Information

How exactly can dsRNA be introduced to a cell?

How exactly can dsRNA be introduced to a cell?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Is it just by viruses or are there other means by which it gets into cells, such as plasmid uptake?


Besides viral infections there are different pathways for cells to take up dsRNA. Inside the cells these dsRNA are processed by Dicer which processes these RNAs into small interfering RNA, which play an important role in the regulation of gene activity.

These pathways have mostly been researched in Drosophila and C. elegans, I am indicating where evidence for them in humans is available. The different possibilities for the uptake are:

  • Uptake via transmembrane proteins: In C. elegans two different transmembrane proteins have been discovered which passively transport dsRNA into the cells. They are called SID-1 and SID-2 (systemic RNA-interference defective protein), at least for SID-1 a homologue exists in humans as well. These receptors are important for the uptake of systemic (produced by the own organism) dsRNA. See references 1-3 for details.
  • Uptake via Endocytosis: In drosophila cells active transport happens through the scavenger receptors SR-CI and Eater. They bind the dsRNA and are then internalized. Knockdown of these genes inhibited the import of dsRNA and inhibited the endocytosis mediated knockdown of genes. This process seems to be evolutionary conserved, as knockdown of the orthologous genes in C. elegans led to similar results. This mechanism seems to be more important for the uptake of environmental dsRNA. See references 4 and 5 for more details.
  • Uptake via Phagocytosis: At least for drosophila, there is evidence for a phagocytosis mediated uptake mechanism, which is independent of endocytosis. See reference 6 for details.

References

  1. SID-1 is a dsRNA-selective dsRNA-gated channel.
  2. Uptake of extracellular double-stranded RNA by SID-2
  3. RNA interference: a mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells.
  4. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.
  5. Double-stranded RNA Is Internalized by Scavenger Receptor-mediated Endocytosis in Drosophila S2 Cells
  6. A phagocytic route for uptake of double-stranded RNA in RNAi

Biologists first to observe direct inheritance of gene-silencing RNA

In this image of a roundworm (Caenorhabditis elegans), a recently fertilized egg cell (arrow) contains particles of double-stranded RNA (dsRNA, small magenta dots) that are capable of silencing specific genes. A new University of Maryland study shows, for the first time, that these dsRNA molecules pass directly from the parent worm's circulatory system to the egg, revealing a possible mechanism for non-genetic inheritance. Credit: Antony Jose

The basics of genetic inheritance are well known: parents each pass half of their DNA to their offspring during reproduction. This genetic recipe is thought to contain all of the information that a new organism needs to build and operate its body.

But recent research has shown that, in some species, parents' life experiences can alter their offspring. Being underfed, exposed to toxins or stricken by disease can cause changes in a parent's gene expression patterns, and in some cases, these changes can be passed down to the next generation. However, the mechanisms that cause this effect—known as non-genetic inheritance—are a mystery.

New research from the University of Maryland provides a surprising possible explanation. For the first time, developmental biologists have observed molecules of double-stranded RNA (dsRNA)—a close cousin of DNA that can silence genes within cells—being passed directly from parent to offspring in the roundworm Caenorhabditis elegans. Importantly, the gene silencing effect created by dsRNA molecules in parents also persisted in their offspring.

The work, published October 17, 2016 in the online early edition of the Proceedings of the National Academy of Sciences, suggests that the mechanisms for non-genetic inheritance might be simpler than anyone had suspected.

"This is the first time we've seen a dsRNA molecule passing from one generation to the next," said Antony Jose, an assistant professor in the UMD Department of Cell Biology and Molecular Genetics and senior author on the study. "The assumption has been that dsRNA changes the parent's genetic material and this altered genetic material is transmitted to the next generation. But our observations suggest that RNA is cutting out the middle man."

Jose and his team, including graduate student and lead author Julia Marré and former research technician Edward Traver, introduced dsRNA marked with a fluorescent label into the circulatory system of C. elegans worms. They then watched as these fluorescent RNA molecules physically moved from the parent's circulatory system into an egg cell waiting to be fertilized.

In this sequence of microscopic images, taken at the University of Maryland, an immature egg cell from the roundworm Caenorhabditis elegans gets ready for fertilization. The egg already contains half the DNA needed to create a new worm. But as it picks up nutrients from its parent, the egg also picks up particles of double-stranded RNA -- seen here as small magenta dots. This marks the first time that scientists have directly observed particles of double-stranded RNA being transferred to a new generation, revealing new details about non-genetic inheritance. Credit: Antony Jose/University of Maryland College of Computer, Mathematical, and Natural Sciences

In a surprising turn of events, some of the dsRNA molecules could not silence genes in the parent because the dsRNA sequence did not match any of the parent's genes. But the dsRNA molecules did silence genes in the offspring, when the new worm gained a copy of the matching gene from its other parent. This suggests that, in some cases, gene silencing by dsRNA might be able to skip an entire generation.

"It's shocking that we can see dsRNA cross generational boundaries. Our results provide a concrete mechanism for how the environment in one generation could affect the next generation," Jose said. "But it's doubly surprising to see that a parent can transmit the information to silence a gene it doesn't have."

Jose and his colleagues did not expect dsRNA to play such a direct role in the transmission of information across generations. Because dsRNA factors into the life cycle of many viruses, Jose explained, it is reasonable to assume that a living cell's natural defenses would prevent dsRNA from invading the next generation.

"It's very surprising. One would think the next generation would be protected, but we are seeing all of these dsRNA molecules being dumped into the next generation," Jose added. "Egg cells use the same mechanism to absorb nutrients as they prepare for fertilization. The next generation is not only getting nutrition, it's also getting information."

Jose and his colleagues hope to learn more about the precise mechanisms by which dsRNA silences genes across multiple generations.

"There are hints that similar things could be happening in humans. We know that RNA exists in the human bloodstream. But, we don't know where the RNA molecules are coming from, where they're going or exactly what they're doing," Jose said. "Our work reveals an exciting possibility—they could be messages from parents to their offspring."


Naturally occurring siRNAs have a well-defined structure that is a short (usually 20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. The Dicer enzyme catalyzes production of siRNAs from long dsRNAs and small hairpin RNAs. [2] siRNAs can also be introduced into cells by transfection. Since in principle any gene can be knocked down by a synthetic siRNA with a complementary sequence, siRNAs are an important tool for validating gene function and drug targeting in the post-genomic era.

In 1998, Andrew Fire at Carnegie Institution for Science in Washington DC and Craig Mello at University of Massachusetts in Worcester discovered the RNAi mechanism while working on the gene expression in the nematode, Caenorhabditis elegans. [3] They won the Nobel prize for their research with RNAi in 2006. siRNAs and their role in post-transcriptional gene silencing(PTGS) was discovered in plants by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England and reported in Science in 1999. [4] Thomas Tuschl and colleagues soon reported in Nature that synthetic siRNAs could induce RNAi in mammalian cells. [5] In 2001, the expression of a specific gene was successfully silenced by introducing chemically synthesized siRNA into mammalian cells (Tuschl et al). These discoveries led to a surge in interest in harnessing RNAi for biomedical research and drug development. Significant developments in siRNA therapies have been made with both organic (carbon based) and inorganic (non-carbon based) nanoparticles, which have been successful in drug delivery to the brain, offering promising methods to deliver therapeutics into human subjects. However, human applications of siRNA have had significant limitations to its success. One of these being off-targeting. There is also a possibility that these therapies can trigger innate immunity. [3] Animal models have not been successful in accurately representing the extent of this response in humans. Hence, studying the effects of siRNA therapies has been a challenge.

In recent years, siRNA therapies have been approved and new methods have been established to overcome these challenges. There are approved therapies available for commercial use and several currently in the pipeline waiting to get approval. [ citation needed ] [6]

The mechanism by which natural siRNA causes gene silencing through repression of translation occurs as follows:

  1. Long dsRNA (which can come from hairpin, complementary RNAs, and RNA-dependent RNA polymerases) is cleaved by an endo-ribonuclease called Dicer. Dicer cuts the long dsRNA to form short interfering RNA or siRNA this is what enables the molecules to form the RNA-Induced Silencing Complex (RISC).
  2. Once siRNA enters the cell it gets incorporated into other proteins to form the RISC.
  3. Once the siRNA is part of the RISC complex, the siRNA is unwound to form single stranded siRNA.
  4. The strand that is thermodynamically less stable due to its base pairing at the 5´end is chosen to remain part of the RISC-complex
  5. The single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA
  6. Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage.
  7. The mRNA is now cut and recognized as abnormal by the cell. This causes degradation of the mRNA and in turn no translation of the mRNA into amino acids and then proteins. Thus silencing the gene that encodes that mRNA.

siRNA is also similar to miRNA, however, miRNAs are derived from shorter stemloop RNA products, typically silence genes by repression of translation, and have broader specificity of action, while siRNAs typically work by cleaving the mRNA before translation, and have 100% complementarity, thus very tight target specificity. [7]

Gene knockdown by transfection of exogenous siRNA is often unsatisfactory because the effect is only transient, especially in rapidly dividing cells. This may be overcome by creating an expression vector for the siRNA. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is a short hairpin RNA (shRNA), which can be processed into a functional siRNA by Dicer in its usual fashion. [8] Typical transcription cassettes use an RNA polymerase III promoter (e.g., U6 or H1) to direct the transcription of small nuclear RNAs (snRNAs) (U6 is involved in gene splicing H1 is the RNase component of human RNase P). It is theorized that the resulting siRNA transcript is then processed by Dicer.

The gene knockdown efficiency can also be improved by using cell squeezing. [9]

The activity of siRNAs in RNAi is largely dependent on its binding ability to the RNA-induced silencing complex (RISC). Binding of the duplex siRNA to RISC is followed by unwinding and cleavage of the sense strand with endonucleases. The remaining anti-sense strand-RISC complex can then bind to target mRNAs for initiating transcriptional silencing. [10]

It has been found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. It has been shown that dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms. [11]

The siRNA-induced post transcriptional gene silencing starts with the assembly of the RNA-induced silencing complex (RISC). The complex silences certain gene expression by cleaving the mRNA molecules coding the target genes. To begin the process, one of the two siRNA strands, the guide strand (anti-sense strand), will be loaded into the RISC while the other strand, the passenger strand (sense strand), is degraded. Certain Dicer enzymes may be responsible for loading the guide strand into RISC. [12] Then, the siRNA scans for and directs RISC to perfectly complementary sequence on the mRNA molecules. [13] The cleavage of the mRNA molecules is thought to be catalyzed by the Piwi domain of Argonaute proteins of the RISC. The mRNA molecule is then cut precisely by cleaving the phosphodiester bond between the target nucleotides which are paired to siRNA residues 10 and 11, counting from the 5’end. [14] This cleavage results in mRNA fragments that are further degraded by cellular exonucleases. The 5' fragment is degraded from its 3' end by exosome while the 3' fragment is degraded from its 5' end by 5' -3' exoribonuclease 1(XRN1). [15] Dissociation of the target mRNA strand from RISC after the cleavage allow more mRNA to be silenced. This dissociation process is likely to be promoted by extrinsic factors driven by ATP hydrolysis. [14]

Sometimes cleavage of the target mRNA molecule does not occur. In some cases, the endonucleolytic cleavage of the phosphodiester backbone may be suppressed by mismatches of siRNA and target mRNA near the cleaving site. Other times, the Argonaute proteins of the RISC lack endonuclease activity even when the target mRNA and siRNA are perfectly paired. [14] In such cases, gene expression will be silenced by an miRNA induced mechanism instead. [13]

Piwi-interacting RNAs are responsible for the silencing of transposons and are not siRNAs. [16]

Because RNAi intersects with a number of other pathways, it is not surprising that on occasion nonspecific effects are triggered by the experimental introduction of an siRNA. [17] [18] When a mammalian cell encounters a double-stranded RNA such as an siRNA, it may mistake it as a viral by-product and mount an immune response. Furthermore, because structurally related microRNAs modulate gene expression largely via incomplete complementarity base pair interactions with a target mRNA, the introduction of an siRNA may cause unintended off-targeting. Chemical modifications of siRNA may alter the thermodynamic properties that also result in a loss of single nucleotide specificity. [19]

Innate immunity Edit

Introduction of too many siRNA can result in nonspecific events due to activation of innate immune responses. [20] Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid-inducible gene I (RIG-I) may also be involved. [21] The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. Chemical modification of siRNA is employed to reduce in the activation of the innate immune response for gene function and therapeutic applications. One promising method of reducing the nonspecific effects is to convert the siRNA into a microRNA. [22] MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize nonspecific effects.

Off-targeting Edit

Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool. [18] Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (in effect, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. This, however, can be partly addressed by designing appropriate control experiments, and siRNA design algorithms are currently being developed to produce siRNAs free from off-targeting. Genome-wide expression analysis, e.g., by microarray technology, can then be used to verify this and further refine the algorithms. A 2006 paper from the laboratory of Dr. Khvorova implicates 6- or 7-basepair-long stretches from position 2 onward in the siRNA matching with 3'UTR regions in off-targeted genes. [23]

Adaptive immune responses Edit

Plain RNAs may be poor immunogens, but antibodies can easily be created against RNA-protein complexes. Many autoimmune diseases see these types of antibodies. There haven't yet been reports of antibodies against siRNA bound to proteins. Some methods for siRNA delivery adjoin polyethylene glycol (PEG) to the oligonucleotide reducing excretion and improving circulating half-life. However recently a large Phase III trial of PEGylated RNA aptamer against factor IX had to be discontinued by Regado Biosciences because of a severe anaphylactic reaction to the PEG part of the RNA. This reaction led to death in some cases and raises significant concerns about siRNA delivery when PEGylated oligonucleotides are involved. [24]

Saturation of the RNAi machinery Edit

siRNAs transfection into cells typically lowers the expression of many genes, however, the upregulation of genes is also observed. The upregulation of gene expression can partially be explained by the predicted gene targets of endogenous miRNAs. Computational analyses of more than 150 siRNA transfection experiments support a model where exogenous siRNAs can saturate the endogenous RNAi machinery, resulting in the de-repression of endogenous miRNA-regulated genes. [25] Thus, while siRNAs can produce unwanted off-target effects, i.e. unintended downregulation of mRNAs via a partial sequence match between the siRNA and target, the saturation of RNAi machinery is another distinct nonspecific effect, which involves the de-repression of miRNA-regulated genes and results in similar problems in data interpretation and potential toxicity. [26]

siRNAs have been chemically modified to enhance their therapeutic properties, such as enhanced activity, increased serum stability, fewer off-targets and decreased immunological activation. Commonly, siRNA has been encapsulated in a nanolipid particle to prevent degradation in the blood. A detailed database of all such chemical modifications is manually curated as siRNAmod in scientific literature. [27] Chemical modification of siRNA can also inadvertently result in loss of single-nucleotide specificity. [28]

Given the ability to knock down, in essence, any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic [29] and applied biology.

One of the biggest challenges to siRNA and RNAi based therapeutics is intracellular delivery. [30] siRNA also has weak stability and pharmacokinetic behavior. [31] Delivery of siRNA via nanoparticles has shown promise. [30] siRNA oligos in vivo are vulnerable to degradation by plasma and tissue endonucleases and exonucleases [32] and have shown only mild effectiveness in localized delivery sites, such as the human eye. [33] Delivering pure DNA to target organisms is challenging because its large size and structure prevents it from diffusing readily across membranes. [30] siRNA oligos circumvent this problem due to their small size of 21-23 oligos. [34] This allows delivery via nano-scale delivery vehicles called nanovectors. [33]

A good nanovector for siRNA delivery should protect siRNA from degradation, enrich siRNA in the target organ and facilitate the cellular uptake of siRNA. [32] The three main groups of siRNA nanovectors are: lipid based, non-lipid organic-based, and inorganic. [32] Lipid based nanovectors are excellent for delivering siRNA to solid tumors, [32] but other cancers may require different non-lipid based organic nanovectors such as cyclodextrin based nanoparticles. [32] [35]

siRNAs delivered via lipid based nanoparticles have been shown to have therapeutic potential for central nervous system (CNS) disorders. [36] Central nervous disorders are not uncommon, but the blood brain barrier (BBB) often blocks access of potential therapeutics to the brain. [36] siRNAs that target and silence efflux proteins on the BBB surface have been shown to create an increase in BBB permeability. [36] siRNA delivered via lipid based nanoparticles is able to cross the BBB completely. [36]

A huge difficulty in siRNA delivery is the problem of off-targeting. [30] [33] Since genes are read in both directions, there exists a possibility that even if the intended antisense siRNA strand is read and knocks out the target mRNA, the sense siRNA strand may target another protein involved in another function. [37]

Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 that siRNAs are well tolerated and have suitable pharmacokinetic properties. [38]

In a phase 1 clinical trial, 41 patients with advanced cancer metastasised to liver were administered RNAi delivered through lipid nanoparticles. The RNAi targeted two genes encoding key proteins in the growth of the cancer cells, vascular endothelial growth factor, (VEGF), and kinesin spindle protein (KSP). The results showed clinical benefits, with the cancer either stabilized after six months, or regression of metastasis in some of the patients. Pharmacodynamic analysis of biopsy samples from the patients revealed the presence of the RNAi constructs in the samples, proving that the molecules reached the intended target. [39] [40]

Proof of concept trials have indicated that Ebola-targeted siRNAs may be effective as post-exposure prophylaxis in humans, with 100% of non-human primates surviving a lethal dose of Zaire Ebolavirus, the most lethal strain. [41]

Delivering siRNA intracellularly continues to be a challenge. There are three main techniques of delivery for siRNA that differ on efficiency and toxicity.

Transfection Edit

In this technique siRNA first must be designed against the target gene. Once the siRNA is configured against the gene it has to be effectively delivered through a transfection protocol. Delivery is usually done by cationic liposomes, polymer nanoparticles, and lipid conjugation. [42] This method is advantageous because it can deliver siRNA to most types of cells, has high efficiency and reproducibility, and is offered commercially. The most common commercial reagents for transfection of siRNA are Lipofectamine and Neon Transfection. However it is not compatible with all cell types, and has low in vivo efficiency. [43] [44]

Electroporation Edit

Electrical pulses are also used to intracellularly deliver siRNA into cells. The cell membrane is made of phospholipids which makes it susceptible to an electric field. When quick but powerful electrical pulses are initiated the lipid molecules reorient themselves, while undergoing thermal phase transitions because of heating. This results in the making of hydrophilic pores and localized perturbations in the lipid bilayer cell membrane also causing a temporary loss of semipermeability. This allows for the escape of many intracellular contents, such as ions and metabolites as well as the simultaneous uptake of drugs, molecular probes, and nucleic acids. For cells that are difficult to transfect electroporation is advantageous however cell death is more probable under this technique. [45]

This method has been used to deliver siRNA targeting VEGF into the xenografted tumors in nude mice, which resulted in a significant suppression of tumor growth. [46]

Viral-mediated delivery Edit

The gene silencing effects of transfected designed siRNA are generally transient, but this difficulty can be overcome through an RNAi approach. Delivering this siRNA from DNA templates can be done through several recombinant viral vectors based on retrovirus, adeno-associated virus, adenovirus, and lentivirus. [47] The latter is the most efficient virus that stably delivers siRNA to target cells as it can transduce nondividing cells as well as directly target the nucleus. [48] These specific viral vectors have been synthesized to effectively facilitate siRNA that is not viable for transfection into cells. Another aspect is that in some cases synthetic viral vectors can integrate siRNA into the cell genome which allows for stable expression of siRNA and long-term gene knockdown. This technique is advantageous because it is in vivo and effective for difficult to transfect cell. However problems arise because it can trigger antiviral responses in some cell types leading to mutagenic and immunogenic effects.

This method has potential use in gene silencing of the central nervous system for the treatment of Huntington's disease. [49]

A decade after the discovery of RNAi mechanism in 1993, the pharmaceutical sector heavily invested in the research and development of siRNA therapy. There are several advantages that this therapy has over small molecules and antibodies. It can be administered quarterly or every six months. Another advantage is that, unlike small molecule and monoclonal antibodies that need to recognize specific conformation of a protein, siRNA functions by Watson-Crick basepairing with mRNA. Therefore, any target molecule that needs to be treated with high affinity and specificity can be selected if the right nucleotide sequence is available. [31] One of the biggest challenges researchers needed to overcome was the identification and establishment of a delivery system through which the therapies would enter the body. And that the immune system often mistakes the RNAi therapies as remnants of infectious agents, which can trigger an immune response. [3] Animal models did not accurately represent the degree of immune response that was seen in humans and despite the promise in the treatment investors divested away from RNAi. [3]

However, there were a few companies that continued with the development of RNAi therapy for humans. Alnylam Pharmaceuticals, Sirna Therapeutics and Dicerna Pharmaceuticals are few of the companies still working on bringing RNAi therapies to market. It was learned that almost all siRNA therapies administered in the bloodstream accumulated up in the liver. That is why most of the early drug targets were diseases that affected the liver. Repeated developmental work also shed light on improving the chemical composition of the RNA molecule to reduce the immune response, subsequently causing little to no side effects. [50] Listed below are some of approved therapies or therapies in pipeline.

Alnylam Pharmaceuticals Edit

In 2018, Alnylam pharmaceuticals became the first company to have a siRNA therapy approved by the FDA. Onpattro (patisiran) was approved for the treatment of polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults. hATTR is a rare, progressively debilitating condition. It affects 50,000 people worldwide. To deliver the drug directly to the liver, siRNA is encased in a lipid nanoparticle. The siRNA molecule halts the production of amyloid proteins by interfering with the RNA production of abnormal TTR proteins. This prevents the accumulation of these proteins in different organs of the body and helps the patients manage this disease. [51] [52]

Other treatment options for hATTR is an orthotopic liver transplant (OLT) that could potentially help if the disease is still in the early stage. However, OLT can only slow the progression of the disease and not treat it. There are also small molecule medications that provide temporary relief. Before Onpattro was released, the treatment options for hATTR were limited. After the approval of Onpattro, FDA awarded Alnylam with the Breakthrough Therapy Designation, which is given to drugs that are intended to treat a serious condition and are a substantial improvement over any available therapy. It was also awarded Orphan Drug Designations given to those treatments that are intended to safely treat conditions affecting less than 200,000 people. [53]

In 2019, FDA approved the second RNAi therapy, Givlaari (givosiran) used to treat acute hepatic porphyria (AHP). The disease is caused due to the accumulation of toxic porphobilinogen (PBG) molecules which are formed during the production of heme. These molecules accumulate in different organs and this can lead to the symptoms or attacks of AHP.

Givlaari is an siRNA drug that downregulates the expression of aminolevulinic acid synthase 1 (ALAS1), a liver enzyme involved in an early step in heme production. The downregulation of ALAS1 lowers the levels of neurotoxic intermediates that cause AHP symptoms. [31]

Years of research has led to a greater understanding of siRNA therapies beyond those affecting the liver. Alnylam Pharmaceuticals is currently involved in therapies that may treat amyloidosis and CNS disorders like Huntington’s disease and Alzheimer’s disease. [3] They have also recently partnered with Regeneron Pharmaceuticals to develop therapies for CNS, eye and liver diseases.

As of 2020, ONPATTRO and GIVLAARI, are available for commercial application, and two siRNAs, lumasiran (ALN-GO1) and inclisiran, have been submitted for new drug application to the FDA. Several siRNAs are undergoing phase 3 clinical studies, and more candidates are in the early developmental stage. [31] In 2020, Alnylam and Vir pharmaceuticals announced a partnership and have started working on a RNAi therapy that would treat severe cases of COVID-19. [54]

Other companies that have had success in developing a pipeline of siRNA therapies are Dicerna Pharmaceuticals, partnered with Eli Lily and Arrowhead Pharmaceuticals partnered with Johnson and Johnson. Several other big pharmaceutical companies such as Amgen and AstraZeneca have also invested heavily in siRNA therapies as they see the potential success of this area of biological drugs. [55]


Observing Direct Inheritance of Gene-Silencing RNA

The basics of genetic inheritance are well known: parents each pass half of their DNA to their offspring during reproduction. This genetic recipe is thought to contain all the information that a new organism needs to build and operate its body.

But recent research has shown that, in some species, parents’ life experiences can alter their offspring. Being underfed, exposed to toxins or stricken by disease can cause changes in a parent’s gene expression patterns, and in some cases, these changes can be passed down to the next generation. However, the mechanisms that cause this effect—known as non-genetic inheritance—are a mystery.

New research from the University of Maryland provides a surprising possible explanation. For the first time, developmental biologists have observed molecules of double-stranded RNA (dsRNA)—a close cousin of DNA that can silence genes within cells—being passed directly from parent to offspring in the roundworm Caenorhabditis elegans. Importantly, the gene silencing effect created by dsRNA molecules in parents also persisted in their offspring.

The work, published October 17, 2016 in the online early edition of the Proceedings of the National Academy of Sciences, suggests that the mechanisms for non-genetic inheritance might be simpler than anyone had suspected.

“This is the first time we’ve seen a dsRNA molecule passing from one generation to the next,” said Antony Jose, an assistant professor in the UMD Department of Cell Biology and Molecular Genetics and senior author on the study. “The assumption has been that dsRNA changes the parent’s genetic material and this altered genetic material is transmitted to the next generation. But our observations suggest that RNA is cutting out the middle man.”

Jose and his team, including graduate student and lead author Julia Marré and former research technician Edward Traver, introduced dsRNA marked with a fluorescent label into the circulatory system of C. elegans worms. They then watched as these fluorescent RNA molecules physically moved from the parent’s circulatory system into an egg cell waiting to be fertilized.

In a surprising turn of events, some of the dsRNA molecules could not silence genes in the parent because the dsRNA sequence did not match any of the parent’s genes. But the dsRNA molecules did silence genes in the offspring, when the new worm gained a copy of the matching gene from its other parent. This suggests that, in some cases, gene silencing by dsRNA might be able to skip an entire generation.

“It’s shocking that we can see dsRNA cross generational boundaries. Our results provide a concrete mechanism for how the environment in one generation could affect the next generation,” Jose said. “But it’s doubly surprising to see that a parent can transmit the information to silence a gene it doesn’t have.”

Jose and his colleagues did not expect dsRNA to play such a direct role in the transmission of information across generations. Because dsRNA factors into the life cycle of many viruses, Jose explained, it is reasonable to assume that a living cell’s natural defenses would prevent dsRNA from invading the next generation.

“It’s very surprising. One would think the next generation would be protected, but we are seeing all of these dsRNA molecules being dumped into the next generation,” Jose added. “Egg cells use the same mechanism to absorb nutrients as they prepare for fertilization. The next generation is not only getting nutrition it’s also getting information.”

Jose and his colleagues hope to learn more about the precise mechanisms by which dsRNA silences genes across multiple generations.

“There are hints that similar things could be happening in humans. We know that RNA exists in the human bloodstream. But, we don’t know where the RNA molecules are coming from, where they’re going or exactly what they’re doing,” Jose said. “Our work reveals an exciting possibility—they could be messages from parents to their offspring.”


In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line

The farming of baitfish, fish used by anglers to catch predatory species, is of economic and ecological importance in North America. Baitfish, including the fathead minnow (Pimephales promelas), are susceptible to infection from aquatic viruses, such as viral hemorrhagic septicemia virus (VHSV). VHSV infections can cause mass mortality events and have the potential to be spread to novel water bodies through baitfish as a vector. In this study, a novel skin cell line derived from fathead minnow (FHMskin) is described and its use as a tool to study innate antiviral immune responses and possible therapies is introduced. FHMskin grows optimally in 10% fetal bovine serum and at warmer temperatures, 25-30 °C. FHMskin is susceptible and permissive to VHSV-IVb infection, producing high viral titres of 7.35 × 10 7 TCID50/mL after only 2 days. FHMskin cells do not experience significant dsRNA-induced death after treatment with 50-500 ng/mL of in vitro transcribed dsRNA for 48 h and respond to dsRNA treatment by expressing high levels of three innate immune genes, viperin, ISG15, and Mx1. Pretreatment with dsRNA for 24 h significantly protected cells from VHSV-induced cell death, 500 ng/mL of dsRNA reduced cell death from 70% to less than 15% at a multiplicity of infection of 0.1. Thus, the novel cell line, FHMskin, represents a new method for producing high tires of VHSV-IVb in culture, and for studying dsRNA-induced innate antiviral responses, with future applications in dsRNA-based antiviral therapeutics.

Keywords: Antiviral immunity Fathead minnow Interferon-stimulated genes Viral hemorrhagic septicemia virus dsRNA.


Contents

RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression. [4] [5] [6] RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20–30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification. [7] [8] [9] In the context in which the phenomenon was first studied, small RNA was found to play an important role in defending plants against viruses. For example, these studies demonstrated that enzymes detect double-stranded RNA (dsRNA) not normally found in cells and digest it into small pieces that are not able to cause disease. [10] [11] [12] [13] [2]

While some functions of RNA silencing and its machinery are understood, many are not. For example, RNA silencing has been shown to be important in the regulation of development and in the control of transposition events. [14] RNA silencing has been shown to play a role in antiviral protection in plants as well as insects. [15] Also in yeast, RNA silencing has been shown to maintain heterochromatin structure. [16] However, the varied and nuanced role of RNA silencing in the regulation of gene expression remains an ongoing scientific inquiry. A range of diverse functions have been proposed for a growing number of characterized small RNA sequences—e.g., regulation of developmental, neuronal cell fate, cell death, proliferation, fat storage, haematopoietic cell fate, insulin secretion. [17]

RNA silencing functions by repressing translation or by cleaving messenger RNA (mRNA), depending on the amount of complementarity of base-pairing. RNA has been largely investigated within its role as an intermediary in the translation of genes into proteins. [18] More active regulatory functions, however, only began to be addressed by researchers beginning in the late-1990s. [19] The landmark study providing an understanding of the first identified mechanism was published in 1998 by Fire et al., [1] demonstrating that double-stranded RNA could act as a trigger for gene silencing. [19] Since then, various other classes of RNA silencing have been identified and characterized. [4] Presently, the therapeutic potential of these discoveries is being explored, for example, in the context of targeted gene therapy. [20] [21]

While RNA silencing is an evolving class of mechanisms, a common theme is the fundamental relationship between small RNAs and gene expression. [8] It has also been observed that the major RNA silencing pathways currently identified have mechanisms of action which may involve both post-transcriptional gene silencing (PTGS) [22] as well as chromatin-dependent gene silencing (CDGS) pathways. [4] CDGS involves the assembly of small RNA complexes on nascent transcripts and is regarded as encompassing mechanisms of action which implicate transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) events. [23] This is significant at least because the evidence suggests that small RNAs play a role in the modulation of chromatin structure and TGS. [24] [25]

Despite early focus in the literature on RNA interference (RNAi) as a core mechanism which occurs at the level of messenger RNA translation, others have since been identified in the broader family of conserved RNA silencing pathways acting at the DNA and chromatin level. [26] RNA silencing refers to the silencing activity of a range of small RNAs and is generally regarded as a broader category than RNAi. While the terms have sometimes been used interchangeably in the literature, RNAi is generally regarded as a branch of RNA silencing. To the extent it is useful to craft a distinction between these related concepts, RNA silencing may be thought of as referring to the broader scheme of small RNA related controls involved in gene expression and the protection of the genome against mobile repetitive DNA sequences, retroelements, and transposons to the extent that these can induce mutations. [27] The molecular mechanisms for RNA silencing were initially studied in plants [12] but have since broadened to cover a variety of subjects, from fungi to mammals, providing strong evidence that these pathways are highly conserved. [28]

At least three primary classes of small RNA have currently been identified, namely: small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA).

Small interfering RNA (siRNA) Edit

siRNAs act in the nucleus and the cytoplasm and are involved in RNAi as well as CDGS. [4] siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA (ssRNA) precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors. [29] The volume of literature on siRNA within the framework of RNAi is extensive.

MicroRNA (miRNA) Edit

The majority of miRNAs act in the cytoplasm and mediate mRNA degradation or translational arrest. [30] However, some plant miRNAs have been shown to act directly to promote DNA methylation. [31] miRNAs come from hairpin precursors generated by the RNaseIII enzymes Drosha and Dicer. [32] Both miRNA and siRNA form either the RNA-induced silencing complex (RISC) or the nuclear form of RISC known as RNA-induced transcriptional silencing complex (RITS). [33] The volume of literature on miRNA within the framework of RNAi is extensive.

Three prime untranslated regions and microRNAs Edit

Three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

As of 2014, the miRBase web site, [34] an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes). [35] Freidman et al. [35] estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs. [36] Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold). [37] [38]

The effects of miRNA dysregulation of gene expression seem to be important in cancer. [39] For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes. [40]

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders. [41] [42] [43]

Piwi-interacting RNA (piRNA) Edit

piRNAs represent the largest class of small non-coding RNA molecules expressed in animal cells, deriving from a large variety of sources, including repetitive DNA and transposons. [44] However, the biogenesis of piRNAs is also the least well understood. [45] piRNAs appear to act both at the post-transcriptional and chromatin levels. They are distinct from miRNA due to at least an increase in terms of size and complexity. Repeat associated small interfering RNA (rasiRNAs) are considered to be a subspecies of piRNA. [3]

The most basic mechanistic flow for RNA Silencing is as follows: (For a more detailed explanation of the mechanism, refer to the RNAi:Cellular mechanism article.)

1: RNA with inverted repeats hairpin/panhandle constructs --> 2: dsRNA --> 3: miRNAs/siRNAs --> 4: RISC --> 5: Destruction of target mRNA

  1. It has been discovered that the best precursor to good RNA silencing is to have single stranded antisense RNA with inverted repeats which, in turn, build small hairpin RNA and panhandle constructs. [6] The hairpin or panhandle constructs exist so that the RNA can remain independent and not anneal with other RNA strands.
  2. These small hairpin RNAs and/or panhandles then get transported from the nucleus to the cytosol through the nuclear export receptor called exportin-5, and then get transformed into a dsRNA, a double stranded RNA, which, like DNA, is a double stranded series of nucleotides. If the mechanism didn't use dsRNAs, but only single strands, there would be a higher chance for it to hybridize to other "good" mRNAs. As a double strand, it can be kept on call for when it is needed.
  3. The dsRNA then gets cut up by a Dicer into small (21-28 nt = nucleotides long) strands of miRNAs (microRNAs) or siRNAs (short interfering RNAs.) A Dicer is an endoribonucleaseRNase, which is a complex of a protein mixed with strand(s) of RNA.
  4. Lastly, the double stranded miRNAs/siRNAs separate into single strands the antisense RNA strand of the two will combine with another endoribonuclease enzyme complex called RISC (RNA-induced silencing complex), which includes the catalytic component Argonaute, and will guide the RISC to break up the "perfectly complementary" target mRNA or viral genomic RNA so that it can be destroyed. [2][6]
  5. It means that based on a short sequence specific area, a corresponding mRNA will be cut. To make sure, it will be cut in many other places as well. (If the mechanism only worked with a long stretch, then there would be higher chance that it would not have time to match to its complementary long mRNA.) It has also been shown that the repeated-associated short interference RNAs (rasiRNA) have a role in guiding chromatin modification. [2]

For an animated explanation of the mechanism of RNAi by Nature Reviews, see the External Links section below.

Immunity against viruses or transposons Edit

RNA silencing is the mechanism that our cells (and cells from all kingdoms) use to fight RNA viruses and transposons (which originate from our own cells as well as from other vehicles). [2] In the case of RNA viruses, these get destroyed immediately by the mechanism cited above. In the case of transposons, it's a little more indirect. Since transposons are located in different parts of the genome, the different transcriptions from the different promoters produce complementary mRNAs that can hybridize with each other. When this happens, the RNAi machinery goes into action, debilitating the mRNAs of the proteins that would be required to move the transposons themselves. [46]

Down-regulation of genes Edit

For a detailed explanation of the down-regulation of genes, see RNAi:downregulation of genes

Up-regulation of genes Edit

For a detailed explanation of the up-regulation of genes, see RNAi:upregulation of genes

RNA silencing also gets regulated Edit

The same way that RNA silencing regulates downstream target mRNAs, RNA silencing itself is regulated. For example, silencing signals get spread between cells by a group of enzymes called RdRPs (RNA-dependent RNA polymerases) or RDRs. [2]

Growing understanding of small RNA gene-silencing mechanisms involving dsRNA-mediated sequence-specific mRNA degradation has directly impacted the fields of functional genomics, biomedicine, and experimental biology. The following section describes various applications involving the effects of RNA silencing. These include uses in biotechnology, therapeutics, and laboratory research. Bioinformatics techniques are also being applied to identify and characterize large numbers of small RNAs and their targets.

Biotechnology Edit

Artificial introduction of long dsRNAs or siRNAs has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms. [2] Structural and functional resolution of small RNAs as the effectors of RNA silencing has had a direct impact on experimental biology. For example, dsRNA may be synthesized to have a specific sequence complementary to a gene of interest. Once introduced into a cell or biological system, it is recognized as exogenous genetic material and activates the corresponding RNA silencing pathway. This mechanism can be used to effect decreases in gene expression with respect to the target, useful for investigating loss of function for genes relative to a phenotype. That is, studying the phenotypic and/or physiologic effects of expression decreases can reveal the role of a gene product. The observable effects can be nuanced, such that some methods can distinguish between “knockdown” (decrease expression) and “knockout” (eliminate expression) of a gene. [47] RNA interference technologies have been noted recently as one of the most widely utilized techniques in functional genomics. [48] Screens developed using small RNAs have been used to identify genes involved in fundamental processes such as cell division, apoptosis and fat regulation.

Biomedicine Edit

Since at least the mid-2000s, there has been intensifying interest in developing short interfering RNAs for biomedical and therapeutic applications. [49] Bolstering this interest is a growing number of experiments which have successfully demonstrated the clinical potential and safety of small RNAs for combatting diseases ranging from viral infections to cancer as well as neurodegenerative disorders. [50] In 2004, the first Investigational New Drug applications for siRNA were filed in the United States with the Food and Drug Administration it was intended as a therapy for age-related macular degeneration. [48] RNA silencing in vitro and in vivo has been accomplished by creating triggers (nucleic acids that induce RNAi) either via expression in viruses or synthesis of oligonucleotides. [51] Optimistically many studies indicate that small RNA-based therapies may offer novel and potent weapons against pathogens and diseases where small molecule/pharmacologic and vaccine/biologic treatments have failed or proved less effective in the past. [49] However, it is also warned that the design and delivery of small RNA effector molecules should be carefully considered in order to ensure safety and efficacy.

The role of RNA silencing in therapeutics, clinical medicine, and diagnostics is a fast developing area and it is expected that in the next few years some of the compounds using this technology will reach market approval. A report has been summarized below to highlight the many clinical domains in which RNA silencing is playing an increasingly important role, chief among them are ocular and retinal disorders, cancer, kidney disorders, LDL lowering, and antiviral. [51] The following table displays a listing of RNAi based therapy currently in various phases of clinical trials. The status of these trials can be monitored on the ClinicalTrials.gov website, a service of the National Institutes of Health (NIH). [52] Of note are treatments in development for ocular and retinal disorders, that were among the first compounds to reach clinical development. AGN211745 (sirna027) (Allergan) and bevasiranib (Cand5) (Opko) underwent clinical development for the treatment of age-related macular degeneration, but trials were terminated before the compounds reached the market. Other compounds in development for ocular conditions include SYL040012 (Sylentis) and QPI-007 (Quark). SYL040012 (bamosinan) is a drug candidate under clinical development for glaucoma, a progressive optic neurdegeneration frequently associated to increased intraocular pressure QPI-007 is a candidate for the treatment of angle-closure glaucoma and Non-arteritic anterior ischaemic optic neuropathy both compounds are currently undergoing phase II clinical trials. Several compounds are also under development for conditions such as cancer and rare diseases.

Clinical domain Drug Indication Target
Ocular and retinal disorders TD101 Pachyonychia congenita Keratin 6A N171K mutant
Ocular and retinal disorders QPI-1007 Non-arteritic anterior ischaemic optic neuropathy Caspase 2
Ocular and retinal disorders AGN211745 Age-related macular degeneration, choroidal neovascularization VEGFR1
Ocular and retinal disorders PF-655 Diabetic macular oedema, age-related macular degeneration RTP801
Ocular and retinal disorders SYL040012 Glaucoma β2 adrenergic receptor
Ocular and retinal disorders Bevasiranib Diabetic macular oedema VEGF
Ocular and retinal disorders Bevasiranib Macular degeneration VEGF
Cancer CEQ508 Familial adenomatous polyposis β-catenin
Cancer ALN-PLK1 Liver tumor PLK1
Cancer FANG Solid tumor Furin
Cancer CALAA-01 Solid tumor RRM2
Cancer SPC2996 chronic lymphocytic leukemia BCL-2
Cancer ALN-VSP02 Solid tumor VEGF, kinesin spindle protein
Cancer NCT00672542 Metastatic melanoma LMP2, LMP7, and MECL1
Cancer Atu027 Solid malignancies PKN3
Kidney disorders QPI-1002/I5NP Acute kidney injury p53
Kidney disorders QPI-1002/I5NP Graft dysfunction kidney transplant p53
Kidney disorders QPI-1002/I5NP Kidney injury acute renal failure p53
LDL lowering TKM-ApoB Hypercholesterolaemia APOB
LDL lowering PRO-040,201 Hypercholesterolaemia APOB
Antiviral miravirsen Hepatitis C virus miR-122
Antiviral pHIV7-shI-TAR-CCR5RZ HIV HIV Tat protein, HIV TAR RNA, human CCR5
Antiviral ALN-RSV01 RSV RSV nucleocapsid
Antiviral ALN-RSV01 RSV in lung transplant patients RSV nucleocapsid

Main challenge Edit

As with conventional manufactured drugs, the main challenge in developing successful offshoots of the RNAi-based drugs is the precise delivery of the RNAi triggers to where they are needed in the body. The reason that the ocular macular degeneration antidote was successful sooner than the antidote with other diseases is that the eyeball is almost a closed system, and the serum can be injected with a needle exactly where it needs to be. The future successful drugs will be the ones who are able to land where needed, probably with the help of nanobots. Below is a rendition of a table [51] that shows the existing means of delivery of the RNAi triggers.

Laboratory Edit

The scientific community has been quick to harness RNA silencing as a research tool. The strategic targeting of mRNA can provide a large amount of information about gene function and its ability to be turned on and off. Induced RNA silencing can serve as a controlled method for suppressing gene expression. Since the machinery is conserved across most eukaryotes, these experiments scale well to a range of model organisms. [53] In practice, expressing synthetic short hairpin RNAs can be used to reach stable knock-down. [54] If promoters can be made to express these designer short hairpin RNAs, the result is often potent, stable, and controlled gene knock-down in both in vitro and in vivo contexts. [55] Short hairpin RNA vector systems can be seen as roughly analogous in scope to using cDNA overexpression systems. [56] Overall, synthetic and natural small RNAs have proven to be an important tool for studying gene function in cells as well as animals. [57]

Bioinformatics approaches to identify small RNAs and their targets have returned several hundred, if not thousands of, small RNA candidates predicted to affect gene expression in plants, C. elegans, D. melanogaster, zebrafish, mouse, rat, and human. [58] These methods are largely directed to identifying small RNA candidates for knock-out experiments but may have broader applications. One bioinformatics approach evaluated sequence conservation criteria by filtering seed complementary target-binding sites. The cited study predicted that approximately one third of mammalian genes were to be regulated by, in this case, miRNAs. [59]

Ethics & Risk-Benefit Analysis Edit

One aspect of RNA silencing to consider is its possible off-target affects, toxicity, and delivery methods. If RNA silencing is to become a conventional drug, it must first pass the typical ethical issues of biomedicine. [60] Using risk-benefit analysis, researchers can determine whether RNA silencing conforms to ethical ideologies such as nonmaleficence, beneficence, and autonomy. [61]

There is a risk of creating infection-competent viruses that could infect non-consenting people. [62] There is also a risk of affecting future generations based on these treatments. These two scenarios, in respect to autonomy, is possible unethical. At this moment, unsafe delivery methods and unintended aspects of vector viruses add to the argument against RNA silencing. [61]

In terms of off-target effects, siRNA can induce innate interferon responses, inhibit endogenous miRNAs through saturation, and may have complementary sequences to other non-target mRNAs. These off-targets could also have target up-regulations such as oncogenes and antiapoptotic genes. The toxicity of RNA silencing is still under review as there are conflicting reports. [61] [62] [63]

RNA silencing is quickly developing, because of that, the ethical issues need to be discussed further. With the knowledge of general ethical principles, we must continuously perform risk-benefit analysis. [61]


UMD Biologists First to Observe Direct Inheritance of Gene-Silencing RNA

The basics of genetic inheritance are well known: parents each pass half of their DNA to their offspring during reproduction. This genetic recipe is thought to contain all of the information that a new organism needs to build and operate its body.

But recent research has shown that, in some species, parents’ life experiences can alter their offspring. Being underfed, exposed to toxins or stricken by disease can cause changes in a parent’s gene expression patterns, and in some cases, these changes can be passed down to the next generation. However, the mechanisms that cause this effect—known as non-genetic inheritance—are a mystery.

/>New research from the University of Maryland provides a surprising possible explanation. For the first time, developmental biologists have observed molecules of double-stranded RNA (dsRNA)—a close cousin of DNA that can silence genes within cells—being passed directly from parent to offspring in the roundworm Caenorhabditis elegans. Importantly, the gene silencing effect created by dsRNA molecules in parents also persisted in their offspring.

The work, published October 17, 2016 in the online early edition of the Proceedings of the National Academy of Sciences, suggests that the mechanisms for non-genetic inheritance might be simpler than anyone had suspected.

“This is the first time we’ve seen a dsRNA molecule passing from one generation to the next,” said Antony Jose, an assistant professor in the UMD Department of Cell Biology and Molecular Genetics and senior author on the study. “The assumption has been that dsRNA changes the parent’s genetic material and this altered genetic material is transmitted to the next generation. But our observations suggest that RNA is cutting out the middle man.”

Jose and his team, including graduate student and lead author Julia Marré and former research technician Edward Traver, introduced dsRNA marked with a fluorescent label into the circulatory system of C. elegans worms. They then watched as these fluorescent RNA molecules physically moved from the parent’s circulatory system into an egg cell waiting to be fertilized.

In a surprising turn of events, some of the dsRNA molecules could not silence genes in the parent because the dsRNA sequence did not match any of the parent’s genes. But the dsRNA molecules did silence genes in the offspring, when the new worm gained a copy of the matching gene from its other parent. This suggests that, in some cases, gene silencing by dsRNA might be able to skip an entire generation.

“It’s shocking that we can see dsRNA cross generational boundaries. Our results provide a concrete mechanism for how the environment in one generation could affect the next generation,” Jose said. “But it’s doubly surprising to see that a parent can transmit the information to silence a gene it doesn’t have.”

Jose and his colleagues did not expect dsRNA to play such a direct role in the transmission of information across generations. Because dsRNA factors into the life cycle of many viruses, Jose explained, it is reasonable to assume that a living cell’s natural defenses would prevent dsRNA from invading the next generation.

“It’s very surprising. One would think the next generation would be protected, but we are seeing all of these dsRNA molecules being dumped into the next generation,” Jose added. “Egg cells use the same mechanism to absorb nutrients as they prepare for fertilization. The next generation is not only getting nutrition, it’s also getting information.”

Jose and his colleagues hope to learn more about the precise mechanisms by which dsRNA silences genes across multiple generations.

“There are hints that similar things could be happening in humans. We know that RNA exists in the human bloodstream. But, we don’t know where the RNA molecules are coming from, where they’re going or exactly what they’re doing,” Jose said. “Our work reveals an exciting possibility—they could be messages from parents to their offspring.”

In addition to Jose, UMD co-authors on the paper included graduate student Julia Marré and former research technician Edward Traver.

The research paper, “Extracellular RNA is transported from one generation to the next in Caenorhabditis elegans ,” Julia Marré, Edward Traver and Antony Jose, appears in the October 17, 2016 online early edition of the Proceedings of the National Academy of Sciences .

This work was supported by the National Institutes of Health (Award No. R01GM111457). The content of this article does not necessarily reflect the views of this organization.

Media Relations Contact: Matthew Wright, 301-405-9267, [email protected]

University of Maryland
College of Computer, Mathematical, and Natural Sciences
2300 Symons Hall
College Park, MD 20742
www.cmns.umd.edu
@UMDscience

About the College of Computer, Mathematical, and Natural Sciences
The College of Computer, Mathematical, and Natural Sciences at the University of Maryland educates more than 7,000 future scientific leaders in its undergraduate and graduate programs each year. The college's 10 departments and more than a dozen interdisciplinary research centers foster scientific discovery with annual sponsored research funding exceeding $150 million.


Summary

In addition to protein transcription factors, eukaryotes use small RNA molecules to regulate gene expression &mdash almost always by repressing it &mdash so the phenomenon is called RNA silencing.

There are two sources of small RNA molecules:

  • small interfering RNAs (siRNAs)
    • Plant cells make these from the double-stranded RNA (dsRNA) of invading viruses.
    • Scientists and pharmaceutical companies make these as agents to turn off the expression of specific genes (called RNA interference or RNAi).
    • These are encoded in the genomes of all plants and animals.
    • Both are generated by Dicer.
    • Both are incorporated into an RNA-induced silencing complex (RISC).
      • If the nucleotide sequence of the small RNA exactly matches that of the mRNA, the mRNA is cut and destroyed.
      • If there is only a partial match (usually in its 3' UTR), translation (i.e., protein synthesis) is repressed. Both of these activities take place in the cytosol &mdash perhaps in P bodies.
      • However, for some small RNAs, the RISC complex enters the nucleus and turns off transcription of the corresponding gene(s) by
        • binding to the unwound DNA sequence (or perhaps the RNA transcript as it is being formed)
        • converting euchromatin to heterochromatin [Link]
        • methylating of lysine-9 histone H3 in the nucleosomes around the gene(s) [Link].

        Aside from their use as laboratory &mdash and perhaps therapeutic &mdash tools, small RNAs are clearly essential to the organisms that make them.


        Other TLR Pathway Defects

        Rebeca Pérez de Diego , Carlos Rodríguez-Gallego , in Stiehm's Immune Deficiencies , 2014

        TLR3 Deficiency

        TLR3 is a type I integral membrane protein which recognizes dsRNA, an intermediate generated during most viral infections. Other dsRNA of non-viral origin, such as cell-endogenous RNA, probably released by necrotic cells 113,114 or ultraviolet-damaged small nuclear U1 RNA (snU1), 115 can also activate TLR3. By contrast, total human cellular RNA cannot activate TLR3. 116 The principal cells expressing TLR3 are peripheral leukocytes, including dendritic cells, CD8+ T cells, and NK cells. Many other cell types, including neurons, oligodendrocytes, astrocytes, and microglia cells also express TLR3. TLR3 is intracellular in most cell types, with the only cells which have been demonstrated to apparently express TLR3 on their cell surface being human lung fibroblasts. 11

        In humans, TLR3 deficiency has been described in three patients with HSE. 20,24 The first report of TLR3 deficiency included two patients who suffered from HSE. These two patients were unrelated and were born to French non-consanguineous parents. 20 The first patient was a girl who suffered from HSE at the age of 5 years. The disease debuted with right-side facial clonic seizures, followed by right brachiofacial paralysis and secondary fever (38.3°C). The patient was treated with acyclovir (60 mg/kg per day IV for 3 weeks) with good recovery. A year and 7 months later, he developed a new episode of HSE. Acyclovir treatment was resumed (60 mg/kg per day IV for 3 weeks followed by oral acyclovir). Clinical symptoms regressed and the child recovered. The second patient was diagnosed with HSE when she was 5 months old. This episode started with high fever (39.5°C), and right hemiclonic seizure. Clinical status improved under intravenous acyclovir therapy (60 mg/kg per day IV for 3 weeks). The third patient was a boy born to non-consanguineous Polish parents. He suffered HSE at the age of 8 years. Clinical signs were well controlled by acyclovir treatment (60 mg/kg per day IV for 3 weeks). The patient presented herpes labialis before and after the episode of HSE. The patient has suffered from major neurological sequelae since the episode of HSE. 24

        Human TLR3 (4q35) has five exons, of which exon 1 and parts of exon 2 and exon 5 are non-coding. TLR3 encodes a protein of 904 amino acids composed of a leader sequence, an LRR domain, a transmembrane domain, and a TIR domain. UNC-93B deficiency was first excluded in the three patients. Two patients were unrelated and they have the same heterozygous substitution in TLR3 at nucleotide position 1660 (c.1660C>T), as a result of independent mutational events. The mutation leads to the replacement of a proline by a serine at residue 554 (P554S), which is located in the LRR domain, a region critical for dsRNA binding to TLR3. The P554S allele encodes a truncated protein, and was shown to be loss of function and to have a dominant negative effect conferring autosomal dominant (AD) TLR3 deficiency. 20,24 The third patient was compound heterozygous for P554S and a substitution c.2236G>T. The mutation c.2236G>T introduces a termination codon at residue 746 (E746X). The E746X protein lacks the TIR domain, and, due to abnormal glycosylation, mutant proteins of two different molecular masses can be detected. 24 E746X was found to be a loss-of-function allele. The patient is thus compound heterozygous for two loss-of-function TLR3 alleles and he has an AR form of complete TLR3 deficiency. Fibroblasts from the AR patient’s mother, who is heterozygous for the E746X mutation, respond normally to poly (I:C), suggesting that E746X is not dominant. 24 Six relatives from the reported patients were heterozygous for the mutation P554S. They were HSV-1 seropositive but had not suffered from HSE, which suggest that the P554S TLR3 mutation confers an AD predisposition to HSE with incomplete clinical penetrance. 20,24

        A fourth TLR3-deficient patient, heterozygous for the TLR3 P554S mutation, was identified in a survey aimed to identify TLR3 variants in 57 patients from Germany with biopsy-proven enteroviral myocarditis. The patient had suffered from coxsackievirus B3 (CVB3) myocarditis at the age of 54 years. 22

        AD and AR TLR3-deficient PBMCs displayed normal production of IFN-α, IFN-β, and/or IFN-λ in response to 11 viruses tested, including HSV-1, and to poly (I:C). 20,24 A normal production of antiviral IFNs or of IFN-inducible genes was also observed in different subsets of monocytes and monocyte-derived macrophages (MDMs) from the AR TLR3-deficient patient stimulated with poly (I:C) or HSV-1. 24 The response to poly (I:C) was also normal in pDCs and myeloid DCs (mDC) from AD TLR3-deficient patients. 20 These data suggest that these leukocytes do not require an intact TLR3 pathway for antiviral IFN induction in response to HSV-1. In addition, it may explain the lack of disseminated disease during the course of HSE and the absence of other viral illnesses in patients with TLR3 deficiency. 20,24 Other cell types, such as monocyte-derived dendritic cells (MDDCs), NK cells, and CD8 α/β T cells, have impaired response to poly (I:C) stimulation in terms of antiviral IFN production. 20 Although affected by the TLR3 mutation, the contribution of NK and CD8 T cells to the pathogenesis of HSE in TLR3-heterozygous children is probably modest, as suggested by the fact that patients with NK and T cell deficiencies, as well as CD8- and HLA-I-deficient patients, are not particularly prone to HSE. 103,117 The production of IFN-β and -λ and of IL-6 in response to stimulation with poly (I:C), HSV-1, and VSV is impaired in AR and AD TLR3-deficient fibroblasts, although a residual response to high concentrations of poly (I:C) is observed in fibroblasts from AD TLR3-deficient patients. Impaired TLR3 signaling also leads to high levels of viral replication and cell death in fibroblasts from the patients following infection with HSV-1 or VSV. Similar results were observed when fibroblasts from the AR TLR3-deficient patient were stimulated with poly (A:U), which apparently stimulates TLR3 more specifically than poly (I:C). 24 A HEK293 cell line (derived from human embryonic kidney cells) transfected with wild-type or P554S TLR3 showed that the mutation also results in a significant increase in CVB3 replication. 22 Overall, the activation of TLR3-independent dsRNA-responsive pathways may contribute to control of viruses other than HSV-1, and possibly CVb3, in patients with deficiencies involving the TLR3 pathway. 109,110,118–120


        Recommended checks and controls for siRNA experiments

        ​An increasing number of labs are using the siRNA knockdown technique as part of the process to assess the function of a protein within cells. The technique is usually used to determine the effect of removing the protein from the cells:

        • Does the cell die? Is it a lethal knockout?
        • Does it affect the expression level of other proteins (particularly within signaling pathways)?
        • Does it affect the location of other proteins within the cell?
        • How does it affect cell phenotype, morphology, function?

        Brief introduction to siRNA

        Small interfering RNA (siRNAs) are 20-25 nucleotide long double-stranded RNA molecules that have a variety of roles in the cell.They are involved in the RNA interference (RNAi) pathway, where they interfere with gene expression by hybridizing to complementary mRNA molecules. This triggers mRNA degradation and suppression of gene expression for a particular gene.

        siRNAs were first discovered by David Baulcombe's lab in 1999. In 2001, synthetic siRNAs were shown to induce RNA interference (RNAi) in mammalian cells by Thomas Tuschl in the following paper:

        Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494–498. PubMed 11373684.

        ‘Synthetic’ siRNA oligos designed to hybridize to and degrade target sequences of RNA are now commonly used to induce RNAi in cells. The degradation of the targeted mRNA effectively ‘knocks out’ expression of the corresponding protein. The effects of reduced levels of the target protein can then be analyzed.

        How does it work?

        1. Double stranded RNA (dsRNA) is introduced into the cell either using a short oligo siRNA or a DNA plasmid from which a siRNA can be transcribed.
        2. The Dicer protein in the cell digests dsRNA into 21 bp dsRNA (siRNA).
        3. siRNAs are integrated into the RNA Induced Silencing Complex (RISC).
        4. Within this RISC complex, the dsRNAs undergo strand separation. The antisense strand hybridizes to the complementary / target mRNA in the cell.
        5. Nucleases within the activated RISC degrade targeted mRNA.
        6. The fragmented mRNA cannot be translated into protein. This means the protein cannot be expressed, resulting in knockout of the protein.

        Optimization of the siRNA sequence for optimal knockout

        Many online programs are available to help provide the most suitable siRNA sequence from the mRNA sequence of the protein you wish to knock down.
        These computer programs score 21 bp sequences through the full length mRNA of the protein based on the following:

        1. Sequence located within 50-100 nucleotides of the AUG start codon or within 50-100 nucleotides of the termination codon (to ensure transcribed gene is silenced).
        2. siRNA sequence begins with AA (allows use of dTdT at 3’-end of the antisense sequence).This reduces the cost of synthesis and renders the siRNA duplex more resistant to exonuclease activity.
        3. GC content: Ideally the GC content is < 50% (most software defaults range between 40 to 50%)
        4. Stretches of nucleotide repeats.
          Avoids sequences with repeats of three or more G’s or C’s
          (Initiates intra-molecular secondary structures preventing effective hybridization)
        5. Blast Search (to prevent ‘off targeting’).

        Once a target sequence has been chosen, a BLAST search is initiated to ensure that your target sequence is not homologous to other gene sequences.
        As a general rule, choose and try three siRNA sequences with the highest score that the program provides, this should give you a high chance that one would work.
        Note – this will often be expected by editors for journal publications
        Use combinations – start with three SiRNAs and scale down to one
        It is possible to knockdown more than two genes at once, but optimize separately first.

        Recommended controls and checks for siRNA experiments

        The following section provides information on the control samples we would recommend to include in your siRNA experiments. It also includes information on checks that should be carried out when designing and performing the experiment.

        Cell line – include one cell line known to have high transfection efficiency.
        E.g. 292, HeLa, MRC5, U2OS (We advise not to use primary cells – they do not transfect easily).

        An endogenous positive control sample with no siRNA.
        As a positive control for the protein of interest and a negative control for siRNA knockout. All reagents other than the siRNA should be added, this checks any effect from the transfection reagents.

        Use a dose response curve to optimize the amount of siRNA oligo or plasmid.
        To work out an optimal siRNA concentration. There should be enough siRNA to create knockout but at a concentration that does not over-activate the RISC complex or result in toxic effects from other reagents.

        Tagged siRNA - observe for example GFP tag fluorescence to confirm transfection.
        A small percentage of the siRNA added to the cells can be fluorescently tagged (e.g. GFP) to confirm transfection. Only a small percentage of the total siRNA should be tagged, the rest must be untagged because the tag will prevent RNA binding.

        Toxicity controls to check viability of cells.
        Calculate and monitor transfection toxicity as some of the reagents can be toxic. This can be done for example by checking cell viability using various cell stains to detect dead cells, e.g. trypan blue.

        Induced / non induced.
        If the siRNA is being expressed from a plasmid with an inducible promoter, both induced and non induced transfected samples should be tested.

        siRNA negative control (using siRNA with a nonsense / scrambled sequence).
        siRNA intersects with a number of other pathways, so nonspecific effects can be triggered.
        MicroRNAs modulate gene expression largely via incomplete hybridization with a target mRNA and the introduction of an siRNA may cause unintended off-targeting.

        Check the sequence of the siRNA (computer programs) – BLAST search.
        Blast search the siRNA sequences to ensure the siRNA will hybridize only to the mRNA related to the protein you are interested in.

        ‘Mock’ control.
        Use another protein siRNA e.g. GAPDH (with no target protein siRNA) to check activation of RISC signaling pathway and also that it is not affecting overall cell function.

        Check the time for degradation of the mRNA and the existing protein.
        The larger the protein, the longer the half life of both the protein and its associated mRNA. You may need to optimize the time required for the siRNA knockdown to take effect on the cells.

        Rescue experiment control.
        Transfect cells with recombinant protein to re-introduce the protein. Often requested by journals for publication.

        Assessing the results – application control samples (e.g. positive and negative controls).
        Ensure you include all the required controls for the application you use to assess the results. This will include endogenous positive and negative controls.

        Assessing the results:

        RT-PCR
        To check for presence of the mRNA. Is the targeted mRNA still present? Or has the knockdown been successful?
        Very sensitive but doesn’t give an accurate prediction of expected protein levels.

        Western blot
        Indicates the presence or absence of protein. Can also use antibodies to detect several proteins in the sample and therefore observe the effect the knockout of the target protein has on other proteins.

        Immunocytochemistry
        Indicates the presence or absence of the knockdown protein.
        Advantage – dual staining so can also check effect on expression and cellular location of other proteins.

        Troubleshooting summary

        1. How have you selected the sequence? Have you tried more than one siRNA sequence? Check whether the sequence corresponds correctly to the protein.
        2. Have you checked the transfection efficiency and optimized the length of time for knockout to take effect?
        3. Have you checked the transfection by fluorescence tags and the knockout by RT-PCR?
        4. Have you used the correct endogenous positive and negative controls? Have you used scrambled siRNA or standard negative siRNA controls?

        Resources

        • Free online siRNA design and flash tutorial. provides access to biomedical and genomic information, from where mRNA or cDNA sequence for target selection can be obtained.
        • Blat tool on UCSC Genome Website.


        Watch the video: Diseño dsRNA (May 2022).


Comments:

  1. Taro

    In my opinion you are mistaken. I suggest it to discuss. Write to me in PM.

  2. Arashiran

    I mean you are wrong. I can prove it.

  3. Marrok

    You are not right. I'm sure. We will discuss. Write in PM, we will talk.

  4. Seorus

    Judging by the reviews - you need to download.

  5. Gurr

    You hit the mark. It seems to me an excellent thought. I agree with you.

  6. Dit

    Congratulate me my son was born!

  7. Zeleny

    Exactly! I like this idea, I completely agree with you.



Write a message