gene silencing

• Genomic Imprinting
• Paramutation
• Transposon silencing (or Histone Modifications)
• Transgene silencing
• Position effect
• RNA-directed DNA methylation

• RNA interference
• RNA silencing
• Nonsense mediated decay

• Transvection
• Meiotic silencing of unpaired DNA

Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson.{{cite journal | vauthors = Kole R, Krainer AR, Altman S | title = RNA therapeutics: beyond RNA interference and antisense oligonucleotides | journal = Nature Reviews. Drug Discovery | volume = 11 | issue = 2 | pages = 125–40 | date = February 2012 | pmid = 22262036 | doi = 10.1038/nrd3625 | pmc=4743652}} Oligonucleotides, which are short nucleic acid fragments, bind to complementary target mRNA molecules when added to the cell.{{cite journal | vauthors = Dias N, Stein CA | title = Antisense oligonucleotides: basic concepts and mechanisms | journal = Molecular Cancer Therapeutics | volume = 1 | issue = 5 | pages = 347–55 | date = March 2002 | pmid = 12489851 }} These molecules can be composed of single-stranded DNA or RNA and are generally 13–25 nucleotides long.{{cite journal | vauthors = Kurreck J | title = Antisense and RNA interference approaches to target validation in pain research | journal = Current Opinion in Drug Discovery & Development | volume = 7 | issue = 2 | pages = 179–87 | date = March 2004 | pmid = 15603251 }} The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism. RNase H-dependent oligonucleotides cause the target mRNA molecules to be degraded, while steric-blocker oligonucleotides prevent translation of the mRNA molecule. The majority of antisense drugs function through the RNase H-dependent mechanism, in which RNase H hydrolyzes the RNA strand of the DNA/RNA heteroduplex. expression.

File:Ribozyme mechanism.png

Ribozymes are catalytic RNA molecules used to inhibit gene expression. These molecules work by cleaving mRNA molecules, essentially silencing the genes that produced them. Sidney Altman and Thomas Cech first discovered catalytic RNA molecules, RNase P and group II intron ribozymes, in 1989 and won the Nobel Prize for their discovery.{{cite journal|last=Phylactou|first=L.|title=Ribozymes as therapeutic tools for genetic disease|journal=Human Molecular Genetics|date=1 September 1998|volume=7|issue=10|pages=1649–1653|doi=10.1093/hmg/7.10.1649|pmid=9735387|doi-access=free}}{{cite journal | vauthors = Shampo MA, Kyle RA, Steensma DP | title = Sidney Altman--Nobel laureate for work with RNA | journal = Mayo Clinic Proceedings | volume = 87 | issue = 10 | pages = e73 | date = October 2012 | pmid = 23036683 | doi = 10.1016/j.mayocp.2012.01.022 | pmc=3498233}} 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 motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule. Lower eukaryotes and a few bacteria contain group I and group II ribozymes. These motifs can self-splice by cleaving and joining phosphodiester bonds. The last ribozyme motif, the RNase P ribozyme, is found in Escherichia coli and is known for its ability to cleave the phosphodiester bonds of several tRNA precursors when joined to a protein cofactor.

The general catalytic mechanism used by ribozymes is similar to the mechanism used by protein ribonucleases.{{cite journal | vauthors = Doherty EA, Doudna JA | title = Ribozyme structures and mechanisms | journal = Annual Review of Biophysics and Biomolecular Structure | volume = 30 | issue = 1 | pages = 457–75 | date = 1 June 2001 | pmid = 11441810 | doi = 10.1146/annurev.biophys.30.1.457 }} 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. This catalytic mechanism has been increasingly used by scientists to perform sequence-specific cleavage of target mRNA molecules. In addition, attempts are being made to use ribozymes to produce gene silencing therapeutics, which would silence genes that are responsible for causing diseases.{{cite book|editor-last=Tollefsbol|editor-first=Trygve O.|title=Biological aging methods and protocols|year=2007|publisher=Humana Press|location=Totowa, N.J.|isbn=9781597453615}}

File:RNAi-simplified.png

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.{{cite web|title=RNA Interference Fact Sheet |url=http://www.nigms.nih.gov/News/Extras/RNAi/factsheet.htm |publisher=National Institutes of Health |access-date=24 November 2013 |url-status=dead |archive-url=https://web.archive.org/web/20131125205730/http://www.nigms.nih.gov/News/Extras/RNAi/factsheet.htm |archive-date=November 25, 2013 }} 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. The double-stranded molecule is then cut into small double-stranded fragments by an enzyme called Dicer. These small fragments, which include small interfering RNAs (siRNA) and microRNA (miRNA), are approximately 21–23 nucleotides in length. The fragments integrate into a multi-subunit protein called the RNA-induced silencing complex, which contains Argonaute proteins that are essential components of the RNAi pathway. One strand of the molecule, called the "guide" strand, binds to RISC, while the other strand, known as the "passenger" strand is degraded. The guide or antisense strand of the fragment that remains bound to RISC directs the sequence-specific silencing of the target mRNA molecule. The genes can be silenced by siRNA molecules that cause the endonucleatic cleavage of the target mRNA molecules or by miRNA molecules that suppress translation of the mRNA molecule.{{cite journal | vauthors = Wilson RC, Doudna JA | title = Molecular mechanisms of RNA interference | journal = Annual Review of Biophysics | volume = 42 | pages = 217–39 | year = 2013 | pmid = 23654304 | pmc = 5895182 | doi = 10.1146/annurev-biophys-083012-130404 }} With the cleavage or translational repression of the mRNA molecules, the genes that form them are rendered essentially inactive. RNAi is thought to have evolved as a cellular defense mechanism against invaders, such as RNA viruses, or to combat the proliferation of transposons within a cell's DNA. Both RNA viruses and transposons can exist as double-stranded RNA and lead to the activation of RNAi. Currently, siRNAs are being widely used to suppress specific gene expression and to assess the function of genes. Companies utilizing this approach include Alnylam, Sanofi,{{cite news|title = Big Pharma's Turn On RNAi Shows That New Technologies Don't Guarantee R&D Success|url = https://www.forbes.com/sites/johnlamattina/2014/04/15/big-pharmas-turn-on-rnai-shows-that-new-technologies-dont-guarantee-rd-success/|website = Forbes|access-date = 2015-10-11 | last1=Lamattina | first1=John }} Arrowhead, Discerna,{{cite web|title = The Second Coming of RNAi {{!}} The Scientist Magazine®|url = http://www.the-scientist.com/?articles.view/articleNo/40871/title/The-Second-Coming-of-RNAi/|website = The Scientist|access-date = 2015-10-11}} and Persomics,{{cite web|title = Products {{!}} Persomics|url = http://www.persomics.com/products|website = www.persomics.com|access-date = 2015-10-11}} among others.

{{main|Three prime untranslated region}}

{{main|MicroRNA}}

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.{{cn|date=December 2023}}

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.{{cn|date=December 2023}}

As of 2014, the miRBase web site,miRBase.org 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).{{cite journal | vauthors = Friedman RC, Farh KK, Burge CB, Bartel DP | title = Most mammalian mRNAs are conserved targets of microRNAs | journal = Genome Research | volume = 19 | issue = 1 | pages = 92–105 | date = January 2009 | pmid = 18955434 | pmc = 2612969 | doi = 10.1101/gr.082701.108 }} Freidman et al. 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.{{cn|date=December 2023}}

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs.{{cite journal | vauthors = Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM | title = Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs | journal = Nature | volume = 433 | issue = 7027 | pages = 769–73 | date = February 2005 | pmid = 15685193 | doi = 10.1038/nature03315 | bibcode = 2005Natur.433..769L | s2cid = 4430576 }} 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).{{cite journal | vauthors = Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N | title = Widespread changes in protein synthesis induced by microRNAs | journal = Nature | volume = 455 | issue = 7209 | pages = 58–63 | date = September 2008 | pmid = 18668040 | doi = 10.1038/nature07228 | bibcode = 2008Natur.455...58S | s2cid = 4429008 }}{{cite journal | vauthors = Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP | title = The impact of microRNAs on protein output | journal = Nature | volume = 455 | issue = 7209 | pages = 64–71 | date = September 2008 | pmid = 18668037 | pmc = 2745094 | doi = 10.1038/nature07242 | bibcode = 2008Natur.455...64B }}

The effects of miRNA dysregulation of gene expression seem to be important in cancer.{{cite journal | vauthors = Palmero EI, de Campos SG, Campos M, de Souza NC, Guerreiro ID, Carvalho AL, Marques MM | title = Mechanisms and role of microRNA deregulation in cancer onset and progression | journal = Genetics and Molecular Biology | volume = 34 | issue = 3 | pages = 363–70 | date = July 2011 | pmid = 21931505 | pmc = 3168173 | doi = 10.1590/S1415-47572011000300001 }} For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.{{cite journal | vauthors = Bernstein C, Bernstein H | title = Epigenetic reduction of DNA repair in progression to gastrointestinal cancer | journal = World Journal of Gastrointestinal Oncology | volume = 7 | issue = 5 | pages = 30–46 | date = May 2015 | pmid = 25987950 | pmc = 4434036 | doi = 10.4251/wjgo.v7.i5.30 | doi-access = free }}

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.{{cite journal | vauthors = Maffioletti E, Tardito D, Gennarelli M, Bocchio-Chiavetto L | title = Micro spies from the brain to the periphery: new clues from studies on microRNAs in neuropsychiatric disorders | journal = Frontiers in Cellular Neuroscience | volume = 8 | pages = 75 | year = 2014 | pmid = 24653674 | pmc = 3949217 | doi = 10.3389/fncel.2014.00075 | doi-access = free }}{{cite journal | vauthors = Mellios N, Sur M | title = The Emerging Role of microRNAs in Schizophrenia and Autism Spectrum Disorders | journal = Frontiers in Psychiatry | volume = 3 | pages = 39 | year = 2012 | pmid = 22539927 | pmc = 3336189 | doi = 10.3389/fpsyt.2012.00039 | doi-access = free }}{{cite journal | vauthors = Geaghan M, Cairns MJ | title = MicroRNA and Posttranscriptional Dysregulation in Psychiatry | journal = Biological Psychiatry | volume = 78 | issue = 4 | pages = 231–9 | date = August 2015 | pmid = 25636176 | doi = 10.1016/j.biopsych.2014.12.009 | s2cid = 5730697 | doi-access = free | hdl = 1959.13/1335073 | hdl-access = free }}

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.{{cn|date=December 2023}}

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.{{cite journal | vauthors = Chen J, Wall NR, Kocher K, Duclos N, Fabbro D, Neuberg D, Griffin JD, Shi Y, Gilliland DG | title = Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin | journal = The Journal of Clinical Investigation | volume = 113 | issue = 12 | pages = 1784–91 | date = June 2004 | pmid = 15199413 | doi = 10.1172/JCI20673 | pmc=420507}} 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. RNA interference can also be used to target specific mutants. For instance, siRNAs were able to bind specifically to tumor suppressor p53 molecules containing a single point mutation and destroy it, while leaving the wild-type suppressor intact.{{cite journal | vauthors = Martinez LA, Naguibneva I, Lehrmann H, Vervisch A, Tchénio T, Lozano G, Harel-Bellan A | title = Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 23 | pages = 14849–54 | date = November 2002 | pmid = 12403821 | doi = 10.1073/pnas.222406899 | bibcode = 2002PNAS...9914849M | pmc=137507| doi-access = free }}

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.{{cite journal | vauthors = Lapteva N, Yang AG, Sanders DE, Strube RW, Chen SY | title = CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo | journal = Cancer Gene Therapy | volume = 12 | issue = 1 | pages = 84–9 | date = January 2005 | pmid = 15472715 | doi = 10.1038/sj.cgt.7700770 | s2cid = 23402257 }} 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.{{cite journal | vauthors = July LV, Beraldi E, So A, Fazli L, Evans K, English JC, Gleave ME | title = Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells both in vitro and in vivo | journal = Molecular Cancer Therapeutics | volume = 3 | issue = 3 | pages = 223–32 | date = March 2004 | doi = 10.1158/1535-7163.223.3.3 | pmid = 15026542 | s2cid = 37703422 | doi-access = free }}{{cite journal | vauthors = Ning S, Fuessel S, Kotzsch M, Kraemer K, Kappler M, Schmidt U, Taubert H, Wirth MP, Meye A | title = siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth | journal = International Journal of Oncology | volume = 25 | issue = 4 | pages = 1065–71 | date = October 2004 | pmid = 15375557 | doi = 10.3892/ijo.25.4.1065 | doi-broken-date = 1 November 2024 | url = https://www.spandidos-publications.com/ijo/25/4/1065}} 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. In vivo studies are also being increasingly utilized to study the potential use of siRNA molecules in cancer therapeutics. For instance, mice implanted with colon adenocarcinoma cells were found to survive longer when the cells were pretreated with siRNAs that targeted B-catenin in the cancer cells.{{cite journal | vauthors = Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB |author5-link=Richard Gaynor | title = Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells | journal = Clinical Cancer Research | volume = 9 | issue = 4 | pages = 1291–300 | date = April 2003 | pmid = 12684397 }}

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.{{cite journal | vauthors = Dave RS, Pomerantz RJ | title = Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains | journal = Journal of Virology | volume = 78 | issue = 24 | pages = 13687–96 | date = December 2004 | pmid = 15564478 | doi = 10.1128/JVI.78.24.13687-13696.2004 | pmc=533941}}{{cite journal | vauthors = Wilson JA, Jayasena S, Khvorova A, Sabatinos S, Rodrigue-Gervais IG, Arya S, Sarangi F, Harris-Brandts M, Beaulieu S, Richardson CD | title = RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 5 | pages = 2783–8 | date = March 2003 | pmid = 12594341 | doi = 10.1073/pnas.252758799 | bibcode = 2003PNAS..100.2783W | pmc=151418| doi-access = free }} In particular, siRNA was used to silence the primary HIV receptor chemokine receptor 5 (CCR5).{{cite journal | vauthors = Qin XF, An DS, Chen IS, Baltimore D | title = Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 1 | pages = 183–8 | date = January 2003 | pmid = 12518064 | doi = 10.1073/pnas.232688199 | pmc=140921| bibcode = 2003PNAS..100..183Q | doi-access = free }} This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells.{{cite journal | vauthors = Li MJ, Bauer G, Michienzi A, Yee JK, Lee NS, Kim J, Li S, Castanotto D, Zaia J, Rossi JJ | title = Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs | journal = Molecular Therapy | volume = 8 | issue = 2 | pages = 196–206 | date = August 2003 | pmid = 12907142 | doi = 10.1016/s1525-0016(03)00165-5 | doi-access = free }} 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.{{cite journal | vauthors = Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E | title = Small interfering RNA inhibits hepatitis B virus replication in mice | journal = Molecular Therapy | volume = 8 | issue = 5 | pages = 769–76 | date = November 2003 | pmid = 14599810 | doi = 10.1016/s1525-0016(03)00244-2 }} In addition, siRNA techniques used in hepatitis C were able to lower the amount of the virus in the cell by 98%.{{cite journal | vauthors = Randall G, Grakoui A, Rice CM | title = Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 1 | pages = 235–40 | date = January 2003 | pmid = 12518066 | doi = 10.1073/pnas.0235524100 | pmc=140937| bibcode = 2003PNAS..100..235R | doi-access = free }}{{cite journal | vauthors = Randall G, Rice CM | title = Interfering with hepatitis C virus RNA replication | journal = Virus Research | volume = 102 | issue = 1 | pages = 19–25 | date = June 2004 | pmid = 15068876 | doi = 10.1016/j.virusres.2004.01.011 }}

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.{{cite journal | vauthors = Beachy RN, Loesch-Fries S, Tumer NE| title = Coat Protein-Mediated Resistance Against Virus Infection | journal = Annual Review of Phytopathology | date = 1990 | volume = 28 | pages= 451–472 | doi=10.1146/annurev.py.28.090190.002315}} 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.{{cite journal | vauthors = Lindbo JA, Dougherty WG | title = Plant pathology and RNAi: a brief history | journal = Annual Review of Phytopathology | volume = 43 | pages = 191–204 | date = 2005 | pmid = 16078882 | doi = 10.1146/annurev.phyto.43.040204.140228 }}{{cite journal | vauthors = Lindbo JA, Silva-Rosales L, Proebsting WM, Dougherty WG | title = Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance | journal = The Plant Cell | volume = 5 | issue = 12 | pages = 1749–1759 | date = December 1993 | pmid = 12271055 | doi = 10.1105/tpc.5.12.1749 | pmc=160401}} The discovery of small interfering RNAs (the specificity determinant in RNA-mediated gene silencing) also utilized virus-induced post-transcriptional gene silencing in plants.{{cite journal | vauthors = Hamilton AJ, Baulcombe DC | title = A species of small antisense RNA in posttranscriptional gene silencing in plants | journal = Science | volume = 286 | issue = 5441 | pages = 950–2 | date = October 1999 | pmid = 10542148 | doi=10.1126/science.286.5441.950}} 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.{{cite journal | last1 = Kaniewski | first1 = Wojciech K | last2 = Thomas | first2 = Peter E. | name-list-style = vanc | title = The Potato Story | journal = AgBioForum | volume = 7 | issue = 1&2 | pages = 41–46 | year = 2004 }} 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.{{Cite journal | last1 = Ferreira | first1 = S. A. | last2 = Pitz | first2 = K. Y. | last3 = Manshardt | first3 = R. | last4 = Zee | first4 = F. | last5 = Fitch | first5 = M. | last6 = Gonsalves | first6 = D. | doi = 10.1094/PDIS.2002.86.2.101 | title = Virus Coat Protein Transgenic Papaya Provides Practical Control ofPapaya ringspot virusin Hawaii | journal = Plant Disease | volume = 86 | issue = 2 | pages = 101–105 | year = 2002 | pmid = 30823304}} These papayas also remain in use at present, although not without significant public protest,{{cite web|title=Papaya: A GMO success story|url=http://hawaiitribune-herald.com/sections/news/local-news/papaya-gmo-success-story.html|access-date=2016-08-30|archive-url=https://web.archive.org/web/20150610064402/http://hawaiitribune-herald.com/sections/news/local-news/papaya-gmo-success-story.html|archive-date=2015-06-10|url-status=dead}}{{cite web|title=Papaya in the Crosshairs: A Heated Island Battle Over GMOs - Modern Farmer | url = http://modernfarmer.com/2013/12/battleground-hawaii-tiny-island-state-leading-battle-gmos/| date = 19 December 2013}} 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.{{cite journal | vauthors = Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M, Hoppe-Seyler F | title = siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells | journal = Oncogene | volume = 22 | issue = 38 | pages = 5938–45 | date = September 2003 | pmid = 12955072 | doi = 10.1038/sj.onc.1206894 | s2cid = 21504155 }} Plasmid siRNA expression vectors used to target the West Nile virus were also able to prevent the replication of viruses in cell lines.{{cite journal | vauthors = McCown M, Diamond MS, Pekosz A | title = The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses | journal = Virology | volume = 313 | issue = 2 | pages = 514–24 | date = September 2003 | pmid = 12954218 | doi = 10.1016/s0042-6822(03)00341-6 | doi-access = free }} 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.{{cite journal | vauthors = Fan Q, Wei C, Xia M, Jiang X | title = Inhibition of Tulane virus replication in vitro with RNA interference | journal = Journal of Medical Virology | volume = 85 | issue = 1 | pages = 179–86 | date = January 2013 | pmid = 23154881 | doi = 10.1002/jmv.23340 | pmc=3508507}} 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. Although the Tulane virus is species-specific and does not affect humans, it has been shown to be closely related to the human norovirus, which is the most common cause of acute gastroenteritis and food-borne disease outbreaks in the United States.{{cite web|title=Norovirus Overview|url=https://www.cdc.gov/norovirus/about/overview.html|publisher=Center for Disease Control and Prevention|date=2018-12-21}} 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.{{cn|date=December 2023}}

File:Prokaryote cell.svg

Unlike viruses, bacteria are not as susceptible to silencing by siRNA.{{cite journal | vauthors = Lieberman J, Song E, Lee SK, Shankar P | title = Interfering with disease: opportunities and roadblocks to harnessing RNA interference | journal = Trends in Molecular Medicine | volume = 9 | issue = 9 | pages = 397–403 | date = September 2003 | pmid = 13129706 | doi = 10.1016/s1471-4914(03)00143-6 | pmc = 7128953 }} 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. However, bacterial infections can still be suppressed by siRNA by targeting the host genes that are involved in the immune response caused by the infection or by targeting the host genes involved in mediating the entry of bacteria into cells.{{cite journal | vauthors = Leung RK, Whittaker PA | title = RNA interference: from gene silencing to gene-specific therapeutics | journal = Pharmacology & Therapeutics | volume = 107 | issue = 2 | pages = 222–39 | date = August 2005 | pmid = 15908010 | doi = 10.1016/j.pharmthera.2005.03.004 | pmc = 7112686 }} For instance, siRNA was used to reduce the amount of pro-inflammatory cytokines expressed in the cells of mice treated with lipopolysaccharide (LPS).{{cite book | vauthors = Sørensen DR, Sioud M | chapter = Systemic Delivery of Synthetic siRNAs | title = RNA Therapeutics | series = Methods in Molecular Biology | volume = 629 | pages = 87–91 | year = 2010 | pmid = 20387144 | doi = 10.1007/978-1-60761-657-3_6 | isbn = 978-1-60761-656-6 }} 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. In addition, siRNA was used to prevent the bacteria, Psueomonas aeruginosa, from invading murine lung epithelial cells by knocking down the caveolin-2 (CAV2) gene.{{cite journal | vauthors = Zaas DW, Duncan MJ, Li G, Wright JR, Abraham SN | title = Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2 | journal = The Journal of Biological Chemistry | volume = 280 | issue = 6 | pages = 4864–72 | date = February 2005 | pmid = 15545264 | doi = 10.1074/jbc.M411702200 | s2cid = 43122091 | doi-access = free }} 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.{{cn|date=December 2023}}

Ribozymes, antisense oligonucleotides, and more recently RNAi have been used to target mRNA molecules involved in asthma.{{cite journal | vauthors = Popescu FD, Popescu F | title = A review of antisense therapeutic interventions for molecular biological targets in asthma | journal = Biologics: Targets and Therapy | volume = 1 | issue = 3 | pages = 271–83 | date = September 2007 | pmid = 19707336 | pmc=2721314}} These experiments have suggested that siRNA may be used to combat other respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. COPD is characterized by goblet cell hyperplasia and mucus hypersecretion.{{cite journal | vauthors = Pistelli R, Lange P, Miller DL | title = Determinants of prognosis of COPD in the elderly: mucus hypersecretion, infections, cardiovascular comorbidity | journal = The European Respiratory Journal. Supplement | volume = 40 | pages = 10s–14s | date = May 2003 | issue = 40 suppl | pmid = 12762568 | doi = 10.1183/09031936.03.00403403 | s2cid = 19006320 | doi-access = free }} 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.{{cite journal | vauthors = Shao MX, Nakanaga T, Nadel JA | title = Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells | journal = American Journal of Physiology. Lung Cellular and Molecular Physiology | volume = 287 | issue = 2 | pages = L420–7 | date = August 2004 | pmid = 15121636 | doi = 10.1152/ajplung.00019.2004 }} 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.{{cite journal | vauthors = Rennard SI | title = Inflammation and repair processes in chronic obstructive pulmonary disease | journal = American Journal of Respiratory and Critical Care Medicine | volume = 160 | issue = 5 Pt 2 | pages = S12–6 | date = November 1999 | pmid = 10556162 | doi = 10.1164/ajrccm.160.supplement_1.5 }}{{cite journal | vauthors = Sacco O, Silvestri M, Sabatini F, Sale R, Defilippi AC, Rossi GA | title = Epithelial cells and fibroblasts: structural repair and remodelling in the airways | journal = Paediatric Respiratory Reviews | volume = 5 Suppl A | pages = S35–40 | year = 2004 | pmid = 14980241 | doi = 10.1016/s1526-0542(04)90008-5 }} 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.{{cite web|title=Pulmonary Fibrosis|url=http://www.mayoclinic.com/health/pulmonary-fibrosis/DS00927|publisher=Mayo Clinic|access-date=13 December 2013}}{{cite journal | vauthors = Gurujeyalakshmi G, Giri SN | title = Molecular mechanisms of antifibrotic effect of interferon gamma in bleomycin-mouse model of lung fibrosis: downregulation of TGF-beta and procollagen I and III gene expression | journal = Experimental Lung Research | volume = 21 | issue = 5 | pages = 791–808 | date = Sep–Oct 1995 | pmid = 8556994 | doi = 10.3109/01902149509050842 }}

File:PDB 3io4 EBI.png

Huntington's disease (HD) results from a mutation in the huntingtin gene that causes an excess of CAG repeats.{{cite web|title=Gene Silencing|url=http://www.stanford.edu/group/hopes/cgi-bin/wordpress/2012/04/gene-silencing/|work=HOPES - Huntington's Outreach Project for Education, at Stanford|publisher=Stanford University|access-date=13 December 2013|date=2012-04-05}} The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus.{{cite journal | vauthors = Mantha N, Das SK, Das NG | title = RNAi-based therapies for Huntington's disease: delivery challenges and opportunities | journal = Therapeutic Delivery | volume = 3 | issue = 9 | pages = 1061–76 | date = September 2012 | pmid = 23035592 | doi = 10.4155/tde.12.80 }} This disease is incurable and known to cause motor, cognitive, and behavioral deficits.{{cite journal | vauthors = Harper SQ | title = Progress and challenges in RNA interference therapy for Huntington disease | journal = Archives of Neurology | volume = 66 | issue = 8 | pages = 933–8 | date = August 2009 | pmid = 19667213 | doi = 10.1001/archneurol.2009.180 | doi-access = free }} Researchers have been looking to gene silencing as a potential therapeutic for HD.{{cn|date=December 2023}}

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.

Non-allele specific gene silencing using siRNA molecules has also been used to silence the mutant huntingtin proteins. Through this approach, instead of targeting SNPs on the mutated protein, all of the normal and mutated huntingtin proteins are targeted. When studied in mice, it was found that siRNA could reduce the normal and mutant huntingtin levels by 75%. At this level, they found that the mice developed improved motor control and a longer survival rate when compared to the controls. Thus, gene silencing methods may prove to be beneficial in treating HD.

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a motor neuron disease that affects the brain and spinal cord. The disease causes motor neurons to degenerate, which eventually leads to neuron death and muscular degeneration.{{cite web|title=What is ALS?|url=http://www.alsa.org/about-als/what-is-als.html|publisher=The ALS Association}} Hundreds of mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been found to cause ALS.{{cite journal | vauthors = Geng CM, Ding HL | title = Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing allele | journal = Acta Pharmacologica Sinica | volume = 29 | issue = 2 | pages = 211–6 | date = February 2008 | pmid = 18215350 | doi = 10.1111/j.1745-7254.2008.00740.x | s2cid = 24809180 | doi-access = free }} Gene silencing has been used to knock down the SOD1 mutant that is characteristic of ALS.{{cite web|last=Boulis|first=Nicholas|title=Gene Therapy for Motor Neuron Disease|url=http://www.sfn.org/~/media/SfN/Documents/Short%20Courses/2011%20Short%20Course%20I/2011_SC1_Boulis.ashx|publisher=Society for Neuroscience|access-date=13 December 2013}} In specific, siRNA molecules have been successfully used to target the SOD1 mutant gene and reduce its expression through allele-specific gene silencing.{{cite journal | vauthors = Ding H, Schwarz DS, Keene A, Affar el B, Fenton L, Xia X, Shi Y, Zamore PD, Xu Z | title = Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis | journal = Aging Cell | volume = 2 | issue = 4 | pages = 209–17 | date = August 2003 | pmid = 12934714 | doi = 10.1046/j.1474-9728.2003.00054.x | s2cid = 31752201 }}

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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. Thus, researchers have found that they must directly inject the molecules or implant pumps that push them into the brain.

Once inside the brain, however, the molecules must move inside of the targeted cells. In order to efficiently deliver siRNA molecules into the cells, viral vectors can be used. Nevertheless, this method of delivery can also be problematic as it can elicit an immune response against the molecules. In addition to delivery, specificity has also been found to be an issue in gene silencing. Both antisense oligonucleotides and siRNA molecules can potentially bind to the wrong mRNA molecule. Thus, researchers are searching for more efficient methods to deliver and develop specific gene silencing therapeutics that are still safe and effective.{{cn|date=December 2023}}

{{Main|Arctic Apples}}

Arctic Apples are a suite of trademarked[http://www.aphis.usda.gov/brs/aphisdocs/10_16101p.pdf Petition for Determination of Nonregulated Status: Arctic™ Apple (Malus x domestica) Events GD743 and GS784]. United States Department of Agriculture – Animal and Plant Health Inspection Service. Retrieved 2012-08-03. 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.{{cite web|url=http://www.arcticapples.com/arctic-apples-story/how-we-keep-apples-from-turning-brown|title=Apple-to-apple transformation|work=Okanagan Specialty Fruits|access-date=2012-08-03|archive-date=2013-09-25|archive-url=https://web.archive.org/web/20130925060233/http://www.arcticapples.com/arctic-apples-story/how-we-keep-apples-from-turning-brown|url-status=dead}}

• CRISPR
• DNA-directed RNA interference
• Gene drive
• Gene knockdown
• PPRHs

{{reflist|2}}

• [http://rnaiatlas.ethz.ch/ RNAiAtlas - database of siRNA libraries and their target analysis results]{{webarchive |url=https://web.archive.org/web/20150210230607/http://rnaiatlas.ethz.ch/ |date=February 10, 2015 }}.
• [https://web.archive.org/web/20110721173426/http://www.gmo-safety.eu/database/1030.transgenic-apple-varieties-approaches-preventing-outcrossing-possible-effects-micro-organisms.html Science project: Transgenic apple varieties] Approaches to preventing outcrossing – possible effects on micro-organisms
• {{MeshName|Gene+silencing}}
• [https://web.archive.org/web/20080829180955/http://www.4engr.com/research/catalog/237/index.html Research project: New Cost-effective method for gene silencing]
• {{cite book | vauthors = van Leeuwen F, Gottschling DE | title = Guide to Yeast Genetics and Molecular and Cell Biology - Part B | chapter = Assays for gene silencing in yeast | series = Methods in Enzymology | volume = 350 | pages = 165–86 | year = 2002 | pmid = 12073311 | doi = 10.1016/S0076-6879(02)50962-9 | isbn = 9780121822538 }}

{{Regulation of gene expression}}

Category:Gene expression

Category:Epigenetics

Category:DNA

Category:RNA

Category:Post-translational modification

Category:Australian inventions