²²

As with other oligonucleotide-based therapeutic candidates, nucleotide analogs can be used to improve the activities and half-lives of miRNA mimics. A variety of sugar and phosphate modifications can be incorporated in both the active and passenger strand, including 2′-O-methyl, 2′F, 2′NH₂, 2′H, phosphorothioates and locked nucleic acids. Terminal modifications such as inverted bases, biotin, alkyl groups and others can be added in the passenger strand without negatively affecting the activity of the miRNA mimic (unpublished data). Although nucleotide analogs can significantly enhance the half-lives of the synthetic miRNAs, they can also affect the hybridization kinetics and melting temperatures and, consequently, target specificities of the molecules. As miRNA replacement therapy depends on providing patients with miRNA activities that are identical to those that are reduced or missing in the disease cells, measuring target specificity is an essential component when developing a miRNA mimic (Box 1). In general, 3–10-fold improvements in miRNA potency can be achieved by incorporating 2′-modified nucleotides into the internal regions of the active strand and the 5′ and 3′ flanks of the passenger strand (unpublished data). Changes in miRNA activity and target specificity are often a result from terminal modifications in the active strand and a high degree of modification in the central regions of both strands. As the specific, pharmacologically optimal combinations of modifications vary for each miRNA, creating custom-made chemistries for each active pharmaceutical ingredient candidate is highly recommended.

Box 1. Process of API development.

Modification walk—Incorporate a nucleotide analog at each position along both the active and passenger strand and test the resulting miRNAs for activity using cell culture models. This will reveal those positions that are sensitive to modification.

Candidate development—Create mimics with nucleotide analogs at multiple positions that are insensitive to change and test for activity using cell culture models, stability in serum and target specificity by mRNA array analysis. As each miRNA produces a unique mRNA profile in a given cell type due to its ability to affect the expression of many different genes, the mRNA array fingerprint that results from the transfection of a miRNA mimic can be used to establish whether a combination of nucleotide analogs has altered the target specificity of a miRNA mimic.

Pharmacokinetics and pharmacodynamics—Inject miRNA mimic candidates selected in Step 2 into an animal model and evaluate the pharmacokinetic and pharmacodynamic properties of the molecules. Select those candidates that provide accumulation and target effects in the target tissue that are superior to the unmodified miRNA mimic without causing undue toxicity.

Pharmacology and toxicity—Inject a range of doses of the miRNA mimics selected in Step 3 into an animal model and evaluate the pharmacological and toxicological effects of the API candidates. Select the API candidate that shows maximal pharmacological activity and minimal toxicity.

Abbreviations: API, active pharmaceutical ingredient; miRNA, microRNA.

DELIVERY OF THERAPEUTIC microRNAs

Increasing evidence demonstrates that miRNAs are promising agents in cancer therapy. However, similar to other therapeutic oligonucleotides, the main challenge remains the successful delivery of the therapeutic miRNAs to the target tissue without compromising the integrity of the miRNA (reviewed in Akhtar and Benter,²³ Castanotto and Rossi,²⁴ Kaasgaard and Andresen,²⁵ Whitehead et al.²⁶ and Davis et al.²⁷). Naked ribonucleic acids are subject to rapid nuclease dependent degradation and are therefore inherently unstable in biofluids. Thus, many therapeutic applications of RNAi are limited to local administration for which exposure of the RNAi agent to potential degradation mechanisms is restricted to a minimum. However, local administration is merely applicable to a short list of target tissues and frequently does not facilitate exposure of all disease cells to the drug. Systemic delivery is therefore a better route of administration because—in theory—it provides a much more efficient dissemination of the therapeutic to target tissues. Next to rapid degradation by serum nucleases, however, the miRNA will have to overcome many obstacles before it reaches the target (Box 2). In addition, systemic delivery of miRNAs may induce similar adverse events that have been reported for other oligonucleotide-based therapies, such as aggregation and complement activation, liver toxicity and stimulation of an immune response by the nonspecific activation of toll-like receptors.²⁸

Box 2. In vivo barriers to systemic delivery of miRNAs.

Degradation by serum and tissue nucleases

Renal clearance (renal filtration of particles <50 kDa)

Failure to cross the capillary endothelium (problematic for particles >5 nm in Ø; few organs absorb particles ≤ 200 nm: liver, spleen, certain tumors)

Uptake by scavenging macrophages

Limited passage through extra-cellular matrix: polysaccharides, phagocytes, fibrous proteins

Inefficient endocytosis by target cells

Ineffective endosomal release

Abbreviation: miRNA, microRNA.

As miRNA antagonists and mimics are chemically similar to antisense oligonucleotides and therapeutic siRNAs, many technologies developed for these may also be applicable to miRNAs. Therefore, the development of clinically relevant miRNA formulations frequently involves a thorough evaluation of existing technologies to identify those that are amenable to the miRNA and its chemistry. Criteria critical in the evaluation process are (i) sufficient delivery to induce a therapeutic effect in disease models and (ii) a significant safety margin at therapeutic levels. Several technologies have proven effective in delivering therapeutic miRNAs to tumor tissues in vivo. These include vector-based systems that were originally developed for gene therapy.²⁹ For instance, adenoviral or lentiviral delivery was employed to evaluate the therapeutic activity of let-7 in the KRAS^G12D transgenic mouse model of non-small-cell lung cancer. This model is based on an activating KRAS mutation that is also prevalent in human cancers and leads to tumors that resemble those in man.³⁰ The respiratory delivery of virus-encoded let-7 inhibited lung tumorigenesis prophylactically before oncogene activation, but also in mice that carried well-established tumors.^31,32 Similarly, adenoviral delivery of miR-26a to MYC-induced murine hepatocellular carcinomas led to inhibition of tumor cell proliferation and induction of tumor-specific apoptosis.³³ In this case, the adenoviral load was injected systemically into the blood stream, which facilitated satisfactory miR-26a expression in normal liver and liver tumor cells. Of note, MYC is not a direct target of miR-26a and, therefore, the inhibitory activity of miR-26a is attributed to the repression of other genes presumably downstream of oncogenic MYC.³³ Neither lung delivery of let-7 nor systemic delivery of miR-26a led to noticeable toxicities in these animals.^31,33

Another example for in vivo delivery of a miRNA-based therapeutic is provided by miR-10b. This miRNA is transcriptionally induced by TWIST, a transcription factor that contributes to epithelial–mesenchymal transitions and functions in high-grade malignancies.³⁴ In accord, miR-10b is highly expressed in metastatic cancer cells and tumor tissues, and can induce a metastatic phenotype in cells that otherwise lack metastatic potential.³⁵ Thus, the therapeutic application around miR-10b involves an antagonistic single-stranded oligo-nucleotide, such as an antagomiR, to silence miR-10b in tumor tissues. As predicted, systemic delivery of the miR-10b antagomiR prevented the formation of metastases that are usually produced by the primary orthotopic 4T1 breast cancer xenograft.³⁶ Another miRNA that was studied in the context of metastasis is miR-16. In contrast to miR-10b, however, miR-16 functions as a tumor suppressor that is downregulated in tumor cells of the prostate and—when re-introduced—induces apoptosis.³⁷ Systemic delivery of a miR-16 mimic inhibited metastasis of PC-3M prostate cancer cells intra-cardially injected 4 days before treatment.³⁷ The therapeutic delivery was facilitated using atelocollagen, a cationic polymer that associates with RNA through electrostatic interactions and forms particles in the nanometer diameter range. As atelocollagen is a natural product, these nanoparticles are considered to be highly biocompatible and seem to achieve tumor-specific delivery via enhanced permeability and retention.³⁸ Although both the miR-10b antagomiR and the MiR-16 mimic inhibited the formation of metastases, the cellular mechanisms of inhibition are likely to be substantially different. Although miR-10b seems to play a role in pathways that control invasion and early stages of metastasis, the effects of miR-16 appear to be due to a repression of cell cycle genes.^36,37 It is interesting to note that the therapeutic delivery of the miR-10b antagomiR did not inhibit the growth of existing metastases and suggests that inhibition of miR-10b may be used to prevent primary tumors from becoming metastatic.³⁶ In contrast, miR-16 blocked the growth of disseminated prostate cancer cells and might therefore prove useful in treating metastatic disease.³⁷ Thus, miR-10b and miR-16 illustrate the diversity of miRNA function in cancer and how miRNAs can create unique opportunities for therapeutic intervention. It is possible that a combination of the miR-10b antagonist and miR-16 mimic might prove particularly effective in treating cancer patients.

A candidate that has attracted much interest for therapeutic development is miR-34a. It is downregulated in a broad variety of different cancer types. miR-34a is transcriptionally induced by the tumor suppressor protein p53 and phenocopies p53 by blocking cancer cell cycle progression as well as by inducing apoptosis and senescence.³⁹ miR-34a also represses genes that block p53 activity, including SIRT1 and YY1, thereby establishing a positive feedback loop back to p53 to maintain endogenous miR-34a expression and inhibitory pathways regulated by p53 only.^40,41 However, p53 activity is not a mandatory event downstream of miR-34a—miR-34a can also inhibit cancer cells devoid of p53.¹⁴ Therefore, the p53-independent, but nevertheless p53-alike, functions qualify miR-34a for becoming a promising anticancer therapeutic that is in direct opposition to p53 gene therapy. Proof of concept for its therapeutic activity was demonstrated in mouse models of non-small cell lung cancer.¹⁴ Therapeutic delivery of a miR-34a mimic using a neutral lipid emulsion, either by direct injections into the tumor or by systemic tail vein injections, prevented the outgrowth of viable subcutaneous lung tumor xenografts.¹⁴ Inhibition of tumor growth correlated with an accumulation of exogenous miR-34a in the tumor, as well as a specific repression of transcripts regulated by miR-34a. In addition, miR-34a mimics formulated in the neutral lipid emulsion failed to induce elevated levels of neither cytokines nor liver and kidney enzymes in serum, suggesting that tumor inhibition was a specific effect of the mimic and that treatment was well tolerated.¹⁴

PRECLINICAL DEVELOPMENT OF THERAPEUTIC microRNAs

The discovery and characterization of miRNAs has provided exceptional opportunities for the development of new therapeutics for human diseases. Regulatory guidelines, however, have not kept pace with the rapid growth of this new class of therapeutics. Although specific references are rare, the International Conference on Harmonization (ICH) guideline M3 (R2) noted that ‘for products using innovative therapeutic modalities (e.g., siRNA), particular studies can be abbreviated, deferred, omitted, or added’. This lack of definition creates an opportunity for a case-by-case approach in designing the non-clinical development programs. The goal of any non-clinical testing program is to provide the rationale for conducting human studies, characterize the pharmacokinetic profile, identify potential adverse drug effects and define a safety margin for human clinical studies. As miRNAs are chemically synthesized, the expectation is that miRNAs will be reviewed under the guidelines for small molecules.

ICH-S9, Non-clinical Evaluation of Anticancer Pharmaceuticals, provides information on the program design of non-clinical studies to support the development of anticancer pharmaceuticals. As noted in this guidance, genotoxicity studies and separate safety pharmacology assessments are not a requirement to support an initial clinical trial for an oncology indication. It is generally expected that the toxicology studies will be conducted in a rodent and non-rodent species. As miRNA sequences are highly conserved across species, there are no apparent restrictions on species selection or a requirement to develop analogues or surrogates, as has been the case with non-conserved oligonucleotides. The dose levels can be established according to data obtained from pharmacology studies and non-GLP dose-ranging studies in the selected tox species. Dosing route and regimen should be consistent with the proposed clinical trial. The duration of the initial clinical trial will determine the duration of the non-clinical studies, and examples of dosing regimens are included in the guideline. Considering that the vehicle components may produce or enhance/alter toxicity, preclinical studies should feature the same formulation as will be used in the clinic. Metabolism and excretion studies may not be needed as therapeutic miRNAs will be metabolized by endo- and exo-nucleases to the nucleotide components.

There are no assured outcomes in the evaluation of a new molecular entity and it should be recognized that the systemic delivery of miRNA mimics and antagonists may induce adverse events that have been reported for other oligonucleotide-based therapies in higher species, such as complement activation, liver toxicity and the stimulation of an immune response by the nonspecific activation of toll-like receptors.^24,28,42 A continuing dialog among regulators, academia, industry and specialty groups (for example, Oligonucleotide Safety Working Group, BioSafe) is recommended to characterize the potential risks, develop acceptable methodologies to assess the risks and define non-clinical regulatory guidelines. Although the non-clinical development of a miRNA is similar to that of other new molecular entities, it is reasonable to operate ‘case-by-case’ and implement new tests when required and give up others that have become unnecessary. Thus, it is recommended to partner early with the Food and Drug Administration and to take advantage of the pre-investigational new drug meeting to discuss investigational new drug-enabling strategies and mandatory studies.

THE MARKET PROMISE OF microRNAs-BASED THERAPEUTICS

The path from drug discovery, preclinical development and clinical trials to market approval and market entry is known to be a long road hampered by many challenges, obstacles and disappointments, notably in cancer. On average, only about 8% of all investigational cancer drugs that enter Phase I clinical testing will ultimately enter the market; the failure rate of a cancer compound in Phase III clinical trials is ~50%.⁴³ Nevertheless, developing an investigational product for a cancer indication has its benefits due to the high unmet medical needs and the promise of bringing an innovative drug to market. In addition, the US Food and Drug Administration tolerates a greater latitude on the safety or toxicity testing requirements due to the serious and life-threatening nature of many cancer types. Another added benefit might be the availability of different regulatory approval pathways, such as accelerated approval, fast track and priority review, which allows biopharmaceutical companies to shorten the overall time to market. However, even when being granted one or more of the aforementioned shortened regulatory pathways, the clinical development and Food and Drug Administration review and approval time-line for investigational products in cancer takes on average 7 years.

In many cases, the successful regulatory approval of new cancer drugs hinges on the design of clinical trials and the definition of its endpoints. Targeted therapies are most effective in patients who harbor the underlying genetic aberration; however, this patient population is usually a small fraction of all cancer patients and needs to be carefully selected for clinical trials to be successful. As therapeutic miRNAs constitute another form of targeted therapies, therapeutic miRNAs are likely no exception. Tools and diagnostics already promoted for personalized medicine and used to accurately characterize a patient’s cancer genotype will also become valuable resources for implementing miRNA-based therapies in man. However, miRNAs might nevertheless benefit from an accelerated route to market. Given the broad range of cancer types affected by certain miRNAs, the strong anti-oncogenic activity of miRNA-based therapies in animal models and the presumably safe and unencumbered translation of miRNA replacement into the clinic, therapeutic miRNAs may stand up to its promise. In addition, recent implications of miRNAs in cancer stem cells as well as their ability to sensitize cancer cells to radiation and conventional chemotherapy create new opportunities to treat cancer more effectively.^15,44–46

CLOSING REMARKS

Within a decade, miRNAs have quickly moved from discovery into therapeutic development programs. This rapid progress reflects the importance of miRNAs in cancer and is based on the thorough validation of key miRNAs as ideal candidates for therapeutic intervention. Although there is little doubt about the therapeutic potential, the challenge remains to translate this potential into readily available medicines. The main focus in bringing miRNAs to cancer patients is the enablement of pharmacological drug delivery, a task that has hampered the progress of related antisense and siRNA therapeutics. Yet, the recent clinical success of existing delivery technologies^47,48 and the continuous emergence of new ones suggest that miRNA therapeutics for cancer is within the realms of possibilities. As many current delivery systems show distinct biodistribution profiles, the choice of cancer type may largely depend on the performance of the underlying delivery system. However, given that miRNAs can function as cellular master regulators, show broad activity across multiple cancer types and appear to specifically inhibit metastasis,^36,37,46 the goal is to treat cancers for which there are no or insufficient treatment options.

ABBREVIATIONS

ICH

International Conference on Harmonization

miRNA

microRNA

RISC

RNA-induced silencing complex

siRNA

small interfering RNA