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Abstract Rationale Lower respiratory tract infections due to respiratory syncytial virus (RSV) are associated with development of bronchiolitis obliterans syndrome in lung transplant (LTX) recipients. ALN-RSV01 is a small interfering RNA targeting RSV replication. Objectives To determine the safety and explore the efficacy of ALN-RSV01 in RSV infection. Methods We performed a randomized, double-blind, placebo-controlled trial in LTX recipients with RSV respiratory tract infection. Patients were permitted to receive standard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebo was administered daily for 3 days. Viral load was determined by quantitative reverse transcriptase–polymerase chain reaction on serial nasal swabs. Patients completed symptom score cards twice daily. Lung function, including the incidence of new-onset or progressive bronchiolitis obliterans syndrome, was recorded at Day 90. Measurements and Main Results We enrolled 24 patients (ALN-RSV01, n = 16; placebo, n = 8); randomization was stratified by ribavirin use. ALN-RSV01 was well tolerated, with no drug-related serious adverse events or post-inhalation perturbations in lung function. Interpretation of viral measures was confounded by baseline differences between the two groups in viral load and time from symptom onset to first dose. Mean daily symptom scores were lower in subjects receiving ALN-RSV01, and the mean cumulative daily total symptom score was significantly lower with ALN-RSV01 (114.7 ± 63.13 vs. 189.3 ± 99.59, P = 0.035). At Day 90, incidence of new or progressive bronchiolitis obliterans syndrome was significantly reduced in ALN-RSV01 recipients compared with placebo (6.3% vs. 50%, P = 0.027). Conclusions ALN-RSV01 was safe and may have beneficial effects on long-term allograft function in LTX patients infected with RSV. Clinical trial registered with www.clinicaltrials.gov (NCT00658086).

Background The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab. Methods NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index. Results EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA. Conclusions EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream \"resistance\" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.

MicroRNAs (miRNAs) are ∼22 nt non‐coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non‐coding antisense transcripts in human cells. Specifically, we show that miR‐671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration‐Related protein 1 ( CDR1 ) locus in an Ago2‐slicer‐dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels. Natural antisense transcripts appear to have widespread roles in gene regulation. This study provides the first example of miRNA targeting of an antisense transcript. Nuclear miR‐671 targets and cleaves a circular antisense transcript expressed from the CDR1 locus, reducing CDR1 mRNA levels.

Key Points MicroRNAs (miRNAs) have important roles in many aspects of human diseases, and their targeted inhibition may have substantial therapeutic impact. Inhibition of miRNAs can be achieved through a variety of methods and chemically modified antisense oligonucleotides (anti-miRs) have shown the most prominent effects. Targeted delivery of anti-miRs is crucial to achieve intended therapeutic effects, and further efforts are warranted to develop more efficient delivery systems. MicroRNAs (miRNAs) — 21- to 23-nucleotide single-stranded RNAs that regulate gene expression — have roles in numerous diseases, and are therefore attractive therapeutic targets. Li and Rana discuss strategies in the design of miRNA-targeting oligonucleotides with increased efficacy and improved in vivo delivery characteristics, and highlight some of the challenges that lie ahead in the clinical development of these therapeutics. MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo . Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.

Summary Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents.

Key Points All RNA-targeted therapeutic technologies exploit oligonucleotides that bind to target RNA, but they differ in their mechanism of action and produce different effects. Small interfering RNAs, antisense oligonucleotides and external guide sequences lead to enzyme-dependent degradation of targeted mRNA. Drugs involving these approaches are designed to reduce the level of harmful gene products such as viral or bacterial proteins or disease-promoting cellular proteins. They could be useful against cancer as well as viral and bacterial infections, or used to prevent the accumulation of high levels of cholesterol in the bloodstream. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading RNA. Splice-switching oligonucleotides are discussed in detail in this Review; these oligonucleotides redirect alternative splicing, repair defective RNA or restore the production of proteins that are missing because of genetic defects. Splice-switching oligonucleotide-based drugs should be useful for the treatment of genetic diseases such as Duchenne muscular dystrophy, spinal muscular atrophy and β-thalassaemia. Compared to classical small-molecule drugs, it is much more difficult to achieve intracellular delivery with oligonucleotides; this is still a major issue for this class of drugs. The advantage of oligonucleotides is their high specificity, which results from sequence-specific base pairing to target RNA. The oligonucleotide-based drug fomivirsen was approved by the US Food and Drug Administration in 1998 for the treatment of viral retinitis in patients with AIDS. Oligonucleotide-based drugs are now in advanced clinical trials for the treatment of cancer and Duchenne muscular dystrophy as well as for lowering high cholesterol levels. Here, the authors highlight how RNA-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression, and so may be useful for treating disorders such as Duchenne muscular dystrophy, spinal muscular atrophy and β-thalassaemia. Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degradation of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression. Moreover, they can be extensively chemically modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to achieving its clinical potential.

Human RNAi therapy The ability to downregulate specific genes using systemically delivered short RNA molecules and the cellular mechanism known as RNA interference has been shown previously in mouse and non-human primate models. Davis et al . have now demonstrated for the first time in humans that a short interfering RNA (siRNA) molecule can be systemically delivered using nanoparticles to a solid tumour. The siRNA mediates directed cleavage of its target mRNA, thereby also reducing the protein level. This proof-of-principle study confirms the potential of this technology as a human therapeutic. It has previously been shown in mice and non-human primates that systemically delivered short RNA molecules can inhibit gene expression. Here it is shown that a short interfering RNA (siRNA) can be systemically delivered, using nanoparticles, to a solid tumour in humans. The siRNA mediates cleavage of its target mRNA, thereby also reducing levels of the encoded protein. This proof-of-principle study confirms the potential of this technology for treating human disease. Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients 1 , 2 . Long, double-stranded RNAs were first shown to mediate RNAi in Caenorhabditis elegans 3 , and the potential use of RNAi for human therapy has been demonstrated by the finding that small interfering RNAs (siRNAs; approximately 21-base-pair double-stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response 4 . We are at present conducting the first in-human phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumour biopsies from melanoma patients obtained after treatment show the presence of intracellularly localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is, to our knowledge, a first for systemically delivered nanoparticles of any kind). Furthermore, a reduction was found in both the specific messenger RNA (M2 subunit of ribonucleotide reductase ( RRM2 )) and the protein (RRM2) levels when compared to pre-dosing tissue. Most notably, we detect the presence of an mRNA fragment that demonstrates that siRNA-mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. Together, these data demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action.

The WRKY transcription factor family has 109 members in the rice genome, and has been reported to be involved in the regulation of biotic and abiotic stress in plants. Here, we demonstrated that a rice OsWRKY74 belonging to group III of the WRKY transcription factor family was involved in tolerance to phosphate (Pi) starvation. OsWRKY74 was localized in the nucleus and mainly expressed in roots and leaves. Overexpression of OsWRKY74 significantly enhanced tolerance to Pi starvation, whereas transgenic lines with down-regulation of OsWRKY74 were sensitive to Pi starvation. Root and shoot biomass, and phosphorus (P) concentration in rice OsWRKY74-overexpressing plants were ~16% higher than those of wild-type (WT) plants in Pi-deficient hydroponic solution. In soil pot experiments, >24% increases in tiller number, grain weight and P concentration were observed in rice OsWRKY74-overexpressing plants compared to WT plants when grown in P-deficient medium. Furthermore, Pi starvation-induced changes in root system architecture were more profound in OsWRKY74-overexpressing plants than in WT plants. Expression patterns of a number of Pi-responsive genes were altered in the OsWRKY74-overexpressing and RNA interference lines. In addition, OsWRKY74 may also be involved in the response to deficiencies in iron (Fe) and nitrogen (N) as well as cold stress in rice. In Pi-deficient conditions, OsWRKY74-overexpressing plants exhibited greater accumulation of Fe and up-regulation of the cold-responsive genes than WT plants. These findings highlight the role of OsWRKY74 in modulation of Pi homeostasis and potential crosstalk between P starvation and Fe starvation, and cold stress in rice.

Key Points RNA interference (RNAi) is a fundamental pathway in eukaryotic cells by which sequence-specific small interfering RNA (siRNA) is able to silence genes through the destruction of complementary mRNA. RNAi is an important therapeutic tool that can be used to silence aberrant endogenous genes or to knockdown genes essential to the proliferation of infectious organisms. Delivery remains the central challenge to the therapeutic application of RNAi technology. Before siRNA can take effect in the cytoplasm of a target cell, it must be transported through the body to the target site without undergoing clearance or degradation. Currently, the most effective synthetic, non-viral delivery agents of siRNA are lipids, lipid-like materials and polymers. Various cationic agents including stable nucleic acid–lipid particles, lipidoids, cyclodextrin polymers and polyethyleneimine polymers have been used to achieve the successful systemic delivery of siRNA in mammals without inducing significant toxicity. Direct conjugation of delivery agents to siRNA can facilitate delivery. For example, cholesterol-modified siRNA enables targeting to the liver. RNAi therapeutics have progressed to the clinic, where studies are being conducted to determine siRNA efficacy in treating several diseases, including age-related macular degeneration and respiratory syncytial virus. Moving forward, it will be important to pay close attention to the potential nonspecific immunostimulatory effects of siRNA. Modifications to siRNA can be used to minimize stimulation of the immune system, and an increased emphasis must be placed on performing proper controls to ensure that therapeutic effects are sequence-specific. RNA interference holds vast potential as a therapeutic strategy for both disease prevention and treatment, but its use has so far been hampered by a lack of safe and effective delivery techniques. In their Review, Anderson and colleagues discuss the challenges associated with small interfering RNA delivery and highlight promising novel synthetic delivery agents. In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.

Fleshy fruits are classically divided into climacteric and nonclimacteric types. It has long been thought that the ripening of climacteric and nonclimacteric fruits is regulated by ethylene and abscisic acid (ABA), respectively. Here, we report that sucrose functions as a signal in the ripening of strawberry (Fragaria × ananassa), a nonclimacteric fruit. Pharmacological experiments, as well as gain- and loss-of-function studies, were performed to demonstrate the critical role of sucrose in the regulation of fruit ripening. Fruit growth and development were closely correlated with a change in sucrose content. Exogenous sucrose and its nonmetabolizable analog, turanose, induced ABA accumulation in fruit and accelerated dramatically fruit ripening. A set of sucrose transporters, FaSUT1–7, was identified and characterized, among which FaSUT1 was found to be a major component responsible for sucrose accumulation during fruit development. RNA interference-induced silencing of FaSUT1 led to a decrease in both sucrose and ABA content, and arrested fruit ripening. By contrast, overexpression of FaSUT1 led to an increase in both sucrose and ABA content, and accelerated fruit ripening. In conclusion, this study demonstrates that sucrose is an important signal in the regulation of strawberry fruit ripening.