Small interfering RNA

thumb|right|Mediating RNA interference in cultured mammalian cells.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading messenger RNA (mRNA) after transcription, preventing translation. It was discovered in 1998 by Andrew Fire at the Carnegie Institution for Science in Washington, D.C. and Craig Mello at the University of Massachusetts in Worcester.

Structure

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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.

As of 2001 it had been suggested taht anenzyme referred to as Dicer catalyzed "the initiation step of RNA interference" (production of siRNAs from long dsRNAs and small hairpin RNAs).

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".

History

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. The two won the Nobel prize for their research with RNAi in 2006. siRNA and its role in post-transcriptional gene silencing (PTGS) was discovered in plants by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, a discovery reported in Science in 1999. Thomas Tuschl and colleagues soon reported in Nature that synthetic siRNAs could induce RNAi in mammalian cells.  These discoveries led to a surge in interest in harnessing RNAi for biomedical research and drug development.

As of 2017, human applications of siRNA had faced significant roadblocks to their success, one of these being "off-targeting".

Mechanism

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

thumb|300x300px|siRNA Mechanism

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).
Once siRNA enters the cell it gets incorporated into other proteins to form the RISC.
Once the siRNA is part of the RISC complex, the siRNA is unwound to form single stranded siRNA.
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
The single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA
Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage.
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. miRNAs typically silence genes by repression of translation and have broader specificity of action, while siRNAs typically work with higher specificity by cleaving the mRNA before translation, with 100% complementarity.

RNAi induction using siRNAs or their biosynthetic precursors

thumb|right|Dicer protein colored by [[protein domain.]]

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. Typical transcription cassettes use an RNA polymerase III promoter (e.g., U6 or H1) to direct the transcription of small nuclear RNAs (snRNAs, where U6 is involved in RNA 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.

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.

RNA activation

In addition to their role in RNAi, siRNAs can also activate gene expression, a phenomenon termed "RNA activation" or RNAa. This was first observed when synthetic siRNAs, termed "small activating RNA" (saRNA), targeting gene promoters were found to induce potent transcriptional activation of target genes. RNAa has been demonstrated to be a conserved mechanism, observed across species from insects, C. elegans, and plants, to mammals (including humans).

The mechanism of RNAa involves the targeting of promoter regions by saRNAs, leading to the recruitment of transcriptional machinery and epigenetic changes that promote gene expression. This process often involves the RNA-induced transcriptional activation (RITA) complex, which includes Argonaute proteins (particularly Ago2), RNA helicase A (RHA), and CTR9. Endogenous miRNAs can also mediate RNAa, expanding the regulatory roles of these small RNAs beyond gene silencing.

Several saRNA-based therapeutics are currently in clinical development. MTL-CEBPA, developed by MiNA Therapeutics, targets the CEBPA gene and is in Phase II trials for liver cancer. RAG-01, developed by Ractigen Therapeutics, targets the p21 gene and is in Phase I trials for non-muscle invasive bladder cancer (NMIBC). These clinical trials represent a significant step towards translating the RNAa phenomenon into novel therapeutic strategies.

Post-transcriptional gene silencing

As Richard Carthew and coworkers state in relation to that group's 2004 study of siRNA/miRNA silencing pathways in Drosophila, there, siRNA-induced post transcriptional gene silencing is initiated by the assembly of the RNA-induced silencing complex (RISC), which silences expression of certain genes by cleaving the mRNA molecules coding those genes. Then, in a perspective from 2009, the view is offered that "siRNA scans for and directs RISC" to perfectly complementary sequence on the mRNA molecules.

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. 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.

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. In particular, as of 2019 they were characterised as not being siRNAs and as being responsible for the silencing of transposons.

Transcriptional Gene Silencing

Many model organism, such as plants (Arabidopsis thaliana), yeast (Saccharomyces cerevisiae), flies (Drosophila melanogaster) and worms (C. elegans), have been used to study small non coding RNA-directed Transcriptional gene silencing. In human cell, RNA-directed transcriptional gene silencing was observed a decade ago when exogenous siRNAs silenced a transgenic elongation factor 1 α promoter driving a Green Fluorescent Protein (GFP) reporter gene. The main mechanisms of transcriptional gene silencing (TGS) involving the RNAi machinery include DNA methylation, histone post-translational modifications, and subsequent chromatin remodeling around the target gene into a heterochromatic state. 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.

Innate immunity

Introduction of too many siRNA can result in nonspecific events due to activation of innate immune responses. 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. 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. 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, to minimize nonspecific effects.

Off-targeting

Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool. The tool of siRNA off-target predition is available at http://crdd.osdd.net/servers/aspsirna/asptar.php and published as ASPsiRNA resource.

Saturation of the RNAi machinery

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. 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.

Chemical modification

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siRNAs have been chemically modified to enhance their therapeutic properties, Short interfering RNA (siRNA) must be delivered to the site of action in the cells of target tissues in order for RNAi to fulfill its therapeutic promise. A detailed database of all such chemical modifications is manually curated as siRNAmod in scientific literature. Chemical modification of siRNA can also inadvertently result in loss of single-nucleotide specificity.

Therapeutic applications

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 and applied biology.

One of the biggest challenges to siRNA and RNAi based therapeutics is intracellular delivery. siRNA also has weak stability and pharmacokinetic behavior. Delivery of siRNA via nanoparticles has shown promise. and have shown only mild effectiveness in localized delivery sites, such as the human eye. Delivering pure DNA to target organisms is challenging because its large size and structure prevents it from diffusing readily across membranes. This allows delivery via nano-scale delivery vehicles called nanovectors.

siRNAs delivered via lipid based nanoparticles have been shown to have therapeutic potential for central nervous system (CNS) disorders. Central nervous disorders are not uncommon, but the blood brain barrier (BBB) often blocks access of potential therapeutics to the brain.

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.

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.

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.

Legal categorization and legal issues in a near future

Currently, SiRNA are currently chemically synthesized and so, are legally categorized inside EU and in USA as simple medicinal products. But as bioengineered siRNA (BERAs) are in development, these would be classified as biological medicinal products, at least in EU. The development of the BERAs technology raises the question of the categorization of drugs having the same mechanism of action but being produced chemically or biologically. This lack of consistency should be addressed.

Intracellular delivery

There is great potential for RNA interference (RNAi) to be used therapeutically to reversibly silence any gene. For RNAi to realize its therapeutic potential, small interfering RNA (siRNA) must be delivered to the site of action in the cells of target tissues. But finding safe and efficient delivery mechanisms is a major obstacle to achieving the full potential of siRNA-based therapies. Unmodified siRNA is unstable in the bloodstream, has the potential to cause immunogenicity, and has difficulty readily navigating cell membranes. As a result, chemical alterations and/or delivery tools are needed to safely transfer siRNA to its site of action. 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.

Electroporation

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 hydrophilic pores and localized perturbations in the lipid bilayer of the cell membrane, and temporary loss of semipermeability. This allows for the escape of intracellular contents (ions, metabolites, etc.) as well as for the uptake of drugs, molecular probes, and nucleic acids.

For cells that are difficult to transfect, electroporation is advantageous; however, cell death is more likely under this technique. The 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.

Viral-mediated delivery

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. As of primary research work in 2006, the latter virus was the most efficient for stable delivery of siRNA to target cells, as it could be used to transduce nondividing cells, as well as to directly target the nucleus. These specific viral vectors have been synthesized to effectively facilitate siRNA that is not viable for transfection into cells.

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 an in vivo method, and can be effective for difficult to transfect cells. However, problems arise because it can trigger antiviral responses in some cell types, leading to mutagenic and immunogenic effects.

This method has been shown to have potential for use in gene silencing of the central nervous system genes, e.g., in the treatment of Huntington's disease.

Therapies

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. Listed below are some of approved therapies or therapies in pipeline.

Alnylam Pharmaceuticals

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. During hATTR amyloidosis, misfolded transthyretin (TTR) protein is deposited in the extracellular space. Under typical folding conditions, TTR tetramers are made up of four monomers. Hereditary ATTR amyloidosis is caused by a fault or mutation in the transthyretin (TTR) gene which is inherited. Changing just one amino-acid changes the tetrameric transthyretin proteins, resulting in unstable tetrameric transthyretin protein that aggregates in monomers and forms insoluble extracellular amyloid deposits. Amyloid buildup in various organ systems causes cardiomyopathy, polyneuropathy, gastrointestinal dysfunction. 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.

Traditionally, liver transplantation has been the standard treatment for hereditary transthyretin amyloidosis, however its effectiveness may be limited by the persistent deposition of wild-type transthyretin amyloid after transplantation. 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.

Along with Onpattro, another RNA interference therapeutic drug has also been discovered (Partisiran) which has property of inhibiting hepatic synthesis of transthyretin. Target messenger RNA (mRNA) is cleaved as a result by tiny interfering RNAs coupled to the RNA-induced silencing complex. Patisiran, an investigational RNAi therapeutic drug, uses this process to decrease the production of mutant and wild-type transthyretin by cleaving on 3-untranslated region of transthyretin mRNA.

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.

Other companies that have had success in developing a pipeline of siRNA therapies are Dicerna Pharmaceuticals, partnered Eli Lilly and Company 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.