Gene silencing

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. When genes are silenced, their expression is reduced. since they allow researchers to study essential genes that are required for the animal models to survive and cannot be removed. In addition, they provide a more complete view on the development of diseases since diseases are generally associated with genes that have a reduced expression. Oligonucleotides, which are short nucleic acid fragments, bind to complementary target mRNA molecules when added to the cell. These molecules can be composed of single-stranded DNA or RNA and are generally 13–25 nucleotides long. The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs are generally found in viruses or viroid RNAs. These catalytic RNA molecules bind to a specific site and attack the neighboring phosphate in the RNA backbone with their 2' oxygen, which acts as a nucleophile, resulting in the formation of cleaved products with a 2'3'-cyclic phosphate and a 5' hydroxyl terminal end.

RNA interference

thumb|left|330px|Left:Overview of RNA interference.

RNA interference (RNAi) is a natural process used by cells to regulate gene expression. It was discovered in 1998 by Andrew Fire and Craig Mello, who won the Nobel Prize for their discovery in 2006. The process to silence genes first begins with the entrance of a double-stranded RNA (dsRNA) molecule into the cell, which triggers the RNAi pathway. With the cleavage or translational repression of the mRNA molecules, the genes that form them are rendered essentially inactive. Arrowhead, Discerna, and Persomics, among others.

Three prime untranslated regions and microRNAs

The three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause gene silencing. 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, a large number of specific miRNAs decrease gene expression of their particular target mRNAs by either inhibiting translation or directly causing degradation of the transcript, using a mechanism similar to RNA interference (see MicroRNA). The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of an mRNA.

The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind and cause gene silencing. 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, 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 each have an average of about four hundred target mRNAs (causing gene silencing of several hundred genes). Freidman et al. 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).

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

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.

Applications

Medical research

Gene silencing techniques have been widely used by researchers to study genes associated with disorders. These disorders include cancer, infectious diseases, respiratory diseases, and neurodegenerative disorders. Gene silencing is also currently being used in drug discovery efforts, such as synthetic lethality, high-throughput screening, and miniaturized RNAi screens.

Cancer

RNA interference has been used to silence genes associated with several cancers. In in vitro studies of chronic myelogenous leukemia (CML), siRNA was used to cleave the fusion protein, BCR-ABL, which prevents the drug Gleevec (imatinib) from binding to the cancer cells. Cleaving the fusion protein reduced the amount of transformed hematopoietic cells that spread throughout the body by increasing the sensitivity of the cells to the drug.

Receptors involved in mitogenic pathways that lead to the increased production of cancer cells there have also been targeted by siRNA molecules. The chemokine receptor chemokine receptor 4 (CXCR4), associated with the proliferation of breast cancer, was cleaved by siRNA molecules that reduced the number of divisions commonly observed by the cancer cells. Researchers have also used siRNAs to selectively regulate the expression of cancer-related genes. Antiapoptotic proteins, such as clusterin and survivin, are often expressed in cancer cells. Clusterin and survivin-targeting siRNAs were used to reduce the number of antiapoptotic proteins and, thus, increase the sensitivity of the cancer cells to chemotherapy treatments.

Infectious disease

Viruses

Viral genes and host genes that are required for viruses to replicate or enter the cell, or that play an important role in the life cycle of the virus are often targeted by antiviral therapies. RNAi has been used to target genes in several viral diseases, such as the human immunodeficiency virus (HIV) and hepatitis. In particular, siRNA was used to silence the primary HIV receptor chemokine receptor 5 (CCR5). This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells. A similar technique was used to decrease the amount of the detectable virus in hepatitis B and C infected cells. In hepatitis B, siRNA silencing was used to target the surface antigen on the hepatitis B virus and led to a decrease in the number of viral components. In addition, siRNA techniques used in hepatitis C were able to lower the amount of the virus in the cell by 98%.

RNA interference has been in commercial use to control virus diseases of plants for over 20 years (see Plant disease resistance). In 1986–1990, multiple examples of "coat protein-mediated resistance" against plant viruses were published, before RNAi had been discovered. In 1993, work with tobacco etch virus first demonstrated that host organisms can target specific virus or mRNA sequences for degradation, and that this activity is the mechanism behind some examples of virus resistance in transgenic plants. The discovery of small interfering RNAs (the specificity determinant in RNA-mediated gene silencing) also utilized virus-induced post-transcriptional gene silencing in plants. By 1994, transgenic squash varieties had been generated expressing coat protein genes from three different viruses, providing squash hybrids with field-validated multiviral resistance that remain in commercial use at present. Potato lines expressing viral replicase sequences that confer resistance to potato leafroll virus were sold under the trade names NewLeaf Y and NewLeaf Plus, and were widely accepted in commercial production in 1999–2001, until McDonald's Corp. decided not to purchase GM potatoes and Monsanto decided to close their NatureMark potato business. Another frequently cited example of virus resistance mediated by gene silencing involves papaya, where the Hawaiian papaya industry was rescued by virus-resistant GM papayas produced and licensed by university researchers rather than a large corporation. These papayas also remain in use at present, although not without significant public protest, which is notably less evident in medical uses of gene silencing.

Gene silencing techniques have also been used to target other viruses, such as the human papilloma virus, the West Nile virus, and the Tulane virus. The E6 gene in tumor samples retrieved from patients with the human papilloma virus was targeted and found to cause apoptosis in the infected cells. Plasmid siRNA expression vectors used to target the West Nile virus were also able to prevent the replication of viruses in cell lines. In addition, siRNA has been found to be successful in preventing the replication of the Tulane virus, part of the virus family Caliciviridae, by targeting both its structural and non-structural genes. By targeting the NTPase gene, one dose of siRNA 4 hours pre-infection was shown to control Tulane virus replication for 48 hours post-infection, reducing the viral titer by up to 2.6 logarithms. Human noroviruses are notorious for being difficult to study in the laboratory, but the Tulane virus offers a model through which to study this family of viruses for the clinical goal of developing therapies that can be used to treat illnesses caused by human norovirus.

Bacteria

thumbnail|Structure of a typical Gram-positive bacterial cell

Unlike viruses, bacteria are not as susceptible to silencing by siRNA. This is largely due to how bacteria replicate. Bacteria replicate outside of the host cell and do not contain the necessary machinery for RNAi to function. For instance, siRNA was used to reduce the amount of pro-inflammatory cytokines expressed in the cells of mice treated with lipopolysaccharide (LPS). The reduced expression of the inflammatory cytokine, tumor necrosis factor α (TNFα), in turn, caused a reduction in the septic shock felt by the LPS-treated mice. Thus, though bacteria cannot be directly targeted by siRNA mechanisms, they can still be affected by siRNA when the components involved in the bacterial infection are targeted.

Respiratory diseases

Ribozymes, antisense oligonucleotides, and more recently RNAi have been used to target mRNA molecules involved in asthma. These experiments have suggested that siRNA may be used to combat other respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. Mucus secretion was found to be reduced when the transforming growth factor (TGF)-α was targeted by siRNA in NCI-H292 human airway epithelial cells. In addition to mucus hypersecretion, chronic inflammation and damaged lung tissue are characteristic of COPD and asthma. The transforming growth factor TGF-β is thought to play a role in these manifestations. As a result, when interferon (IFN)-γ was used to knock down TGF-β, fibrosis of the lungs, caused by damage and scarring to lung tissue, was improved.

Neurodegenerative disorders

Huntington's disease

thumbnail|right|Crystallographic structure of the N-terminal region of the human huntingtin protein.

Huntington's disease (HD) results from a mutation in the huntingtin gene that causes an excess of CAG repeats. The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus. This disease is incurable and known to cause motor, cognitive, and behavioral deficits. Researchers have been looking to gene silencing as a potential therapeutic for HD.

Gene silencing can be used to treat HD by targeting the mutant huntingtin protein. The mutant huntingtin protein has been targeted through gene silencing that is allele specific using allele specific oligonucleotides. In this method, the antisense oligonucleotides are used to target single nucleotide polymorphism (SNPs), which are single nucleotide changes in the DNA sequence, since HD patients have been found to share common SNPs that are associated with the mutated huntingtin allele. It has been found that approximately 85% of patients with HD can be covered when three SNPs are targeted. In addition, when antisense oligonucleotides were used to target an HD-associated SNP in mice, there was a 50% decrease in the mutant huntingtin protein. Hundreds of mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been found to cause ALS. Gene silencing has been used to knock down the SOD1 mutant that is characteristic of ALS. In specific, siRNA molecules have been successfully used to target the SOD1 mutant gene and reduce its expression through allele-specific gene silencing.

Therapeutics challenges

thumbnail|Basic mechanism used by viral vectors to deliver genes to target cells. Example shown is a lentiviral vector.

There are several challenges associated with gene silencing therapies, including delivery and specificity for targeted cells. For instance, for treatment of neurodegenerative disorders, molecules for a prospective gene silencing therapy must be delivered to the brain. The blood–brain barrier makes it difficult to deliver molecules into the brain through the bloodstream by preventing the passage of the majority of molecules that are injected or absorbed into the blood. apples that contain a nonbrowning trait created by using gene silencing to reduce the expression of polyphenol oxidase (PPO). It is the first approved food product to use this technique.

References

External links

RNAiAtlas - database of siRNA libraries and their target analysis results.
Science project: Transgenic apple varieties Approaches to preventing outcrossing – possible effects on micro-organisms
Research project: New Cost-effective method for gene silencing