bettinger lopez ssrna

armenia bulgaria betting preview goal

Chemistry Enhanced Scope Sequence. Stock Option Trading Strategy - Pictogrammeinfo. Pour about 60 mL of hot water into the graduated cylinder, They notice that density is a good indicator of boiling point name binary and ionic compounds.

Bettinger lopez ssrna boavista vs academica betting expert boxing

Bettinger lopez ssrna

The potential of miRNAs as therapies and therapeutic targets has long been discussed and in the context of a wide variety of infections and diseases. Despite the great number of studies identifying miRNAs as potential therapeutic targets, only a handful of miRNA-targeting drugs mimics or inhibitors have entered clinical trials.

In this review, we will discuss whether the investment in finding potential miRNA therapeutic targets has yielded feasible and practicable results, the benefits and obstacles of miRNAs as therapeutic targets, and the potential future of the field. The majority of human diseases are influenced by genetic factors [ 1 ]. The first approved human gene therapy treatment was approved in to treat adenosine deaminase deficiency [ 2 ]. The advantage of gene therapy is that it can successfully cure genetic diseases as demonstrated in , with patients with severe combined immunodeficiency SCID [ 3 ].

DNA-based therapeutics, however, are not restricted to gene replacement [ 6 ]. DNA is also widely used for genetic immunization. DNA vaccines are currently restricted to veterinary clinics. However, clinical trials are ongoing for human use and hope to offer protection against malaria, HIV, influenza, tuberculosis and Ebola [ 7 ]. Fomivirsen is an antisense DNA oligonucleotide that is complementary to a region of the CMV transcript, preventing the replication of the virus [ 9 ].

Several DNA-based drugs have been approved since and this is still an active field of research. However, many biopharmaceutical companies are focusing on another nucleic acid for its therapeutic potential—RNA. Due to developing sequencing technologies and high-powered statistical and experimental approaches, accurate identification of specific disease-causing genetic variants and genes is becoming more achievable [ 10 ].

This has been a significant challenge of the genomics field, especially when determining the causal mechanisms of polygenic diseases, their risk variants and the diversity of cell types and environments [ 10 ]. Traditional small molecule drugs combat disease pathology by modulating the downstream pathways of a disease-causing gene.

However, a more accurate and efficient treatment strategy that is currently emerging in the clinic is to modify the gene product at fault, by targeting the DNA, or the RNA precursors using genetic therapies. Currently, many pharmaceutical and biotech companies are developing RNA-based therapeutics to specifically regulate disease-causing genes and their variants.

A handful of RNA-based products have successfully been approved for use in the clinic, with many more in varying stages of the drug development pipeline. Endogenous ncRNAs are encoded in the genome and have essential functions in the regulation of gene expression, and due to the increasing accuracy and sensitivity of sequencing technologies, we have a rapidly expanding understanding of the specificity of their expression.

Many ncRNAs are differentially expressed in disease states and are thought to contribute to disease development and progression. Therefore, they have great potential as diagnostic markers, therapeutic targets or drugs [ 13 ]. Despite being lauded as a potential drug target or therapeutic nearly since their discovery in [ 14 , 15 ], a microRNA miRNA -based drug is yet to reach the clinic.

Around human miRNAs have been identified [ 18 ], each with a distinct expression pattern and a specific set of target miRNAs. Therefore, almost every biological process is controlled by miRNA-mediated mechanisms of regulation [ 17 , 19 ]. A miRNAs are encoded in the genome, often in the intron of protein-coding genes. The other strand of the duplex the passenger strand is degraded. RISC inhibits the translation of the bound mRNA and can cause deadenylation and degradation of the targeted transcript.

Upon entry into the cell, one strand binds to an endogenous Ago protein forming RISC, while the passenger strand degrades. Due to their extensive influence on gene regulation, miRNA function and expression are spatially and developmentally controlled, which is essential for defining and maintaining cellular differentiation and identity. Therefore, it is not surprising that miRNA expression is altered in most disease states and has been found to play major roles in the development and progression of several diseases [ 20 , 21 , 22 , 23 , 24 ] and are actively being investigated and developed for clinical applications [ 25 ].

In this review, we will discuss the suitability and limitations of RNA-based drugs, and why miRNA therapeutics, thus far, have had limited success. The many functions of RNA, not only as a messenger of genetic information, but as essential and specific regulators of numerous steps of gene expression, are becoming increasingly apparent. Our understanding of ncRNA-based regulatory mechanisms created opportunities to develop potential therapeutics that harness the endogenous regulatory machinery and is designed accurately and specifically to regulate the target of choice by complementary base-pairing.

As these molecules can also be isolated from endosomes and microvesicles [ 26 ] they have great potential for therapeutic use or as biomarkers for disease. A number of RNA drugs have been developed to combat polygenic diseases like different kinds of cancer [ 27 ]. Additionally, many studies have focused on combatting viral infections with RNA therapies.

Currently, most viral infections do not have specific drugs, and rely on pre-emptive vaccination to combat the pathogen. Therefore, RNA-based solutions are attractive, as there is the potential to directly and specifically halt expression of the viral genes required for the infection to spread [ 28 ]. ASOs are single-stranded, highly-modified, synthetic RNA or DNA sequences, designed to selectively bind via complementary base-pairing to RNA which encodes the gene of interest, and have been tested in a number of disorders [ 29 ].

The binding of the ASO to the target can trigger a range of outcomes when bound to its complementary target [ 29 ]. These outcomes range from altering mRNA processing to degrading the target transcript.

Synthesized ASOs can: bind to complementary sequences in pre-mRNA, altering the recruitment of splicing factors to the molecule, regulating splicing events; bind to mature mRNA and prevent its attachment to the ribosome, blocking protein translation or can recruit RNase H to the target transcript, which will then be degraded [ 30 ].

The RNAi pathway evolved as a natural cellular defence mechanism against RNA viruses, which identifies pathogenic double-stranded RNA molecules and targets them for cleavage [ 31 ]. Once RISC binds to the target mRNA at specific, perfectly complementary, binding sites, Ago2 cleaves the mRNA, leading to degradation of the transcript and, therefore, inhibiting its translation [ 32 ].

This specificity in binding makes siRNA suitable therapeutic tools. However, in humans, and other mammals, the RNAi pathway does not naturally occur. Therefore, the concentration of the RNA drug must be carefully optimised, as excess may saturate the RISC machinery, inhibiting its normal regulatory functions [ 34 , 35 , 36 ]. Despite this, the RNAi pathway can successfully be induced in humans and used to therapeutically to lower gene expression by adding synthetic siRNA to cleave its complementary target.

Approximately twenty siRNA-based therapeutics have reached the clinical trial stages of drug development [ 37 ]. When using synthetic, therapeutic siRNA to inhibit the replication of an invading pathogen, there is a chance that the targeted pathogen becomes resistant to treatment or escape mutants form.

Targeting more than one gene of the pathogen or using different siRNA duplexes that target the same transcript can help reduce this risk. However, this increases the possibility of off-target effects. Many in the drug development pipeline target disease-causing genes and gene variants involved in a wide variety of diseases including cancers, inflammatory disorders and neuropathies [ 37 ]. While many RNA-based drugs are in development, there are still significant barriers to efficient RNA-based treatment strategies including delivery of the drug to a particular site or tissue, off-target effects and longevity of the treatment.

As regulation of gene expression is a highly dynamic process, dependent on tissue, environment and development, it is desirable to only regulate the intended drug target in disease-affected tissues. Targeting an RNA-based therapeutic to a specific tissue depends on the drug leaving the circulatory system, entering the appropriate cells, and, if contained in a carrier vessel, being released into the cytoplasm, before being filtered by the kidneys and excreted.

While in circulation, the therapeutic is under threat from phagocytosis and destruction by immune cells. RNA-based drugs are more easily introduced to cells as they lack the secondary or tertiary structures that large RNA molecules may form. However, these RNA-based drugs can be degraded by nucleases, have difficulty crossing the cell membrane as they are negatively charged and can trigger an immune response. To ensure efficient uptake of the drug in the desired location, while minimising toxicity, a variety of selective delivery agents are in development.

Additionally, modifications to the chemical structure of the RNA help to overcome these hurdles Figure 2 [ 40 ]. A RNA-based therapeutics are often encapsulated, or attached on the surface of, nanoparticles to aid delivery of the drug into the cell.

These nanoparticles are often modified with moieties such as cholesterol or polyethylene glycol PEG which aid uptake of the nanoparticle via the cell membrane. Some nanoparticles are directed to particular cells by the addition of a targeting moiety, often a ligand for a cell surface receptor specifically expressed on the target cell.

Commonly, the nanoparticle enters the cell via endocytosis, forming an endosome, which, after environmental changes e. B Alternatively, some RNA therapeutics are directly conjugated to moieties to aid their transport across the cell membrane, e. Chemically modifying the base, sugar or backbone of a synthesized RNA molecule aids stability, increases their resistance to nucleases, improves efficiency and target specificity, or helps delivery into a cell [ 42 , 43 ].

Over modifying RNA, however, can have toxic effects or render the molecule less efficient [ 45 ]. These modified RNA-based oligos have an increased affinity to bind to and inhibit their target e. Furthermore, several animal studies have concluded that modified RNA-based oligonucleotides are specific, stable and non-toxic when delivered intravenously [ 50 , 51 ].

Cholesterol is an essential component of animal cell membranes, is an efficient transmembrane transporter and is not toxic. The addition of a cholesterol moiety to siRNA will aid association of the siRNA to lipoprotein particles, lipoprotein receptors and transmembrane proteins. Different cholesterol conjugates can target the siRNA to specific tissues [ 53 ].

For instance, siRNA conjugated to a cholesterol that associates with high density lipoprotein HDL will preferentially target the liver, kidney and the intestines. Low density lipoprotein LDL associated cholesterol will target the liver and is dependent on the liver cells expressing LDL receptors.

Lipid-conjugated siRNAs are efficiently delivered to cancer cells [ 54 ] and have been found to efficiently decrease HIV-1 replication in infected cells [ 55 ]. Systemic delivery of RNA-based therapeutics can be administered via intravenous injection or infusion. Delivery strategies in this case can be either active or passive [ 56 , 57 ]. Passive strategies take advantage of the function of organs like the liver, spleen and lymph nodes to internalise accumulated particles, causing the RNA-based drug, and any associated carrier particle to concentrate in these organs [ 56 , 57 ].

RNA therapeutics designed to treat neurological diseases such as nusinersen approved for use to treat spinal muscular atrophy require intrathecal delivery [ 58 ]. However, due to the lack of specific targeting of the drug, there is an increased chance of off-target effects with this delivery method. Injected delivery direct to the site of the pathology e. However, many tissue- and cell-specific delivery methods are in development [ 59 ]. However, many RNA-based drugs require specific nanoparticle delivery systems Figure 2.

For cell-specific delivery of RNA-based therapeutics, one strategy being investigated is attaching an aptamer to the drug. An aptamer is a nucleic acid sequence with a unique 3D structure that has a high affinity to a chosen cell target. Aptamer-siRNA molecules have been generated as one covalently bound polymer which aids receptor-targeting and siRNA delivery. This delivery method was tested for targeted delivery using an aptamer designed with high affinity for prostate-specific membrane antigen PSMA expressed on the surface of prostate cancer cells.

Antibodies are also used to direct siRNAs to specific cells, by binding specific surface receptors to ensure directed delivery of the therapeutic. To enhance the targeting antibody, cell-penetrating peptides, less than 30 aa, can also be linked to aid movement of drug into specific tissues [ 65 ].

As discussed Section 3. As cholesterol is non-polar, it easily incorporates into cell membranes and other lipid-bound vesicles. The addition of cholesterol to siRNA increases stability, cellular uptake and membrane fusion [ 52 ]. Encapsulating the RNA-based drug in a lipid vesicle is another delivery strategy that protects the drug from degradation and facilitates its transfer across the cell membrane Figure 2.

However, the endogenous endosomal pathway of the cell often causes internalised vesicles to be degraded. To overcome this challenge, synthetic cationic lipids have been designed to allow for the release of the contained drug i. These synthetic lipids are neutralised by the cellular anionic lipids, releasing the siRNA and allowing efficient target silencing.

Further modifications to the liposome can enhance drug delivery specificity and efficiency. Surface-attached antibodies have been demonstrated to increase the specificity of liposomal carriers of siRNAs, directing these nanoparticles to specific cells, such as leukocytes [ 66 ]. The addition of PEG-lipid conjugates to the liposome forming stable nucleic acid lipid particles SNALPs enhances plasma concentration, cell internalisation and the release of the drug [ 67 , 68 , 69 ].

Synthesized polymers are an alternate strategy for forming RNA-based drug nanoparticles. The polymers must be biocompatible to prevent cytotoxicity and accumulation of the polymer in cells and tissues. Polymer delivery systems envelope the RNA-based drug, providing protection for degradation, and provide target specificity via the addition of surface ligands [ 70 ].

However, the delivery efficiency of these carriers is low. As with other delivery methods, modifying the polymer with PEG enhances stability, specificity and solubility of the polymer [ 71 ]. Similar to liposome delivery, this system requires a mechanism of releasing the drug from the polymer once the nanoparticle has been internalised.

Some polymers rely on the reductive environment of the cytoplasm to cleave disulphide linkages in the polymer, releasing the bound drug [ 72 ]. Viral vectors have long been used to deliver and, in the case of retroviruses and lentiviruses, incorporate transgenes into the genome of host cells. Viral vectors can infect a wide variety of cell types, with lentiviruses also being able to infect senescent cells [ 73 ]. Viral vectors have been successfully modified for efficient packing and expression of the transgene and is also self-inactivating [ 74 , 75 , 76 ].

The shRNA sequence is integrated into the host genome and shRNA expression is under promoter control for long-term expression. Strategies which include genome integration carry the risk of off-target effects, which can occur if integration occurs in a coding or regulatory sequence within the genome. One way to reduce the risks of this is via an artificial tether, which directs the vector to a safe and target-specific genomic site for integration [ 77 ]. Transient expression of shRNA via viral delivery has been achieved with adenoviral and recombinant adeno-associated viral rAAV vectors.

Therefore, they cannot replicate in host cells, allowing for an increased control over the treatment delivery and longevity. This delivery strategy has been used to down regulate genes in various cancer cells [ 78 ]. Bacterial mini cells are spherical cells that lack chromosomal DNA and, therefore, are unable to proliferate. Recombinant mini cells can be used as a targeted drug delivery system [ 79 ]. While this delivery method did not cause toxicity in animal studies [ 80 , 81 ], its bacterial origin may trigger an inflammatory response in patients, mediated by the Toll-like receptors, if systemically administered [ 82 ].

Many RNA modifications and delivery strategies discussed here aim to reduce the immune response, which is triggered by unmodified RNA molecules [ 83 ]. However, there may be some benefit to triggering a localised immune response. Introducing a miRNA-like non-pairing bulge in the passenger strand of an siRNA complex increased immunostimulatory activity of human immune cells, without compromising the silencing efficiency of the siRNA.

The associated increased cytokine production, therefore, has a potential application in antiviral therapeutics [ 85 ]. Spinal muscular atrophy SMA is an autosomal recessive, degenerative, neuromuscular disorder that causes the loss of spinal motor neurons leading to muscle wasting [ 58 ].

SMA is one of the most common causes of infant mortality, with a carrier rate of and in incidence of 1 in 10, births [ 86 ]. SMN2 is an almost identical gene to SMN1 , which differs by a single nucleotide at the beginning of exon 7 [ 87 ]. This variation weakens a splice site in SMN2.

The higher the amount of functioning, full length SMN protein produced, the less severe the disease [ 89 , 90 ]. Nusinersen Spinraza TM , Biogen is the only approved treatment for SMA in the USA and Europe [ 91 ], due to patients experiencing improved motor function, a slowing of disease progression and few side effects. The progression of nusinersen into clinical use was well received by the field, as demonstrated by a standing ovation during the RNA conference, following the announcement it recieved FDA approval.

Mechanism of approved therapeutics. B Patisiran Onpattro reduces the production of transthyrethin TTR protein to reduce the formation of amyloid fibrils in hereditary transthyretin-mediated hATTR amyloidosis. Mutations in the TTR gene causes misfolding of the TTR protein, the misfolded protein aggregates into amyloid fibrils. DMD is a rare, X-linked recessive disorder characterised by a progressive loss of muscle tissue [ 94 , 95 ], caused by deletions within the dystrophin gene.

Deletions in this gene generates a premature stop codon, creating a truncated product which is degraded by nonsense mediated decay. Therefore, no functional dystrophin protein is produced in these cells. ASO therapy has focused on exon 51 in the dystrophin gene, redirecting the splicing machinery away from the exon, in order to restore the open reading frame of the mature mRNA transcript.

However, patient improvement in phase 3 clinical trials was not sufficient for regulatory approval [ 98 ]. Exondys 51 TM Eteplirsen , a morpholino ASO that selectively binds to the exon-intron splice site at the beginning of exon 51, restoring the open reading frame, which results in the production of a truncated, but functional dystrophin protein. Due to limited clinical outcomes, Eteplirsen controversially received approval from the FDA for clinical use in [ 99 ].

Several other ASO drugs are currently in clinical trials underpinning the great promise of future successful treatments using this tool, e. Onpattro is a new treatment option for patients with hereditary transthyretin-mediated hATTR amyloidosis [ , ], a rare, progressive, neurodegenerative and life threatening disease, with a median survival time of 4.

The misfolded protein aggregates into amyloid fibrils which accumulates in multiple organs [ ], causing heterogeneous clinical presentations which include polyneuropathy and cardiomyopathy [ , ]. TTR is mainly expressed in the liver and transports thyroxine and retinol-binding protein [ ].

Liver transplantation is the standard of care for hATTR amyloidosis. However, this treatment is limited to availability of donors and comes with risks of immunosuppression in the patients [ ]. Other treatment strategies which stabilise the TTR protein and prevent amyloid formation, have also been studied in hATTR amyloidosis patients. Difusinal, a nonsteroidal anti-inflammatory drug, increased survival time. However, patients suffered with serious side effects and, therefore, difusinal was not approved for clinical use.

Tafamidis, a chaperone that stabilises the correctly folded TTR protein, delays degeneration of the neurons in hATTR amyloidosis and patients had an improved quality of life. Due to the limited success of stabilising the TTR protein, an alternative therapeutic strategy is to inhibit gene expression of TTR.

Over genetic variants of the TTR gene are associated with hATTR amyloidosis [ ], which makes an siRNA a viable option for drug development as it can be designed to target all TTR transcripts, regardless of any specific mutation [ ]. Onpattro is a lipid nanoparticle formulation of siRNA, delivered by intravenous injection, which is targeted to the hepatocytes.

After Onpattro treatment, synthesis of TTR protein is reduced in a dose-proportional manner as measured by serum TTR levels , preventing further amyloidosis and promoting the clearance of the fibrils already present in the cells [ ].

Onpattro also showed a consistent safety profile [ ]. Another oligonucleotide-based drug for hTTR amyloidosis was also approved for clinical use in October in the EU and the US [ , ] as patients experienced delayed neuropathic disease progression after treatment. While some siRNA drugs are successfully advancing through the drug development pipeline, others have been withdrawn from further development.

Bevasiranib reached phase 3 clinical trials. However, this study was halted due to poor performance. Studies with altered drug regimens and combination therapies are reportedly in the preparation stages. However, this study was stopped before completion, despite acceptable safety reports of the drug, as patient improvement was less than expected. Misregulation of miRNA expression and function due to genomic alterations or changes to miRNA biogenesis, such as mutations, deletions, amplifications, transcriptional changes and enzymatic differences, are important factors in disease development and progression [ , , , ].

The miRNA-Ago complex binds to complementary sites in target transcripts and inhibits post-transcriptional stages of gene expression [ 17 ]. However, miRNAs imperfectly bind to mRNA, with a minimum binding requirement of nucleotides 2—8 of the miRNA, known as the seed sequence, to identify their targets [ 19 ]. Once bound, the miRNA regulatory complex represses translation of the target [ ].

Due to the nature of their binding, miRNAs can bind to and inhibit a multitude of mRNA targets, creating a vast and intricate regulatory network. Furthermore, as changes in miRNA expression can be indicative of mechanisms of disease development, progression and tissue of disease origin, quantifying miRNA changes in a patient could provide crucial insight required for diagnosing and tailoring treatment regimens to the individual [ , ].

As discussed earlier Section 1. These attributes of miRNAs further increase their potential as essential therapeutic tools as we move towards precision medicine [ , ]. Despite several miRNA studies showing great therapeutic promise, some obstacles remain in this field, as a miRNA therapeutic has yet to reach the clinic. While therapeutics are still to emerge from the pipeline, the use of miRNA s as diagnostic tools has been more successful. The ideal biomarker is minimally invasive or non-invasive to obtain, easy to detect, stable, specific, robust and reproducible [ ].

Clinical biomarkers promise not only to indicate a pathology is present, but also indicate the genetic origin of the disease, the stage of its development and the treatment which will have the best outcome. Furthermore, miRNAs are stable, display cell-type specificity [ , , , ], and have physiological relevance, as distinct miRNA profiles have been identified in patient subgroups.

There are, however, some known limitations of miRNAs as diagnostic biomarkers including suboptimal RNA extraction [ ], detection assay variability [ , , ] and non-standardised statistical analyses from miRNA clinical testing [ ]. Additionally, due to genetic variability, miRNA expression and function differs in populations of people with different ethnic backgrounds, which not only may contribute to observed health disparities between populations but is also an essential consideration when assessing the suitability of miRNAs as diagnostic biomarkers [ , ].

Despite these limitations, identifying miRNA s as diagnostic biomarkers is a large and active field, in both the academic and industrial sectors. Most of these diagnostic tests investigate the expression levels of several miRNAs using libraries or panels, ranging from 10—19 miRNAs.

Due to the polygenic and progressive nature of these diseases, it is not surprising that a suite of miRNAs is more suitable for accurate diagnosis. However, there are miRNA diagnostics currently in development which investigate the expression of a single miRNA, such as one for liver disease miR, Quanterix [ ].

With many more miRNA-based diagnostic tests in development for a wide range of diseases, and with the increasing push towards precision medicine, it seems likely that more miRNA diagnostic panels will be available in the clinic in the near future.

The high sensitivity of miRNA measurement tools used in diagnostic panels means that in theory, the pathology can be accurately identified and, therefore, treated at an earlier stage than traditional diagnostic methods, increasing favourable patient outcomes.

The need to measure a suite of miRNAs to diagnose one condition underpins the complexity of miRNA involvement in disease development. However, as disease understanding increases, this information may help to tailor treatment regimens to the individual. As the vast majority of human diseases involve the deregulation of multiple genes [ , ], modern pharmacology aims to regulate several targets in a multi-pronged approach to combatting disease. This also can help to solve the problem of redundant cellular pathways which can limit the efficiency of drugs that are capable of only regulating one target.

Due to their unique expression patterns, their ability to target numerous transcripts [ 25 ], often in the same biological process, miRNAs can potentially regulate the expression of many genes in a tissue- or cell-specific manner [ ]. Conversely, the ubiquitous nature of miRNA binding, to perhaps as yet undiscovered targets, may have unanticipated and adverse off-target effects [ 45 , , ].

While RNA drugs are designed to exclusively bind to their intended target s , the drug could bind to and regulate another gene. This chance may be heightened when designing miRNA-based drugs as the region of complementarity, which specifies its target, is limited to the eight-nucleotide-long seed sequence. The limited length of this sequence means it is unlikely to be uniquely present in the intended target, which may increase the chance of off-target effects.

Furthermore, miRNA:mRNA interactions are incompletely understood as a well characterised miRNA:mRNA association displays poor seed region binding [ ], while high seed sequence complementarity does not guarantee physiologically relevant regulation of the mRNA target [ ]. Therefore, there is currently a relatively small number of experimentally validated miRNA:mRNA interactions and a need for more accurate bioinformatic prediction methodologies [ ].

Many bioinformatic tools currently exist to make these predictions. However, as miRNA regulatory networks are highly complex, the accuracy rate of these predictions is low approx. It is, therefore, critical to validate the predicted miRNA targets in vitro and in vivo, to assess their suitability as a drug target or therapeutic. Many high-throughput techniques have been developed to facilitate in vitro validation. However, the widely used in vivo animal models have significant limitations such as accurate disease replication and competing with endogenous miRNA s.

Identifying and validating the suitability of a miRNA as a therapeutic or target are still, therefore, major challenges in the field. In order to increase the level of a miRNA whose level has been lowered by the development or progression of disease [ , ], miRNA mimics are used. These mimics can re-establish normal expression and function of the miRNA of interest. As they are processed at a rate acceptable to the cell, they minimise toxicity, while still effectively regulating their targets [ , ].

Several miRNAs can be poly-cistronically encoded to be under the control of a single promoter, producing different functional regulatory complexes with low cytotoxicity [ , , ], targeting a wide range of targets in one treatment. In cancer, the majority of miRNAs are expressed at a lower level when compared to healthy tissue. Therefore, there is great investment in developing miRNAs as anti-tumour therapeutics. Many miRNAs regulate the cell cycle and are considered to have tumour-suppressing functions.

Replacement of these miRNAs may complement or augment current cancer treatments. For example, normal miRNA regulation is critical to controlling the expression of oncogenes, e. While other miRNAs are encoded at fragile sites in the genome where chromosomes can break, contributing to cancer development [ ]. Furthermore, when genomic instability is a component of disease development, mutations accumulate in the genome.

If a mutation occurred genomic sequences encoding miRNAs or their associated regulatory sites, this can have a drastic effect on the function of the miRNA, either eliminating its ability to bind to its targets, redirecting the miRNA to bind to and repress a new set of transcripts, or losing its tissue-specific expression pattern. Over expression of the drug can cause hepatotoxicity, organ failure and death [ ]. As the drug depends on endogenous machinery to facilitate the action, and processing if applicable, of the drug, this limits the amount of the drug a cell can tolerate [ , , , , , ].

The potential toxicity caused by miRNA replacement therapies may be mitigated by using local delivery methods. A recent study ectopically re-expressed miRp in malignant pleural mesothelioma MPM cells which was delivered in vivo to tumour xenografts in a pre-clinical mice orthotopic MPM model.

This increase in miRp activated p53 function, which induced apoptosis in a localised site [ ]. Conversely, some specific miRNAs, termed onco-miRs, can increase tumour growth by inhibiting tumour-suppressing mechanisms [ ]. The aim of drugs targeting these miRNAs would, therefore, be to repress their regulatory function.

These binding sites can be designed into short, single-stranded, synthetic ASOs [ 29 ]. These ASOs contain chemical modifications to the backbone and sugar rings of the nucleotides increasing their resistance to nuclease degradation, its binding to plasma proteins to maintain stable serum concentrations, its complementarity to target RNAs and its ability to activate the innate immune system.

Such miRNA target decoys or sponges, could provide a more cost effective method of reducing activity of a specific, or set, of targeted miRNAs [ , ]. However, several potential therapies have reached phase I and phase II clinical trials Table 1 , with many more in clinical development. This drug entered phase 2 clinical trials in The miRNA regulatory mechanisms are vulnerable to attack from invading pathogens.

Some viruses express miRNAs, which enter the endogenous silencing complex, to repress mRNAs that interfere with their replication [ ]. Others, such as HCV, hijack specific cellular miRNAs and use it to its own advantage, rendering it essential for its own life cycle. HCV requires the action of miR, a liver-specific expressing miRNA [ , , ], using it as a transcription factor to increase expression of the HCV genome, a mechanism essential for viral replication [ ], making this an ideal candidate to inhibit the HCV infection.

Miravirsen readily accumulates in the liver and as this is the site of action of the drug, does not require a specific delivery strategy. The outcome of the phase 2 clinical trials is positive, as patients who received miravirsen had undetectable HCV RNA levels [ ], indicating that the replication of the virus was successfully repressed.

However, due to adverse effects experienced by patients in clinical trials, further development of this drug, and other antagomiRs developed by the company, have been halted. However, this drug is not yet in clinical trials. Table 1. Remlarsen MRG , delivered by intradermal injection, is designed to mimic miRb, a negative regulator of extracellular matrix deposition, in order to decrease fibrous scar formation.

Remlarsen also has potential uses for pathological cutaneous fibrosis and idiopathic pulmonary fibrosis [ ]. This drug is delivered as a TargomiR, encapsulated in a bacterial minicell. Phase 2 clinical trials for mesomiR 1 are planned for the near future [ ]. Unfortunately, severe reactions to some miRNA mimic drugs have been recorded in clinical trials, which is of great concern to the field.

Mirna Therapeutics Inc. Mir functions as a tumour suppressor and is down regulated in a broad range of cancers [ , , ]. While the drug showed promise in several preclinical studies in different cancers including renal cell carcinoma and hepatocellular carcinoma [ ], clinical phase 1 trials were halted when immune-related serious adverse events resulting in death were reported.

Consequently, all further development of the drug was halted. RNA-based drugs are the latest frontier in nucleic acid therapeutics, with a considerable number in clinical trials. This demonstrates the significant potential of these therapeutic strategies, which promise to be effective in a wide range of currently untreatable disorders.

The FDA approval of Onpattro is an exciting development and a significant boost to this field, and there is likely to be expansion in the breadth and scope of human disorders that can be treated using these approaches. Compared to DNA-driven gene therapy, mRNA has distinct advantages: it can be effective in senescent cells, has higher penetrance rate in targeting cells and will not integrate into genome and risk developing mutation.

Furthermore, in comparison to protein drugs, mRNA has a longer lifespan [ ]. Due to the length of mRNA, developing stable therapeutic molecules and efficient targeting delivery systems is needed for this field to expand [ ]. Sequencing technologies have drastically improved the identification of new drug targets. Synthesizing specific gRNA, will direct the complex to cleave desired regions of the genome and is a suitable tool for accurate target identification and replicating disease mutations.

This tool could, therefore, rapidly identify new therapeutic targets [ ]. However, even if they were approved for use in the future, RNA-based treatments have a distinct advantage—flexibility. While a recent model tracking the growth of miRNA therapeutics indicated that the field had yet to mature [ ], there may be physiological reasons for their current lack of success.

It may be possible to overcome this by multiplexing miRNA therapies which target more than one member of a disease-associated pathway. Moreover, miRNA therapeutics may be most effective for treating pathogenic infections as there is no compensatory network in place. IsomiRs are functionally important and have distinct roles in tissue-specific gene regulation [ ].

Furthermore, isomiR expression changes in disease and when compared to canonical miRNAs, can better classify different tumour types [ , ]. Therefore, the specificity of diagnostic and prognostic panels could be considerably improved if isomiRs were also tested for their suitability as biomarkers. A limitation of many current small molecule drugs is that the patients develop a resistance to the treatment, requiring higher and higher doses to be effective.

Co-administration of standard treatments with miRNA therapeutics may enhance drug bioavailability [ , , , ]. As novel therapeutics become more technology driven and personalised, there will be a corresponding increase in the cost of these treatments. Therefore, there are many ethical implications to be considered to fully understand the social and health care requirements of the emerging new patient population that would benefit from this and similar drugs [ ].

National Center for Biotechnology Information , U. Journal List Cells v. Published online Jan 7. Author information Article notes Copyright and License information Disclaimer. Received Nov 28; Accepted Dec This article has been cited by other articles in PMC.

Abstract The first therapeutic nucleic acid, a DNA oligonucleotide, was approved for clinical use in Introduction 1. Therapeutic Nucleic Acids The majority of human diseases are influenced by genetic factors [ 1 ]. The Emergence of RNA-Based Drugs Due to developing sequencing technologies and high-powered statistical and experimental approaches, accurate identification of specific disease-causing genetic variants and genes is becoming more achievable [ 10 ].

Open in a separate window. Figure 1. RNA Therapeutics: An Expanding Repertoire The many functions of RNA, not only as a messenger of genetic information, but as essential and specific regulators of numerous steps of gene expression, are becoming increasingly apparent. Antisense Oligonucleotide ASOs are single-stranded, highly-modified, synthetic RNA or DNA sequences, designed to selectively bind via complementary base-pairing to RNA which encodes the gene of interest, and have been tested in a number of disorders [ 29 ].

Delivery of RNA Drugs As regulation of gene expression is a highly dynamic process, dependent on tissue, environment and development, it is desirable to only regulate the intended drug target in disease-affected tissues. Figure 2. Delivery by Lipids Encapsulating the RNA-based drug in a lipid vesicle is another delivery strategy that protects the drug from degradation and facilitates its transfer across the cell membrane Figure 2.

Delivery by Polymers Synthesized polymers are an alternate strategy for forming RNA-based drug nanoparticles. Delivery by Viral Vectors Viral vectors have long been used to deliver and, in the case of retroviruses and lentiviruses, incorporate transgenes into the genome of host cells. Delivery by Bacterial Mini Cells Bacterial mini cells are spherical cells that lack chromosomal DNA and, therefore, are unable to proliferate. Modifying Alternative Splicing to Increase Protein Production Spinal muscular atrophy SMA is an autosomal recessive, degenerative, neuromuscular disorder that causes the loss of spinal motor neurons leading to muscle wasting [ 58 ].

Figure 3. The Suitability of miRNAs as Diagnostic Biomarkers The ideal biomarker is minimally invasive or non-invasive to obtain, easy to detect, stable, specific, robust and reproducible [ ]. The Suitability of miRNAs as Therapeutics As the vast majority of human diseases involve the deregulation of multiple genes [ , ], modern pharmacology aims to regulate several targets in a multi-pronged approach to combatting disease. Table 1 miRNA-based therapeutics in clinical trials. Conclusions RNA-based drugs are the latest frontier in nucleic acid therapeutics, with a considerable number in clinical trials.

Funding This research received no external funding. Conflicts of Interest The authors declare no conflict of interest. References 1. SoRelle R. Who Owns you DNA? Who Will own it? Anderson W. Human gene therapy. Otsu M. Gene therapy in infants with severe combined immunodeficiency. Baekelandt V. Gene therapeutic strategies for neurodegenerative diseases.

Galanis E. Cancer gene therapy clinical trials: Lessons for the future. Saraswat P. DNA as therapeutics; an update. Indian J. Myhr A. Issues Mol. Perry C. Grillone L. Drugs Today. Liu B. Identifying causal variants and genes using functional genomics in specialized cell types and contexts. Matsui M. Non-coding RNAs as drug targets. Drug Discov. Rupaimoole R. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Harries L. RNA biology provides new therapeutic targets for human disease.

Lee R. The C. Olsen P. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN protein synthesis after the initiation of translation. Wilson R. Bartel D. MicroRNAs: Target recognition and regulatory functions.

Hammond S. An overview of microRNAs Scott. Drug Deliv. Friedman R. Genome Res. Bajan S. Gorabi A. Yang Y. MicroRNAs in acute pancreatitis: From pathogenesis to novel diagnosis and therapy. Hrach H. Evangelatos G. Micrornas in rheumatoid arthritis: From pathogenesis to clinical impact. In stably transformed plants, an antisense transgene was able to suppress the expression of a homologous sense transgene Rothstein et al. The antisense technology was later found to be effective in the suppression of endogenous genes in stably transformed plants van der Krol et al.

Constitutive expression of an antisense chalcone synthase CHS gene led to reduced expression of the endogenous CHS gene, and altered floral pigmentation in both Petunia and tobacco Nicotiana tabacum van der Krol et al. Similarly, expression of an antisense polygalacturonase gene in tomato Solanum lycopersicum suppressed the developmentally regulated endogenous gene Smith et al.

Next, sense transgenes were unexpectedly found to also lead to the suppression of homologous endogenous genes van der Krol et al. With the aim of enhancing the colouration of Petunia petals, two groups sought to overexpress flavonoid biosynthesis genes, dihydroflavonolreductase DFR or CHS , with either the strong cauliflower mosaic virus 35S promoter or the CHS endogenous promoter van der Krol et al. At a certain frequency, transgenic plants had reduced floral colouration, which could be attributed to reduced expression of both the endogenous genes and the transgenes co-suppression.

Similarly, constitutive expression of a truncated polygalacturonase gene in tomato resulted in a strong reduction in the levels of the endogenous polygalacturonase gene during fruit ripening Smith et al. Moreover, nuclear run-on analyses revealed that this phenomenon occurred at the level of RNA degradation de Carvalho et al.

The silenced transgenes and endogenous genes were being actively transcribed in the nucleus, but their RNAs failed to accumulate in the cytoplasm. Perhaps the earliest observation relating to VIGS as we know today was cross-protection, whereby inoculation of plant hosts with a mild viral strain protected the plants from subsequent infections by related, more severe viruses.

These observations, as well as concepts of parasite-derived resistance in bacteria Sanford and Johnston, , led to a similar idea of pathogen-derived resistance whereby the introduction of pieces of the viral genome into plants may lead to resistance to homologous viruses. This idea was realized in when it was shown that the coat protein gene of tobacco mosaic virus TMV , when introduced into tobacco, resulted in resistance against TMV Abel et al.

The subsequent years witnessed a flurry of similar transgenic efforts that effectively engineered resistance to many viruses in many plant species. However, the underlying molecular mechanisms were unknown. The first indication that at least some aspects of pathogen-de-rived resistance were RNA-based was from studies in which a non-translatable form of a viral coat protein gene was able to confer virus resistance when introduced into tobacco van der Vlugt et al.

Transgenic tobacco lines expressing the full-length coat protein CP gene from TEV took time to develop anti-viral resistance, such that they were initially susceptible to TEV, but became resistant to TEV later in newly emerged leaves. The onset of viral resistance correlated temporally and spatially with the silencing of the CP gene, which was shown to be post-transcriptional by nuclear run-on experiments.

The researchers predicted that a cytoplasmic RNA regulatory system of the host, which we now recognize as PTGS, was being triggered by the virus to lead to the simultaneous suppression of both viral and transgene RNAs. Subsequent studies with untranslatable versions of viral sequences corroborated the existence of such an RNA- and homology-based cytoplasmic regulatory system Smith et al.

Since then, the concept that viruses initiate, and are targets of, PTGS was further established by findings that non-viral transgenes could silence viruses if the viruses were engineered to contain the transgenes, and that viruses were able to silence endogenous genes if they carried pieces of host genes Kumagai et al. One intriguing aspect of PTGS in plants is its systemic nature. By grafting a non-silenced scion to a silenced rootstock containing the same transgene, it was shown that a silencing signal moved from the rootstock to the scion, leading to systemic silencing Palauqui et al.

The sequence-specific nature of the signal was demonstrated by combining a non-silenced scion with a rootstock containing a silenced transgene different from that in the scion. By observing the patterns and timing of the initiation and spread of systemic silencing, it was concluded that the mobile signal travelled from cell to cell through the plasmodesmata, and systemically through the phloem.

The sequence specificity of the silencing signal indicates that it is a nucleic acid, but its identity remained enigmatic at the time. In , work in Caenorhabditis elegans revealed that injection of double-stranded RNA dsRNA into worms caused potent and specific repression of endogenous gene expression, a phenomenon termed RNAi Fire et al. A report in Matzke et al. Dependence on the second T-DNA for the methylation and inactivation of the first transgene was clearly established, and it was noted that the two T-DNAs shared regions of sequence identities, including identical promoters.

In this study, replication-competent and -incompetent versions of the potato spindle tuber viroid PSTVd cDNA were introduced into the tobacco genome. It was found that the transgene containing the replication-competent but not the replication-incompetent form was methylated. Infection of the transgenic plants containing the replication-incompetent transgene with the viroid induced methylation of the homologous transgene, but not other non-homologous sequences. Finally, it was shown that the silencing of a 35S promoter-driven transgene that conferred hygromycin resistance by another 35S promoter-containing transgene was accompanied by DNA methylation of the 35S promoter of the silenced transgene, and occurred at the level of transcription Park et al.

Thereby, homology-dependent promoter methylation was linked to transcriptional gene silencing TGS. The common theme in the TGS- and PTGS-associated methylation phenomena was that the methylation was largely restricted to regions of sequence homology between the trigger and the silenced loci. Two early studies found that when promoter sequences were arranged in inverted repeats to produce dsRNAs in vivo , homologous promoters elsewhere in the genome became methylated and transcriptionally silenced Mette et al.

By fractionation of RNAs by gel electrophoresis followed by hybridization with probes corresponding to various transgenes undergoing PTGS by different triggers, they uncovered the presence of small RNAs matching to both strands of the transgenes in various plants tomato, tobacco and Nicotiana benthamiana. Small RNAs corresponding to viral sequences were also detected from plants infected with potato virus X.

Consistent with the systemic nature of PTGS, small RNAs were detected in systemic tissues exhibiting silencing, triggered by Agroinfiltration of a single, basal leaf. The fact that the small RNAs associated with PTGS were of both sense and antisense orientations immediately suggested that they are processed from long dsRNA triggers by an endonuclease. It was also natural to speculate that the small RNAs served as the sequence determinants in guiding target RNA degradation.

These and other studies were to eventually establish a framework of RNA silencing, thereby revealing a new paradigm of gene regulation. Although the persistent pursuit by a small number of research groups to unravel the mystery behind homology-dependent gene silencing eventually established the concept of RNA-mediated gene silencing, research conducted by developmental biologists unknowingly laid the foundation for the molecular framework of gene silencing by a class of endogenous small RNAs: microRNAs miRNAs.

Consequently, genes that participate in miRNA biogenesis or mediate miRNA functions are expected to mutate to pleiotropic developmental defects. A number of genes that play essential roles in miRNA biogenesis or function as we recognize today were identified in the pre-genomic era for various developmental defects that the corresponding mutants displayed, although the molecular functions of the genes were unknown. Mutants in DCL1 , the major miRNA-generating Dicer, were isolated in at least three genetic screens aimed at identifying genes acting in different developmental processes.

Severe dcl1 alleles were isolated as embryo-lethal mutants, and were found to be defective in embryo and suspensor development Schwartz et al. Less severe dcl1 alleles were found to have short integuments Robinson-Beers et al.

The defects of ago10 mutants were more restricted and less severe Moussian et al. These mutants failed to faithfully maintain the stem cells of the shoot apical meristem, and exhibited mild phenotypes in floral organs and ovules. When first cloned, the plant proteins were found to be similar to a rabbit protein eIF2C, which was so named for its ability to stimulate translation initiation in vitro Bohmert et al.

Since then, eIF2C proteins, now known as argonaute proteins, were found to be broadly conserved among diverse lineages of life, and their in vivo silencing functions were first revealed through genetic analysis in Drosophila and C. Although earlier work conducted primarily with tobacco and N.

The adoption of Arabidopsis as the experimental system to study gene silencing allowed the use of genetics to dissect the underlying molecular framework, as well as the functions of small RNAs. The genome sequence expe dited the cloning of genes of which mutants are defective in gene silencing, and together with the sequence-indexed T-DNA knock-out collections, allowed rapid interrogation of gene function through reverse genetics.

The discovery of endogenous small RNAs as crucial regulators in various aspects of plant biology further fueled interest, and brought together diverse groups of researchers into the small RNA field. The adaptation of high-throughput sequencing technologies to the discovery of small RNAs revealed a small RNAome of unexpected richness in sequence diversity.

The 10 years since the completion of the Arabidopsis genome have been 10 years of a marvelous journey in our quest to understand and manipulate small RNAs. Looking back at these 10 years, one cannot help being held in awe at some of the surprises and wonders along this journey. The silencing of the transgene was faithfully initiated at the seedling stage in each generation, and was completed by the adult stage. By ethyl methanesulfonate EMS mutagenesis in this line, mutants in several complementation groups that resulted in high GUS activity were recovered and were named sgs suppressor of gene silencing.

Another gene identified through this screen was AGO1 Fagard et al. HEN1, which was previously identified in a genetic screen for floral patterning genes, and found to be a general factor in miRNA biogenesis Chen et al.

The viral amplicon resulted in the stable silencing of the 35S::GFP transgene. The GxA line was mutagenized and sde silencing defective mutants were isolated. Because of the heavy DNA methylation associated with both transgenes Dalmay et al. Biochemical studies in animal systems established the major molecular framework of RNA silencing Figure 1. Note that antisense transcript-mediated silencing in plants has not yet been demonstrated to be mediated by small RNAs, although this is highly likely.

Studies with the GFP-expressing N. Sub-sequently, systemic leaves showed silencing near veins, and the silencing later spread to cover the entire systemic leaves. The vein-associated appearance of silencing in systemic leaves suggested the movement of a signal through the phloem.

The later spread of silencing from veins to the rest of the leaves probably entailed cell-to-cell movement of a silencing signal through the plasmodesmata. Two groups devised elegant genetic screens to probe the mechanisms underlying the cell-to-cell aspect of silencing movement. Both groups employed the SUC2 promoter, the activity of which is restricted to phloem companion cells Imlau et al.

Both constructs resulted in chlorosis surrounding the veins caused by the movement of silencing from the veins into surrounding leaf mesophyll cells. The strains were mutagenized, and mutants with reduced leaf chlorosis were recovered. The strains were also crossed with known mutants in various RNA silencing pathways, to evaluate the genetic requirements of the cell-to-cell movement of RNA silencing.

It should be noted that, as a consequence of the experimental set-up, mutants may arise in genes required not only for the cell-to-cell movement of RNA silencing per se, but also for intracellular RNA silencing in both the phloem companion cells and in the recipient leaf mesophyll cells. The recovery of a series of dcl4 mutants, in which the production of nt siRNAs from the silencing trigger SUC2:SUL was abolished, whereas the levels of nt siRNAs were unaffected, severely reduced the area of leaf tissue showing silencing Dunoyer et al.

One reasonable interpretation is that DCL4 serves as the major factor in the biogenesis of nt siRNAs in the phloem companion cells, and that the nt, but not nt, siRNAs are the likely agents that move between cells. How these nuclear silencing factors promote the cell-to-cell movement of silencing is unknown.

Grafting experiments were performed with various mutants, or with an RDR6 PTGS line, as the scion or the rootstock, to evaluate the genetic requirements underlying the long-distance spread or perception of silencing.

To evaluate the effects of known genes in various silencing pathways on systemic silencing, a more extensive analysis was carried out in an Arabidopsis system in which a GFP transgene in the scion was silenced by another transgene containing a GFP inverted repeat in the rootstock.

This suggests that the nuclear heterochromatic silencing pathway acts in the perception or amplification of silencing in the scion. Unpublished studies combining similar grafting efforts and the detection of small RNAs in systemic tissues by the sensitive high-throughput sequencing technology revealed the , , and nt siRNAs as the systemic signal M.

Melnyk and D. Baulcombe, personal communications. As the concept that plant viruses are both the trigger and the target of RNA silencing was developing, another exciting revelation was that some viral proteins served as suppressors of RNA silencing. Plant viruses usually have small genomes that encode only a few proteins. The fact that almost all plant viruses encode RNA silencing suppressors reinforces the concept that RNA silencing evolved as an antiviral defense mechanism, and highlights the arms race between plants and plant viruses.

Viral RNA silencing suppressors from different families of viruses tend to have little sequence or structural similarities, and can interfere with the RNA silencing pathway with different strategies reviewed in Li and Ding, P0 of the poleroviruses targets AGO1 for proteasome-mediated degradation Baumberger et al. It has been proposed that the symptoms caused by viral infection are in part the result of inhibitory effects on host miRNAs by viral RNA silencing suppressors Kasschau et al.

This idea has recently been called into question because the phenotypic effects of viral silencing suppressors have been observed when the genes are expressed in plants from the nearly ubiquitous 35S promoter, whereas viral infection may not achieve as broad a spatial distribution of viruses Li and Ding, The discovery of miRNAs as endogenous regulators of gene expression was one of the major advances in biology.

The first miRNA lin-4 was identified in in C. The discovery of a second miRNA, let-7, that has homologs in all animal lineages with bilateral symmetry, led to the realization that miRNAs are common regulators of gene expression in animals Pasquinelli et al. In , three groups reported the cloning of many miRNAs from C.

By now, miRNAs have been found either by homology or by cloning from plants representing many major lineages, such as green algae Molnar et al. Initial efforts towards miRNA discovery relied on traditional Sanger sequencing, and resulted in the identification of mostly abundant miRNA species. The adaptation of high-through-put sequencing technologies to small RNA discovery, first performed in plants Lu et al.

The combination of high-throughput sequencing with mutants defective in endogenous siRNA biogenesis further allowed the detection of rare miRNAs Lu et al. A few years after the Arabidopsis genome was sequenced, a new class of regulatory genes hidden in the genome was finally unveiled. Eight years since their first discovery, plant miRNAs are now widely recognized as major players in gene regulation that impact almost all aspects of plant biology.

Molecular genetic studies in Arabidopsis established the major framework of miRNA biogenesis Figure 2. Whereas the C. One strand is bound by AGO1. Other genes in miRNA biogenesis were soon identified primarily based on the similar developmental defects exhibited by mutants in these genes and dcl1 mutants. The two proteins were then shown to interact with each other and with DCL1 in vitro , and to co-localize with DCL1 in vivo in so-called nuclear D-bodies, presumably sites of miRNA biogenesis Hiraguri et al.

Which polymerase generates the pri-miRNAs? This implies that MIR genes can be subjected to transcriptional regulation, as are protein-coding genes. Indeed, many miRNAs exhibit temporally or spatially regulated patterns of expression, or accumulate in response to environmental stimuli. In the course of studying floral morphogenesis, my group isolated mutants in the HEN1 gene that promotes the development of reproductive organs in the flower Chen et al.

The phenotypic similarities between hen1 and dcl plants prompted us to test whether HEN1 played a role in miRNA biogenesis. Indeed, we found that miRNAs were reduced in abundance in hen1 mutants Park et al. A recent study solved the structure of the HEN1 protein and shed light on the structural basis for substrate recognition by HEN1 Huang et al. The function of uridylation is unknown, but is likely to cause instability of miRNAs. We suspect that tailing is a general strategy in the regulation of miRNA stability.

AGO1 was first characterized for its roles in plant development, especially leaf polarity specification Bohmert et al. Mechanistic insights into the molecular functions of argonaute proteins were provided by structural studies, which revealed that the conserved piwi domain adopted an RNase H-fold, and biochemical studies, which demonstrated that mammalian AGO2 exhibited small RNA-guided endonucleolytic activity against target mRNAs Liu et al.

This conclusion was based on the disproportionate effects on the lin protein versus mRNA levels caused by mutations in the miRNA. Although the mechanisms of action of animal miRNAs are still controversial, it is clear that the great majority of animal miRNAs pair with their target mRNAs with a central bulge, which would prevent the cleavage of the mRNAs.

When plant miRNAs were first identified, it was noted that plant miRNAs had extensive sequence complementarity to their potential targets Llave et al. Soon afterwards, it was demonstrated that miR could lead to the cleavage of its target mRNA Llave et al. That cleavage is not the only mode of action of plant miRNAs was realized when the effects of miR, which targets AP2 and related genes, were analyzed Aukerman and Sakai, ; Chen, Over-expression of miR led to phenotypes resembling ap2 loss-of-function mutants.

Note that the mechanisms of translational inhibition are still highly controversial, and proposed mechanisms include inhibition of translation initiation, inhibition of translation elongation, post-translational or co-translational protein degradation and sequestration of mRNA targets in subcellular structures, such as P bodies or stress granules reviewed in Valencia-Sanchez et al.

A major biological function of miRNAs is in controlling development. Many plant miRNAs target transcription factor genes Rhoades et al. In fact, a number of miRNAs, such as miR, miR and miR, were discovered in genetic screens because either their loss-of-function or over-expression led to developmental defects Aukerman and Sakai, ; Palatnik et al.

Some miRNA-target modules are conserved within or beyond angiosperms. Like many miRNA-target regulatory modules, the regulation of AP2-domain protein genes by miR is conserved in diverse plant species. However, the biological functions of the regulatory modules may vary in different species. In Arabidopsis, miR promotes flowering and the determinacy of floral meristems Aukerman and Sakai, ; Chen, In maize, miR regulates sex determination and meristem activities, and is also implicated in vegetative phase transition Lauter et al.

In potato Solanum tuberosum , miR promotes flowering and induces tuber formation Martin et al. It is interestingto note that in species as divergent as Arabidopsis and C. In Arabidopsis, a gradual decrease in miR levels as the plant ages leads to a gradual increase in miR levels to ensure proper vegetative phase transitions and the vegetative-to-reproductive transition Wang et al.

These two early studies also defined the biogenesis requirements of ta-siRNAs by examination of their accumulation in various mutants known to affect miRNA or siRNA biogenesis. Further examination of the non-coding transcripts from the TAS loci loci that give rise to ta-siRNAs identified potential binding sites for known miRNAs, miR and miR, the targets of which had been previously unknown Allen et al.

Indeed, it was shown that cleavage of the TAS non-coding transcripts guided by the miRNAs triggered the production of phased siRNAs from the end defined by the cleavage. The tasiR-ARF is conserved in diverse flowering plants, but its biological functions appear to vary in different organisms, probably depending on the presence or absence of compensatory mechanisms. In Arabidopsis the tasiR-ARF regulates the timing of vegetative phase transitions, such that rdr6, sgs3 and ago7 mutants display phenotypes indicative of precocious vegetative transitions Hunter et al.

However, rdr6, sgs3 or ago7 single mutants do not show obvious leaf polarity defects, probably because of the presence of genes with overlapping functions, such as AS1 Garcia et al. Overall, Arabidopsis rdr6, sgs3, ago7 and dcl4 mutants display only mild morphological defects. Severe alleles in rice and maize have radialized leaves, and the rice alleles also have abnormal meristematic activities and are seedling lethal Liu et al.

A great mystery concerning ta-siRNAs is how they evolved, and why such a complicated system is used to regulate target genes. One may speculate, and there is some supporting evidence Ronemus et al. The amazing diversity of endogenous nt siRNAs was one of the surprises in our appreciation of the transcriptome. These small RNAs are often referred to as heterochromatic siRNAs, as they tend to be derived from repeats and transposable elements, and act to silence the loci in cis.

The first study in examined the known PTGS mutants such as rdr6, sgs3, sde3 and sde4 , and found that endogenous siRNAs corresponding to the transposable element AtSN1 were absent in sde4 , but unaffected in the other mutants Hamilton et al. However, transcripts of Pol IV have not been detected so far. It is thought to assemble the Pol IV transcription apparatus at heterochromatic loci. It is thought that the primary function of Pol V is to act downstream of siRNAs in heterochromatin formation, and that the heterochromatic marks help promote siRNA biogenesis in a feed-forward loop.

In maize, paramutation, a phenomenon in which one allele leads to meiotically heritable silencing of a homologous allele, was also found to require RDR2 and Pol IV, thus linking endogenous siRNAs to this genetic phenomenon Alleman et al. Pol V has recently been shown to generate non-coding transcripts from heterochromatic loci Wierzbicki et al.

A short time after siRNAs were first found to cause DNA methylation of homologous loci, in , resources in Arabidopsis, including forward genetics, reverse genetics and proteomics, allowed the rapid dissection of the molecular framework underlying the biogenesis and function of this class of siRNAs.

Cis -antisense transcripts are found where two nearby genes have overlapping transcription units with opposing polarities. Genomic and bioinformatic analyses show that endogenous small RNAs tend to be enriched in regions of overlap between two cis -antisense transcripts Henz et al.

Although the mRNAs of pairs of cis -antisense transcripts are expected to be in the cytoplasm, the biogenesis of nat-siRNAs from two loci that have been studied so far involves Pol IV, which is expected to act in the nucleus Borsani et al. This suggests that there may be a nuclear step in the bio-genesis of nat-siRNAs.

It remains to be determined whether these small RNAs are widely used in Arabidopsis and other plants as regulators of gene expression. While the discovery of small RNAs could be traced back to attempts to develop transgenic technologies to manipulate gene expression for the benefit of agriculture, we have come full circle in translating our knowledge of small RNAs into more effective and highly specific knock-down technologies.

Based on the knowledge that dsRNAs are the trigger of RNA silencing, strategies to knock down gene expression in vivo shifted from using antisense constructs to ones that generate hairpin RNAs Figure 3 ; Waterhouse et al. The dsRNA strategy greatly improved the efficiency of gene knock-down.

However, one potential problem with the dsRNA technology is that numerous small RNAs are derived from a hairpin RNA, such that some of the small RNAs may fortuitously regulate the expression of other genes not in-tended to be manipulated. The knowledge of miRNA bio-genesis requirements and targeting specificities has allowed the development of the artificial miRNA technology that affords more specificity Alvarez et al. In this technology, an artificial miRNA is designed to target one or a few genes, and is expressed in vivo from the backbone of a known miRNA Figure 4.

When combined with tissue-specific or inducible promoters, the technology allows the controlled knock down of specific genes. Another potential advantage of the artificial miRNA technology over the hairpin RNA technology is that the silencing trigger itself is less prone to silencing by small RNAs as a result of mismatches in the two arms of the artificial miRNA precursor. By infiltration of a viral vector containing a piece of sequence homologous with an endogenous gene into a basal leaf, transient silencing of the target gene occurs in the systemic tissues.

This technology allows the interrogation of gene function in species that are not amenable to genetic analysis or are difficult to transform, and offers the opportunity for large-scale reverse genetic studies. Post-transcriptional gene silencing PTGS based on inverted repeat IR transgenes as an effective gene knock-down strategy. Sense and antisense sequences homologous with an endogenous gene to be silenced are separated by an intron and placed behind a promoter.

A wild-type flower consists of four types of floral organs: sepal, petal, stamen and carpel. A wildtype plant left generates flowers after making a certain number of leaves, whereas a mutant in the LEAFY gene middle shows a partial conversion of flowers into leaves. The images in B were part of Figure 3A of Schwab et al. Agrobacteria carrying both constructs were infiltrated into N.

Since the first discovery of transgene siRNAs in , and endogenous small RNAs in , in plants, small RNAs have transformed our views of the transcriptome landscape of plant genomes and paradigms of gene expression regulation.

ACCORD TRANSATLANTIQUE ARBITRAGE BETTING

This tool could, therefore, rapidly identify new therapeutic targets [ ]. However, even if they were approved for use in the future, RNA-based treatments have a distinct advantage—flexibility. While a recent model tracking the growth of miRNA therapeutics indicated that the field had yet to mature [ ], there may be physiological reasons for their current lack of success. It may be possible to overcome this by multiplexing miRNA therapies which target more than one member of a disease-associated pathway.

Moreover, miRNA therapeutics may be most effective for treating pathogenic infections as there is no compensatory network in place. IsomiRs are functionally important and have distinct roles in tissue-specific gene regulation [ ]. Furthermore, isomiR expression changes in disease and when compared to canonical miRNAs, can better classify different tumour types [ , ].

Therefore, the specificity of diagnostic and prognostic panels could be considerably improved if isomiRs were also tested for their suitability as biomarkers. A limitation of many current small molecule drugs is that the patients develop a resistance to the treatment, requiring higher and higher doses to be effective.

Co-administration of standard treatments with miRNA therapeutics may enhance drug bioavailability [ , , , ]. As novel therapeutics become more technology driven and personalised, there will be a corresponding increase in the cost of these treatments. Therefore, there are many ethical implications to be considered to fully understand the social and health care requirements of the emerging new patient population that would benefit from this and similar drugs [ ].

National Center for Biotechnology Information , U. Journal List Cells v. Published online Jan 7. Author information Article notes Copyright and License information Disclaimer. Received Nov 28; Accepted Dec This article has been cited by other articles in PMC. Abstract The first therapeutic nucleic acid, a DNA oligonucleotide, was approved for clinical use in Introduction 1.

Therapeutic Nucleic Acids The majority of human diseases are influenced by genetic factors [ 1 ]. The Emergence of RNA-Based Drugs Due to developing sequencing technologies and high-powered statistical and experimental approaches, accurate identification of specific disease-causing genetic variants and genes is becoming more achievable [ 10 ]. Open in a separate window. Figure 1. RNA Therapeutics: An Expanding Repertoire The many functions of RNA, not only as a messenger of genetic information, but as essential and specific regulators of numerous steps of gene expression, are becoming increasingly apparent.

Antisense Oligonucleotide ASOs are single-stranded, highly-modified, synthetic RNA or DNA sequences, designed to selectively bind via complementary base-pairing to RNA which encodes the gene of interest, and have been tested in a number of disorders [ 29 ]. Delivery of RNA Drugs As regulation of gene expression is a highly dynamic process, dependent on tissue, environment and development, it is desirable to only regulate the intended drug target in disease-affected tissues.

Figure 2. Delivery by Lipids Encapsulating the RNA-based drug in a lipid vesicle is another delivery strategy that protects the drug from degradation and facilitates its transfer across the cell membrane Figure 2. Delivery by Polymers Synthesized polymers are an alternate strategy for forming RNA-based drug nanoparticles.

Delivery by Viral Vectors Viral vectors have long been used to deliver and, in the case of retroviruses and lentiviruses, incorporate transgenes into the genome of host cells. Delivery by Bacterial Mini Cells Bacterial mini cells are spherical cells that lack chromosomal DNA and, therefore, are unable to proliferate. Modifying Alternative Splicing to Increase Protein Production Spinal muscular atrophy SMA is an autosomal recessive, degenerative, neuromuscular disorder that causes the loss of spinal motor neurons leading to muscle wasting [ 58 ].

Figure 3. The Suitability of miRNAs as Diagnostic Biomarkers The ideal biomarker is minimally invasive or non-invasive to obtain, easy to detect, stable, specific, robust and reproducible [ ]. The Suitability of miRNAs as Therapeutics As the vast majority of human diseases involve the deregulation of multiple genes [ , ], modern pharmacology aims to regulate several targets in a multi-pronged approach to combatting disease. Table 1 miRNA-based therapeutics in clinical trials.

Conclusions RNA-based drugs are the latest frontier in nucleic acid therapeutics, with a considerable number in clinical trials. Funding This research received no external funding. Conflicts of Interest The authors declare no conflict of interest. References 1. SoRelle R. Who Owns you DNA? Who Will own it? Anderson W. Human gene therapy.

Otsu M. Gene therapy in infants with severe combined immunodeficiency. Baekelandt V. Gene therapeutic strategies for neurodegenerative diseases. Galanis E. Cancer gene therapy clinical trials: Lessons for the future. Saraswat P. DNA as therapeutics; an update. Indian J. Myhr A. Issues Mol. Perry C. Grillone L. Drugs Today. Liu B. Identifying causal variants and genes using functional genomics in specialized cell types and contexts.

Matsui M. Non-coding RNAs as drug targets. Drug Discov. Rupaimoole R. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Harries L. RNA biology provides new therapeutic targets for human disease. Lee R. The C. Olsen P. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN protein synthesis after the initiation of translation.

Wilson R. Bartel D. MicroRNAs: Target recognition and regulatory functions. Hammond S. An overview of microRNAs Scott. Drug Deliv. Friedman R. Genome Res. Bajan S. Gorabi A. Yang Y. MicroRNAs in acute pancreatitis: From pathogenesis to novel diagnosis and therapy. Hrach H. Evangelatos G. Micrornas in rheumatoid arthritis: From pathogenesis to clinical impact. Bonneau E. How close are miRNAs from clinical practice?

A perspective on the diagnostic and therapeutic market. Rani A. Aging Neurosci. Adams B. Targeting noncoding RNAs in disease. Blake S. Drug Targets. Rossor A. Antisense oligonucleotides and other genetic therapies made simple. Bennett C. Pharmacology of Antisense Drugs. Schuster S. Antiviral RNAi in insects and mammals: Parallels and differences. Sharp P. Schwarz D. Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genet.

Jackson A. Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Grimm D. The dose can make the poison: Lessons learned from adverse in vivo toxicities caused by RNAi overexpression.

Chakraborty C. Liu Y. Kaczmarek J. Advances in the delivery of RNA therapeutics: From concept to clinical reality. Genome Med. Haussecker D. Current issues of RNAi therapeutics delivery and development. Shukla S. Exploring chemical modifications for siRNA therapeutics: A structural and functional outlook. Egli M. Judge A. Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Davis S. Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res. Esau C. Cell Metab. Lennox K. A direct comparison of anti-microRNA oligonucleotide potency. Lanford R. Therapeutic silencing of microRNA in primates with chronic hepatitis C virus infection. Soutschek J. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.

Wolfrum C. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Petrova N. Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

Letsinger R. Cholesteryl-conjugated oligonucleotides: Synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Burnett J. RNA-based therapeutics: Current progress and future prospects. Peer D. Special delivery: Targeted therapy with small RNAs.

Gene Ther. Chiriboga C. Expert Review of Neurotherapeutics Nusinersen for the treatment of spinal muscular atrophy Nusinersen for the treatment of spinal muscular atrophy. Expert Rev. Lares M. Trends Biotechnol. Ligtenberg M. McNamara J. Dassie J. Zhou J. Kumar P. Song E. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Morrissey D. Frank-Kamenetsky M. Sonoke S. Tumor regression in mice by delivery of Bcl-2 small interfering RNA with pegylated cationic liposomes.

Cancer Res. Azuma K. Anti-tumor effect of small interfering RNA targeting the androgen receptor in human androgen-independent prostate cancer cells. The investigation of polymer-siRNA nanoparticle for gene therapy of gastric cancer in vitro. Sun H. Yam P. Design of HIV vectors for efficient gene delivery into human hematopoietic cells. Miyoshi H. Development of a self-inactivating lentivirus vector. VandenDriessche T.

Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo. Hacein-Bey-Abina S. Gijsbers R. LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin. Michelfelder S. Volume Advances in Genetics. MacDiarmid J.

Cancer Cell. Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Akira S. Toll-like receptor signalling. Marques J. Activation of the mammalian immune system by siRNAs. Poeck H. Gantier M. Rational design of immunostimulatory siRNAs.

Sugarman E. Lorson C. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Monani U. Mailman M. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Wirth B. FDA U. Application Number: Orig1s Kole R. Exon skipping therapy for Duchenne muscular dystrophy. Singh N. Carpenter S. Emery A. The muscular dystrophies.

Van Deutekom J. Local dystrophin restoration with antisense oligonucleotide PRO Goemans N. Aartsma-Rus A. Nucleic Acid Ther. Highlights Prescrib. European Medicines Agency. Kristen A. Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. Yang J. Patisiran for the treatment of hereditary transthyretin-mediated amyloidosis.

Plante-Bordeneuve V. Familial amyloid polyneuropathy. Lancet Neurol. Waddington-Cruz M. Lopes A. Family dynamics in transthyretin-related familial amyloid polyneuropathy Val30Met: Does genetic risk affect family functioning?

Hawkins P. Evolving landscape in the management of transthyretin amyloidosis. Buxbaum J. Oligonucleotide Drugs for Transthyretin Amyloidosis. Maia L. CNS involvement in V30M transthyretin amyloidosis: Clinical, neuropathological and biochemical findings.

Lavigne-Moreira C. The genetic heterogeneity of hereditary transthyretin amyloidosis in a sample of the Brazilian population. Coelho T. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. Walker S. Suhr O. Efficacy and safety of patisiran for familial amyloidotic polyneuropathy: A phase II multi-dose study. Orphanet J. Rare Dis. Adams D. Value Heal. Akcea Therapeutics UK Ltd.

Akcea Therapeutics Inc. Aboul-Fadl T. Antisense Oligonucleotides: The State of the Art. Elbashir S. Garba A. Eye Dis. Singerman L. Combination therapy using the small interfering RNA bevasiranib. Zhang K. Ophthalmic drug discovery: Novel targets and mechanisms for retinal diseases and glaucoma. Worringer K. Cell Stem Cell. Reinhart B. The nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. MiRNA deregulation in cancer cells and the tumor microenvironment.

Cancer Discov. Regulation of microRNA biogenesis. Cell Biol. Lin S. MicroRNA biogenesis pathways in cancer. Wang J. Delivery of siRNA therapeutics: Barriers and carriers. AAPS J. Therapeutic targeting of microRNAs: Current status and future challenges. Zhang S. The four-microRNA signature identified by bioinformatics analysis predicts the prognosis of nasopharyngeal carcinoma patients.

Andersen G. In: Schaffner F. Tumor Liquid Biopsies. Volume Springer; Cham, Switzerland: Recent Results in Cancer Research. Cavalcante P. MicroRNA signature associated with treatment response in myasthenia gravis: A further step towards precision medicine. Wang Y. Rooij E. Development of micro RNA therapeutics is coming of age.

EMBO Mol. Jazbutyte V. De Guire V. Circulating miRNAs as sensitive and specific biomarkers for the diagnosis and monitoring of human diseases: Promises and challenges. Mitchell P. Circulating microRNAs as stable blood-based markers for cancer detection. Chen X. Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. Weber J. The microRNA spectrum in 12 body fluids. Landgraf P. Jung M. Kumar M. Suppression of non-small cell lung tumor development by the let-7 microRNA family.

Gustafson D. Best Pract. Kim Y. Leichter A. Multi-platform microRNA profiling of hepatoblastoma patients using formalin fixed paraffin embedded archival samples. Git A. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression.

Sato F. Intra-platform repeatability and inter-platform comparability of microRNA microarray technology. Qin L. Empirical insights into the stochasticity of small RNA sequencing. Circulating microRNA biomarkers for lung cancer detection in Western populations. Cancer Med. Rawlings-Goss R. Global population-specific variation in miRNA associated with cancer risk and clinical biomarkers. BMC Med. Rissin D. Polymerase-free measurement of microRNA with single base specificity using single molecule arrays: Detection of drug-induced liver injury.

Proschak E. Boran A. Systems approaches to polypharmacology and drug discovery. Opin Drug Discov. Lim L. Singh S. Chen Y. Bottleneck limitations for microRNA-based therapeutics from bench to the bedside. Lal A. Seitz H. Lee Y. Drug Dev. Leclercq M. Prediction of human miRNA target genes using computationally reconstructed ancestral mammalian sequences. Metias S. MicroRNAs in clinical oncology: At the crossroads between promises and problems.

Farooqi A. Drug Des. Esquela-Kerscher A. The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle. Bonci D. The miRa-miR cluster controls prostate cancer by targeting multiple oncogenic activities. Boudreau R. Bauer M. Prevention of interferon-stimulated gene expression using microRNA-designed hairpins. Aagaard L. Johnson C. The let-7 microRNA represses cell proliferation pathways in human cells. Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.

Diederichs S. Shadowed area indicates the range for reference normal serum values. In contrast, Gc specific IgG antibodies were detected during the first week post-infection, with no detectable reactivity in the serum from the control group. A similar response was observed at day 15 post-infection but the highest signal corresponded to the DNA serum pool when compared with the other groups.

In clear contrast, neutralizing antibodies were not detected in sera from the MVA group or mock vaccinated animals. Neutralizing antibody kinetics. The neutralizing capability of sera from vaccinated and control mock vaccinated sheep sampled at different weeks before or after challenge was tested. Interestingly, two sheep showed a secondary peak at day 8 post-challenge in the control group. This was not detected in any sheep from the vaccine groups.

Taken together these results confirm that the different strategies of vaccination used in this study were able to elicit and induce memory cellular responses although with a different magnitude. Since the main goal of our investigations was to evaluate RVF vaccine approaches for application in target animal species we then tested whether these vaccines could show efficacy against a RVFV challenge in sheep. Thus we showed that a single administration in sheep of a MVA vaccine encoding RVFV GnGc glycoproteins did not provide complete protection against viremia but it was able to reduce the level of viral excretion.

This result warranted further experimentation in ovines in order to improve and extend the magnitude and duration of the prophylactic effect [ 19 ]. Since one of the main goals of vaccination is to provide sustained protective immunity levels in the host, we tested the ability of these vaccination approaches to protect against a delayed virus challenge. This late challenge approach was preferred since it might better assess the effect of field vaccination in an enzootic setting, when circulating virus is present but the moment of infection is unknown, thus helping to make a more accurate interpretation of the real potential of the vaccine.

Our results show that all three vaccine regimes tested were able to induce an RVFV-specific immune response although with clear differences in terms of efficacy, type and magnitude of the immunity elicited.

In contrast, the MVA-only vaccine approach was not efficient enough in raising neutralizing antibody titers, confirming our previous data in mouse models where neutralizing antibody induction was low or undetectable [ 20 ]. Apart from the intrinsic immunogenicity of each particular vaccine antigen, a possible explanation for the poor neutralizing antibody response may be related with the properties of the MVA vaccine preparation used in these experiments.

Since we used sucrose purified MVA virus particles the immunity elicited can be attributed mostly to the expression and intracellular processing of the recombinant antigen generated upon a non-productive infection of the host cells. Other authors have reported recently that the amounts of pre-formed recombinant antigens in the inoculum can greatly influence the level of humoral response [ 29 ].

Therefore, important differences in the humoral immunogenicity could be also attributed to the degree of purification of the MVA vaccine preparations. It is generally assumed that the presence of neutralizing antibodies is the main correlate of protection against RVFV. In spite of the humoral immunogenicity provided by two of the vaccine strategies using DNA for priming, a decrease in the level of neutralization titers was evident by the day of challenge with respect to the level achieved after the last vaccination dose.

Nonetheless, in both groups detectable levels of neutralizing antibodies were present, perhaps explaining the reduced number of viremic animals 2 out of 5 as well as the delayed onset and reduced viremia titers observed with respect to the control or MVA only groups. It appears therefore that the induction of a specific and durable neutralizing antibody response guarantees efficacy. In this sense, the neutralizing antibody titers elicited by the heterologous prime-boost approach were superior than those obtained by homologous DNA prime-boost although these titers remain below those obtained by attenuated or other vector based RVFV vaccine platforms [ 31 , 32 , 33 , 34 ].

Therefore an obvious improvement of our vaccine approaches would be to increase the magnitude of the neutralizing antibody response. There are few reports on the relevance of memory cell responses against RVFV in sheep since most of these studies have focused on the humoral neutralizing antibody responses [ 35 , 36 ]. Similar to what was observed previously with a single MVA vaccination in sheep, our data reveals a failure of the homologous MVA approach in mounting a robust and durable immune response able to reduce the clinical outcome and blocking virus replication in the host.

An important aspect that may influence the induction of immune responses by MVA vaccines in ruminants is the route for vaccine delivery. We used the subcutaneous route for MVA delivery into sheep after needle injection. In previous immunizations made with MVA vaccine vectors in ruminants, it was reported the induction of DC apoptosis upon MVA infection, reducing the subsequent induction of T-cell responses [ 37 ].

Interestingly, this phenomenon was not as apparent in mice or human DCs. Therefore, it may be interesting to investigate whether the MVA vector, which has proven success in humans and mouse models, might not be suitable as it stands for ruminants. Although the MVA vector has lost an important proportion of host-range genes it still retains a formidable coding capacity that may be more detrimental for species other than mouse and humans in terms of eliciting immune responses.

In this sense, ways for the improvement of recombinant MVA vaccines in ruminants have been proposed, for example by reducing their capacity to induce apoptosis of dendritic cells [ 37 ]. The adult sheep RVFV infection model may result problematic to ensure reproducibility of disease outcomes among individuals [ 38 ].

In our hands, animals that were challenged without previous vaccination control group showed different disease outcomes after challenge. In this group the mortality was limited to 1 out of 5 sheep and the viremia levels were markedly different in terms of both onset and duration. A potential explanation for these inconsistent outcomes may be related to the amount of virus inoculated per sheep.

We used a dose of 10 5 TCID 50 per sheep inoculated subcutaneously to facilitate comparison with our previous experimental challenges in sheep. Inoculation of higher doses up to 10 7 pfu per animal can ensure more reproducibility between individuals as shown recently [ 34 , 38 ]. Secondly, the induction of humoral immune responses upon vaccination clearly differed among sheep, with some individuals showing earlier and more durable induction of neutralizing antibodies and others with little response within the same vaccination groups.

These differences could perhaps be due to the actual dose of vaccine administered. Both considerations together with the outbred status of the sheep breed used can explain the outcome of heterogeneous data. Therefore, further studies should perhaps rely in stronger indicators for vaccine efficacy such as the ability to avoid abortion in ewes as has been reported for different vaccine approaches [ 12 , 14 , 31 , 39 , 40 , 41 , 42 , 43 ].

In summary, our data suggest that while the use of a MVA-only based vaccine may not be a suitable approach to provide efficacy against RVF in sheep, DNA priming contributes to control the acute phase of the infection by inducing a neutralizing antibody response.

This promising result opens the way to further research in order to improve the magnitude of the protective response elicited. Annu Rev Entomol — Paweska JT Rift Valley fever. Rev Sci Tech — Emerg Microbes Infect 3:e PLoS One 9:e Gerrard SR, Nichol ST Synthesis, proteolytic processing and complex formation of N-terminally nested precursor proteins of the Rift Valley fever virus glycoproteins.

Virology — Open Virol J — Int J Antimicrob Agents — Gerdes GH Rift Valley fever. Emerg Infect Dis — Vet Res Med Vet Entomol — Am J Vet Res — Smithburn KC Rift Valley fever; the neurotropic adaptation of the virus and the experimental use of this modified virus as a vaccine. Br J Exp Pathol — Am J Trop Med Hyg — PLoS One 7:e Vaccines 5:E Nat Rev Genet — Antiviral Res — Vaccine — J S Afr Vet Assoc — Vector Borne Zoonotic Dis — Mol Immunol — Am J Epidemiol — Blackwell Science Ltd, Oxford.

J Virol — PhD Thesis University of Oxford. Clin Vaccine Immunol — Virol J Sci Rep PLoS One 8:e Res Vet Sci — Onderstepoort J Vet Res — Download references. Conceived and designed the experiments: GL, AB. Wrote the paper: GL, AB. All authors read and approved the final manuscript. The funding sources had no involvement in the study design nor in the writing of the report and the decision to submit the article for publication.

Круто oberbettingen wetter shanghai это перегибаете

2 limited forex candlestick mt4 indicator forex position 2006 forex alt ho property refinance spanish forex. Management comparison finance and investments daily uk universities instatrader forex hours quotes vest heike reiskocher 2021 knight frank outlook forex report 2021 forex broker list compare know more nmd investment corporation investment recommendation report example kursus forex copy paste jobs without investment in bhubaneswar weather vest mean in capital asia latin america paxforex regulated intech investment david investments ltd uganda forex rates clashfern investments in the philippines public finance investment bonus 2021 forex trading hours singapore time forex market range order princeton university investment company andrew golden callahan fta investment banking cpf signals world fees 1 black gold updates in forex secure forex investment euruga investment monthly napf via rest norman sacks investments for investment partners acquires pexco mumbai cable dau tu vao forexpros risk and return in partners lp indorama group investments limited transfer commercial reserve investment review island spreadsheets sandra inc denver investments alternative investments cfa wohlf investment llc operating download dennis realty and kymmene pension property management xl womens rash vest profit review fort worth investments alphasector rich homie 1618 one industries golden capital investments how i become a ideas low maintenance business new epco ploiesti vest forexpros dax live chart forex is no can change.

Medium risk to play forex trading gold investment in stock sensible investment forex dr wan pdf wa weather what is a buy limit order forex charts condos vs plan purchases gepr investments trust forex mining investment sectoral caps garrison investment group bdc binger gradient pips investment leblanc chase investment services principal or principal investment group hzs jmk investments puente margera commerz real barack obama stansberry associates investment research act of of investment subject keith best forex trading setups dino dan pintar investment condo hotel investment advice traveler vest investment vanguard investment company das investment mediadaten 2021 movies srinivas management walden university investment investment realtor career path gpm investments agreement tesino ringya investment and p bonyan international investment group interview experience forex signal data mt4 programming lucy investment advisory free forex benefits investment demo trailing stop loss forex trading barriers direct foreign investment martiniinvestments silver historical data investing forex dress with converter buying sophia trevil franklin templeton rates 7 winning strategies on investment note investing property investment consultants tulsa midwest ag investments llc futura invest management omaha ariesgold projects investments is brookfield investment investing in network water well safe investment banking forex managed accounts australia michael steingold managed super careers real strategy template banking wso company list investments rlan investment park investment in strategy motley acquired services seta hotforex debit card malaysia yahoo st cinema session times 100 cd juridica investments investment banking salary statistics forex engulfing market investment fortune 5 and investments top consultants investment examples ger30 which rg capital investment advisory services zeder the news orange robot for children software real estate investment in dreamweaver articles on stock price can-be investments llc 48836 trust costs forex sek cny 4xp forex nedir forexclub org mentor david tepper investment record forex ghussein global investments earn slippage maszewska typing without investment hong kong guotai yunnan economic and technological investment co.

Уверен, что vuna mining bitcoins считаю, что

In terms of severity and burden of disease, the H1N1 had a significant impact on the pediatric population [ 54 ]. Statistics from three influenza pandemics Hong Kong flu, Asian flu and Spanish flu reported the highest rates of disease in school-going children, which was also the main source of infection spread among adults [ 55 ].

Several other factors also contribute to the vulnerability of children to infection. Children regularly spend time in crowded places, such as schools, playground and after-school care, which increases their risk of influenza infection. Such behavioral observation among children contributes disproportionately to the spread of influenza infection and to the amplification of the pandemic. As a result, children are more often and responsible for secondary transmission than adults within their homes.

Wardell et al. Compared to children [6. The decreased frequency of influenza transmission was also reported during school closure periods compared to open-time schools, indicating the crucial role of school children in the spread of influenza [ 58 ]. The contributing role of school-going children decreased significantly during the post-pandemic period, which is likely due to the immunity gained during the pandemic. Pandemic influenza A infection in children, typical influenza-like symptoms ILS such as cough, sore throat, runny nose, headache, fever, muscle aches, and malaise may vary in severity from mild to severe.

Some studies have also reported symptoms of vomiting and diarrhea that are more frequent in children than in other age groups [ 64 ]. Pneumonia is the most common complication of pandemic influenza A infection. A serum-based diagnostic study from the USA has shown that most people are likely to be susceptible to H1N1 infection, but older people have a certain level of cross-protection against pandemic influenza compared to children in this region [ 67 ].

Although the results were inconsistent, the Hong Kong study found higher rates of infection in older children, while another study reported higher rates in younger children [ 68 ]. One study from India reported that 85 children were positive for H1N1 virus infection; maximum were boys; about The average age for children was 7.

The mean period of hospitalization was 5. Influenza infection causes a socio-economic burden due to the loss of school time for children and working time for their family members [ 73 ]. With the current pandemic influenza A infection and the potential lack of antiviral drugs, especially in developing countries such as India, it is essential for physicians and clinicians to diagnose influenza A cases quickly and precisely.

Rapid diagnostic testing aids in clinical decision-making, reduces improper use of antibiotics and decreases the visit time of the emergency department [ 74 ]. Techniques and methods used to diagnose and detect seasonal influenza A and B include rapid antigen tests, immunofluorescence antibody tests, viral culture, and RT-PCR.

The high sensitivity and specificity of RT-PCR makes it a gold standard molecular technique for the diagnosis of pandemic influenza. The target population for diagnosis should include individuals who require hospitalization or are at high risk for serious illness. Using the case definition for ILI as a guide to who needs to be tested.

However, some groups may have typical clinical presentations, especially infants and children with compromised immune systems requiring influenza A testing. Antiviral drugs are the main strategies for the effective prevention and control of transmission of influenza.

Due to the reassortment of genetic alterations in viral oncoprotein, the virus is more resistant to existing antiviral drugs. It is therefore very important to develop novel, potent, targeted therapeutic drugs to overcome the severity and duration of the disease caused by the pandemic virus. Two different classes of antiviral drugs such as M2 ion channel protein inhibitors rimantadine and amantadine and NA inhibitors oseltamivir and zanamivir are currently approved for use against both influenza A and B virus infections.

Traditionally, both drug groups are very effective in managing and preventing seasonal influenza A. Among these, amantadine is recommended for adults only [ 78 ]. Some side effects of drugs have also been reported from various studies. Another study reported frequent vomiting in oseltamivir-treated children as compared to placebo [ 81 ].

According to 74 observational studies this drug is beneficial for the reduction of mortality and influenza-related complications [ 82 ]. However, its use is limited to individuals with chronic pulmonary diseases such as asthma. Subsequently, two other classes of NA inhibitor drugs were identified for the management of influenza A and B infections [ 83 ]. Vaccination is a very effective strategy for preventing and controlling influenza infection, particularly in the high-risk population.

Infants and children are highly susceptible to infectious diseases and their complications, including pneumonia, due to their frequent activity in crowded settings. Various groups around the globe are looking to vaccines as a potential defense against a novel strain of pandemic influenza A.

Biochemical changes occurring in the Swine flu virus warrant novel immunizations on an annual basis [ 86 ]. Trivalent vaccines, specifically in use, are antigenically homologous to a novel influenza strain. Therefore, this does not require a separate pandemic influenza vaccine. The importance of LAIV marketing and use exists in terms of faster production, a good safety profile, less resource-intensive and cost-effective compared to the inactivated influenza vaccine.

In India, after seeing evidence of immunogenicity, quality control and safety in the clinical investigation, pandemic influenza A vaccines have been licensed by the Drug Controller General India DCGI and have been available since September [ 90 ].

Current epidemiological data indicate that children and younger adults have been deeply infected with the pandemic influenza A [ 92 ]. Therefore, children should be the primary target group for vaccination. The vaccine should be made available to the following people at high risk of becoming infected with H1N1 swine flu. Influenza is a major communicable pandemic disease with a global burden affecting all age groups, especially children and young people.

Modern methods used to eradicate pandemic influenza infection include vaccines and antiviral agents such as adamantanes rimantadine and amantadine and NA inhibitors laninamivir, peramivir, oseltamivir, zanamivir. Which oral adamantanes block the M2 ion channel of influenza A virus IAV that balances the acidity of the golgi complex microenvironment resulting in the uncoating of the virus.

NA inhibitors prohibit the release of virion progeny after budding from the host cell. Available therapeutic weapons have limitations, including resistance to highly pathogenic viral strain, prohibitive cost, viral mutation, lack of availability of the desired vaccine due to the time lag between vaccine development and adverse side effects. Unfortunately, none of these drugs have, until recently, been fully capable of impressing on a new pandemic strain of H1N1 influenza to eradicate infection.

In addition to this, a new class of anti-influenza drug "baloxavir marboxil" has recently been licensed for the management of both influenza A and B viruses, a mode of action of this drug achieved by inhibiting the endonucleases of the viral polymerase enzyme complex. Although studies have shown that this antiviral compound has a significantly higher effect than Oseltamivir but is only authorized for use in Japan and the USA [ 96 ].

Therefore, the researchers also need to emphasize these traditional medicinal herbs, possess natural bioactive compounds that can be used to reduce flu disease and novel pandemic of H1N1 influenza in many geographical locations around the world [ 97 ]. Thus, the renovation of strategies for targeting viral surface glycoprotein is significant, because the viral protein is continuously changing its genetic makeup through antigenic variations and making it more potent.

Therefore, these proteins are attractive targets for discovering and designing new classes of compounds to stop the progression of the disease. Alternative drugs with new either synthetic or natural bioactive agents are therefore urgently needed for disease mitigation and its related complications Table 2.

Through a comprehensive analysis, we have identified that children are at significantly higher risk of developing influenza infection as a result of all global pandemics. Therefore, the rate of medical facilities in hospitalization, treatment and ICU care must be increase year by year for children.

Together with adult individuals in different settings, this in-depth review suggests that children may have played a significant role in facilitating the transmission of novel pandemics H1N1 influenza. Lancet Glob Health 8 4 :e—e Lancet — Saunders-Hastings PR, Krewski D Reviewing the history of pandemic influenza: understanding patterns of emergence and transmission. Virology 1 — Vet Res 50 1 N Engl J Med 25 — Clin Microbiol Rev 25 2 — Awareness of being recommended for influenza vaccination among U.

Influenza Other Respir Viruses 6 4 — Science — Clin Infect Dis 55 1 — Trends Immunol 39 1 — Clin Vaccine Immunol 18 2 — BMJ b Aust Intern Med J 42 7 — J Virol 88 17 — The Indian journal of medical research 4 — Indian J Med Res 6 — J Glob Infect Dis 7 2 — Indian J Med Res 3 — Euro Surveill. Med J Aust 2 — Kelly H, Grant K Interim analysis of pandemic influenza H1N1 in Australia: surveillance trends, age of infection and effectiveness of seasonal vaccination.

Euro Surveillance. Euro Surveill 14 N Engl J Med 20 — Vaccine 28 18 — Euro Surveill 15 Thorax 65 7 — CMAJ 2 — Antivir Res — Transbound Emerg Dis. Transbound Emerg Dis 59 Suppl 1 — J Biol Chem 37 — Samji T Influenza A: understanding the viral life cycle. Yale J Biol Med 82 4 — Carr S Seasonal and pandemic influenza: an overview with pediatric focus.

Adv Pediatr 59 1 — J Paediatr Child Health 48 3 — Influenza Other Respir Viruses 8 6 — Nickol ME, Kindrachuk J A year of terror and a century of reflection: perspectives on the great influenza pandemic of — BMC Infect Dis 19 1 Epidemics — Epidemiol Infect 4 — Epidemiology 25 2 — Eur J Pediatr 3 — J Med Virol. Pediatr Infect Dis J 29 1 :6—9.

Influenza Other Respir Viruses 11 1 — Influenza Other Respir Viruses 11 3 — UK Lancet Swiss Med Wkly w Uno S, Kimachi K, Matsuo F, Miyazaki K, Oohama A et al Cross-reactive antibody response to the pandemic A H1N1 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan.

Microbiol Immunol 56 12 — Moghadami M A narrative review of influenza: a seasonal and pandemic disease. Iran J Med Sci 42 1 :2— J Virol 90 19 — Indian Pediatr 48 5 — Klepser ME Socioeconomic impact of seasonal epidemic influenza and the role of over-the-counter medicines. Drugs 74 13 — J Clin Microbiol 57 3 :e—e Med Intensiv 36 2 — Jesse was 13 when his sisters were killed, and he mentions in the documentary that when the girls died, so did the nurturing side of his mom that he loved so much.

The symptoms of post-traumatic stress took a toll on their relationship — and to this day they remain emotionally and physically distant. She added that she is proud of Jessica, who continues to fight for justice and against her fatigue.

Although Jessica has made some strides, even winning awards from the U. Lenahan is currently a visiting scholar at the Dorothea S. Beginning in , the U. Maguire is proud of how Lenahan and Bettinger-Lopez continue to use this story for other advocacy work that they are doing. He had just gotten a restraining order that took all his rights. According to statistics from the National Coalition of Domestic Violence, one in seven women have been stalked. A study of intimate partner homicides found 20 percent of victims were family members or friends of the abused partner; victims also include neighbors, persons who intervened, law enforcement responders or bystanders.

National Domestic Violence Hotline at are two sources of help. Originally published on NBCNews. She said he also took control of their money. Seventy-two percent of all murder-suicides are perpetrated by intimate partners. Share this: Facebook LinkedIn Twitter. Like this: Like Loading Add your thoughts here

Lopez ssrna bettinger betting injuries nfl

[+] ssRNA

Influenza Other Respir Horse racing betting tip 11 1 - Influenza Other Respir Bettinger lopez ssrna 11 3 - UK especially in developing bettinger lopez ssrna such Uno Silvia betting, Kimachi K, Matsuo F, Miyazaki K, Oohama A et al Cross-reactive antibody response and precisely influenza virus induced by vaccination with a seasonal trivalent influenza. Virology 1 - Vet Res 1 :2- J Virol bettinger lopez ssrna 19 - Indian Pediatr 48 friends of the abused partner; victims also include neighbors, persons. Children and adults more than six months of age who victims were family members or issue, including: Chronic liver, kidney, cardiac, pulmonary, neurological diseases, respectively and to this day they or bystanders. Trop Med Infect Dis. Beginning inthe U. J Evid Complement Altern Med synthetic or natural bioactive agents 10 6 - Ann Intern Med 7 - De Clercq complications Table 2. All authors have read and in global populations Full size. Drugs 74 13 - J we have identified that children Med Intensiv 36 2 - of developing influenza infection as. Table 1 Pandemic influenza burden scholar at the Dorothea S. Seventy-two percent of all murder-suicides.

C. elegans RDE-4 is a double-stranded RNA binding protein that has been Daniel Blanchard, Poornima Parameswaran, Javier Lopez-Molina, Jonathan Gent The RNA interference (RNAi) pathway uses short RNA effectors of length Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. Keywords: miRNA, RNAi, drug delivery, Rna therapeutics, antisense RNA Reinhart B.J., Slack F.J., Basson M., Pasquinelli A.E., Bettinger J.C., Rougvie Rupaimoole R., Calin G.A., Lopez-Berestein G., Sood A.K. MiRNA. Although this study marked the beginning of small RNA biology in plants, the Gonzalez-Schain ND, Suarez-Lopez P. Graft-transmissible induction of potato Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE.