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Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge.Oligonucleotide-based drugs have the potential to treat or manage a wide range of diseases. However, the widespread application of such therapies has been hampered by the difficulty in achieving efficient delivery to extrahepatic tissues. Here, Roberts et al. overview oligonucleotide-based drug platforms and assess approaches being employed to improve their delivery.

Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5′ and 3′ untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes. Protein expression from circular RNAs is enhanced several hundred-fold by optimizing vector design.

Key Points Nucleic acid aptamers, often termed chemical antibodies, are short, single-stranded DNA or RNA molecules (20–100 nucleotides in length) with defined structures that can specifically bind to a molecular target via three-dimensional structures. Similarly to the way antibodies bind to antigens, aptamers specifically recognize and bind to their cognate targets through unique three-dimensional structures. SELEX (systematic evolution of ligands by exponential enrichment) is a gold-standard methodology for generating aptamers, in which an iterative selection procedure — including binding, partitioning, recovery and re-amplification steps — is conducted. Specific sequences (that is, aptamers) can be enriched and dominate the population of library species. Aptamer-based therapeutics typically exploit one of three strategies: an aptamer can serve as an antagonist for blocking the interaction of disease-associated targets (for example, receptor–ligand interactions); an aptamer can serve as an agonist for activating the function of target receptors; or a cell-type-specific aptamer can serve as a carrier for delivering other therapeutic agents to the target cells or tissue. There are three aptamers designated for use in ophthalmology, including one drug approved by the US Food and Drug Administration (FDA) (pegaptanib (Macugen)), and two in late-stage development (ACR-1905 and E-10030). Six RNA and four DNA aptamers have undergone clinical trials for the treatment of various conditions, including macular degeneration, coagulation, oncology and inflammation. All aptamers that have entered clinical trials so far act as antagonists. Nucleic acid aptamers offer several advantages over traditional antibodies, but their clinical translation has been delayed by several factors, including insufficient potency, lack of safety data and high production costs. Here, Zhou and Rossi provide an overview of aptamer generation, focusing on recent technological advances and clinical development, as well as challenges and lessons learned. Nucleic acid aptamers, often termed 'chemical antibodies', are functionally comparable to traditional antibodies, but offer several advantages, including their relatively small physical size, flexible structure, quick chemical production, versatile chemical modification, high stability and lack of immunogenicity. In addition, many aptamers are internalized upon binding to cellular receptors, making them useful targeted delivery agents for small interfering RNAs (siRNAs), microRNAs and conventional drugs. However, several crucial factors have delayed the clinical translation of therapeutic aptamers, such as their inherent physicochemical characteristics and lack of safety data. This Review discusses these challenges, highlighting recent clinical developments and technological advances that have revived the impetus for this promising class of therapeutics.

DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometre precision. Here we demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. We show that this structure is a potent viral inhibitor and that it can act as a sensor by including a fluorescent output to report binding. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.

Key Points Many cancer targets are difficult to block with conventional therapies. Although RNA interference (RNAi) as a therapeutic approach is appealing, many challenges to delivery must be overcome. Nanoparticles hold promise for the safe and effective intracellular delivery of RNAi-based molecules. Physiological barriers and systemic toxicity of nanoparticle-based carrier systems create multiple challenges to bringing RNAi-based therapeutics to the clinic. Nanoparticles can be used to help avoid immune-mediated responses to systemic RNAi-based therapy. Solutions to improving tumour specificity and the ability to monitor and control short-term and long-term RNAi-based therapies are crucial next steps before clinical use. As the technology for delivery improves, so we will also need to improve our understanding of the heterogeneity of RNAi processing in different cancer types. Various resistance mechanisms to RNAi-based therapies must be anticipated. Inherent difficulties with blocking many desirable targets using conventional approaches have prompted many to consider using RNA interference (RNAi) as a therapeutic approach. This Review explores current challenges to the development of synthetic RNAi-based therapies and considers new approaches to circumvent biological barriers. Inherent difficulties with blocking many desirable targets using conventional approaches have prompted many to consider using RNA interference (RNAi) as a therapeutic approach. Although exploitation of RNAi has immense potential as a cancer therapeutic, many physiological obstacles stand in the way of successful and efficient delivery. This Review explores current challenges to the development of synthetic RNAi-based therapies and considers new approaches to circumvent biological barriers, to avoid intolerable side effects and to achieve controlled and sustained release.

Several discoveries have paved the way to personalise cancer medicine and a tremendous gain of knowledge in genomics and molecular mechanisms of cancer progression cumulated over the last years. Big stories in biology commonly start in a simple model system. No wonder microRNAs have been identified as regulators of embryonic development in the nematode Caenorhabditis elegans . From the first identification in worms to the first-in-man microRNA-based clinical trial in humans, almost 20 years passed. In this review we follow the story of understanding microRNA alterations in cancer, describe recent developments in the microRNA field and critically discuss their potential as diagnostic, prognostic and therapeutics factors in cancer medicine. We will explain the rationale behind the use of microRNAs in cancer diagnosis and prognosis prediction, but also discuss the limitations and pitfalls associated with this. Novel developments of combined microRNA/siRNA pharmacological approaches will be discussed and most recently data about MXR34, the first-tested microRNA drug will be described.

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss. The human heart regenerates poorly, causing insufficient healing after injury; here, microRNAs screened for the ability to induce cardiomyocyte proliferation are shown to stimulate cardiac regeneration and almost complete recovery of the heart after infarction. MicroRNA boosts heart regeneration The mammalian heart has a poor capacity to regenerate, with the ability of cardiomyocytes to proliferate disappearing soon after birth. In this study, Mauro Giacca and colleagues screened a synthetic microRNA (miRNA) library of human origin for the ability to induce cardiomyocyte proliferation, with aim of identifying potential human therapeutics. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in rodent cardiomyocytes. The two most potent, hsa-miR-590 and hsa-miR-199a, were tested further and shown to induce cardiac regeneration after myocardial infarction in mice. In vivo treatment with these miRNAs led to almost complete and stable recovery of cardiac function.

Manipulating plasma lipids Recent work in mice has shown that microRNA-33a is an important regulator of lipid metabolism and that its inhibition can increase plasma high-density lipoprotein (HDL) and decrease atherosclerosis. Rayner et al . take an important step in translating these findings to non-human primates (African green monkeys), which, like humans and unlike mice, express both miR-33a and miR-33b. They find that anti-miR-33 is effective at inhibiting both miR-33a and miR-33b. As seen in the mouse studies, anti-miR33 raised plasma HDL but had the additional beneficial effect of reducing very low-density lipoprotein triglycerides, making this type of 'antagomir' therapy a candidate method of treating dyslipidaemias that increase cardiovascular disease risk. Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects 1 . MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention 2 . MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3–5 ), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels 3 , 4 , 5 and providing protection against atherosclerosis 6 ; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation ( CROT , CPT1A , HADHB and PRKAA1 ) and reduced the expression of genes involved in fatty acid synthesis ( SREBF1 , FASN, ACLY and ACACA ), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.

Key Points RNA interference (RNAi) is a powerful approach for reducing expression of endogenously expressed proteins for biological applications, or targeting the expression of pathological proteins for therapy. Several delivery methods are available to achieve RNAi in ex vivo and in vivo settings for therapeutic results. The development of RNAi-based therapeutics has advanced sufficiently to allow human clinical trials to begin. Here we outline the broad range of cell-, tissue- and disease-specific approaches under investigation for RNAi therapeutics. The barriers posed by certain cells and tissues are described, as are issues with off-target silencing. RNA interference can elicit specific gene silencing and so holds great potential for treating infectious or genetic diseases. Several small-RNA-based therapies have now reached clinical trials, but further work is still needed to improve delivery and efficacy. RNA interference (RNAi) is a powerful approach for reducing expression of endogenously expressed proteins. It is widely used for biological applications and is being harnessed to silence mRNAs encoding pathogenic proteins for therapy. Various methods — including delivering RNA oligonucleotides and expressing RNAi triggers from viral vectors — have been developed for successful RNAi in cell culture and in vivo. Recently, RNAi-based gene silencing approaches have been demonstrated in humans, and ongoing clinical trials hold promise for treating fatal disorders or providing alternatives to traditional small molecule therapies. Here we describe the broad range of approaches to achieve targeted gene silencing for therapy, discuss important considerations when developing RNAi triggers for use in humans, and review the current status of clinical trials.

Expression of miR-124, a microglial microRNA, reduces disease in a mouse model of multiple sclerosis by inhibiting the transcription factor C/EBP-α and its downstream target PU.1. MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45 low , major histocompatibility complex (MHC) class II low phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo . These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.