Gene silencing

From Wikipedia, the free encyclopedia
Jump to: navigation, search

Gene silencing is a general term used to describe the epigenetic regulation of gene expression.[1] In particular, this term refers to the ability of a cell to prevent the expression of a certain gene.[2] Gene silencing can occur during either transcription or translation and is often used in research.[1][2] In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and diseases, such as infectious diseases and neurodegenerative disorders.

Gene silencing is often confused with gene knockout. Though gene silencing is the same as gene knockdown, it is different from gene knockout.[3][4] When genes are silenced, their expression is reduced.[3][4] In contrast, when genes are knocked out, they are completely erased from the organism's genome and, thus, have no expression.[3][4] Gene silencing is considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.[3] Methods using gene knockdowns are often considered better than gene knockouts since they allow researchers to study essential genes that are required for the animal models to survive and cannot be removed.[3] 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.[3]

Gene silencing in cells[edit]

Transcriptional gene silencing[edit]

Post-transcriptional gene silencing[edit]

Meiotic gene silencing[edit]

Gene silencing methods used in research[edit]

Antisense oligonucleotides[edit]

Antisense oligonucleotides were discovered in 1978 by Paul Zamecnik and Mary Stephenson.[5] Oligonucleotides, which are short nucleic acid fragments, bind to complementary target mRNA molecules when added to the cell.[5][6] These molecules can be composed of single-stranded DNA or RNA and are generally 13-25 nucleotides long.[6][7] The antisense oligonucleotides can affect gene expression in two ways: by using an RNase H-dependent mechanism or by using a steric blocking mechanism.[6][7] RNase H-dependent oligonucleotides cause the target mRNA molecules to be degraded, while steric-blocker oligonucleotides prevent translation of the mRNA molecule.[6][7] 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.[6][7] This mechanism is thought to be more efficient, resulting in an approximately 80% to 95% decrease in the protein and mRNA expression.[6]

Ribozymes[edit]

General mechanism utilized by ribozymes to cleave RNA molecules

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.[8][9] 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.[8] These motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule.[8] Lower eukaryotes and a few bacteria contain group I and group II ribozymes.[8] These motifs can self-splice by cleaving and joining together phosphodiester bonds.[8] 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.[8]

The general catalytic mechanism used by ribozymes is similar to the mechanism used by protein ribonucleases.[10] 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.[10] 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.[11]

RNA interference[edit]

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.[12] 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.[12] The double-stranded molecule is then cut into small double-stranded fragments by an enzyme called Dicer.[12] These small fragments, which include small interfering RNAs (siRNA) and microRNA (miRNA), are approximately 21-23 nucleotides in length.[12][13] The fragments integrate into a multi-subunit protein called the RNAi induced silencing complex (RISC), which contains Argonaute proteins that are essential components of the RNAi pathway.[12][13] One strand of the molecule, called the "guide" strand, binds to RISC, while the other strand, known as the "passenger" strand is degraded.[12][13] The guide or antisense strand of the fragment that remains bound to RISC directs the sequence-specific silencing of the target mRNA molecule.[13] 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.[13] With the cleavage or translational repression of the mRNA molecules, the genes that form them are essentially inactive.[12] 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.[12] Both RNA viruses and transposons can exist as double-stranded RNA and lead to the activation of RNAi.[12] Currently, siRNAs are being widely used to suppress specific gene expression and to assess the function of genes.

Gene silencing in medical research[edit]

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.

Cancer[edit]

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.[14] 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.[14] 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.[15]

Receptors involved in mitogenic pathways that lead to the increased production of cancer cells 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.[16] 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.[17][18] 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.[17][18] 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.[19]

Infectious diseases[edit]

Viruses[edit]

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.[20][21] In particular, siRNA was used to silence the primary HIV receptor chemokine receptor 5 (CCR5).[22] This prevented the virus from entering the human peripheral blood lymphocytes and the primary hematopoietic stem cells.[22][23] 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.[24] In addition, siRNA techniques used in hepatitis C were able to lower the amount of the virus in the cell by 98%.[25][26]

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.[27] Plasmid siRNA expression vectors used to target the West Nile Virus were also able to prevent the replication of viruses in cell lines.[28] In addition, siRNA has been found to be successful in preventing the replication of the Tulane virus, part of the Caliciviridae family of viruses, by targeting both its structural and non-structural genes.[29] 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.[29] 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.[30] 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[edit]

Structure of a typical Gram-positive bacterial cell

Unlike viruses, bacteria are not as susceptible to silencing by siRNA.[31] 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.[31] 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.[31][32] For instance, siRNA was used to reduce the amount of pro-inflammatory cytokines expressed in the cells of mice treated with lipopolysaccharide (LPS).[31][33] 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.[33] 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.[34] 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[edit]

Ribozymes, antisense oligonucleotides, and more recently RNAi have been used to target mRNA molecules involved in asthma.[32][35] These experiments have suggested that siRNA may be used to combat other respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis.[32] COPD is characterized by goblet cell hyperplasia and mucus hypersecretion.[36] 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.[37] 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.[38][39] 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.[40][41]

Neurodegenerative disorders[edit]

Hungtington’s disease[edit]

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.[42] The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus.[43] This disease is incurable and known to cause motor, cognitive, and behavioral deficits.[44] 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.[42] 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.[42] It has been found that approximately 85% of patients with HD can be covered when three SNPs are targeted.[42] 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.[42]

Non-allele specific gene silencing using siRNA molecules has also been used to silence the mutant huntingtin proteins.[42] Through this approach, instead of targeting SNPs on the mutated protein, all of the normal and mutated huntingtin proteins are targeted.[42] When studied in mice, it was found that siRNA could reduce the normal and mutant huntingtin levels by 75%.[42] At this level, they found that the mice developed improved motor control and a longer survival rate when compared to the controls.[42] Thus, gene silencing methods may prove to be beneficial in treating HD.

Amyotrophic lateral sclerosis[edit]

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 their death.[45] Hundreds of mutations in the Cu/Zn superoxide dismutase (SOD1) have been found to cause ALS.[46] Gene silencing has been used to knock down the SOD1 mutant that is characteristic of ALS.[46][47] In specific, siRNA molecules have been successfully used to target the SOD1 mutant gene and reduce its expression through allele-specific gene silencing.[46][48]

Challenges to gene silencing therapeutics[edit]

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 instance, in the case of neurodegenerative disorders, gene silencing molecules must be delivered to the brain. The blood-brain barrier makes it difficult to deliver molecules into the brain by preventing the passage of the majority of molecules that are injected or absorbed into the blood.[42][43] Thus, researchers have found that they must directly inject the molecules or implant pumps that push them into the brain.[42]

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.[42][44] Nevertheless, this method of delivery can also be problematic as it can elicit an immune response against the molecules.[42] 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.[42] Thus, researchers are searching for more efficient methods to deliver and develop specific gene silencing therapeutics that are still safe and effective.

References[edit]

  1. ^ a b Redberry, Grace (2006). Gene silencing : new research. New York: Nova Science Publishers. ISBN 9781594548321. 
  2. ^ a b "Gene Silencing". National Center for Biotechnology Information. Retrieved 11 November 2013. 
  3. ^ a b c d e f Hood, E (Mar 2004). "RNAi: What's all the noise about gene silencing?". Environmental Health Perspectives 112 (4): A224-A229. doi:10.1289/ehp.112-a224. PMC 1241909. 
  4. ^ a b c Mocellin, S; Provenzano, M (Nov 22, 2004). "RNA interference: learning gene knock-down from cell physiology.". Journal of translational medicine 2 (1): 39. doi:10.1186/1479-5876-2-39. PMID 15555080. 
  5. ^ a b Kole, R; Krainer, AR; Altman, S (Jan 20, 2012). "RNA therapeutics: beyond RNA interference and antisense oligonucleotides.". Nature reviews. Drug discovery 11 (2): 125–40. doi:10.1038/nrd3625. PMID 22262036. 
  6. ^ a b c d e f Dias, N; Stein, CA (March 2002). "Antisense oligonucleotides: basic concepts and mechanisms.". Molecular cancer therapeutics 1 (5): 347–55. PMID 12489851. 
  7. ^ a b c d Kurreck, J (March 2004). "Antisense and RNA interference approaches to target validation in pain research.". Current opinion in drug discovery & development 7 (2): 179–87. PMID 15603251. 
  8. ^ a b c d e f Phylactou, L. (1 September 1998). "Ribozymes as therapeutic tools for genetic disease". Human Molecular Genetics 7 (10): 1649–1653. doi:10.1093/hmg/7.10.1649. 
  9. ^ Shampo, Marc A.; Kyle, Robert A.; Steensma, David P. (1 October 2012). "Sidney Altman—Nobel Laureate for Work With RNA". Mayo Clinic Proceedings 87 (10): e73. doi:10.1016/j.mayocp.2012.01.022. 
  10. ^ a b Doherty, Elizabeth A.; Doudna, Jennifer A. (1 June 2001). "Ribozyme structures and mechanisms". Annual Review of Biophysics and Biomolecular Structure 30 (1): 457–475. doi:10.1146/annurev.biophys.30.1.457. PMID 11441810. 
  11. ^ Tollefsbol, edited by Trygve O. (2007). Biological aging methods and protocols. Totowa, N.J.: Humana Press. ISBN 9781597453615. 
  12. ^ a b c d e f g h i "RNA Interference Fact Sheet". National Institutes of Health. Retrieved 24 November 2013. 
  13. ^ a b c d e Wilson, RC; Doudna, JA (2013). "Molecular mechanisms of RNA interference.". Annual review of biophysics 42: 217–39. doi:10.1146/annurev-biophys-083012-130404. PMID 23654304. 
  14. ^ a b Chen, J; Wall, NR; Kocher, K; Duclos, N; Fabbro, D; Neuberg, D; Griffin, JD; Shi, Y; Gilliland, DG (June 2004). "Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin.". The Journal of Clinical Investigation 113 (12): 1784–91. doi:10.1172/JCI20673. PMID 15199413. 
  15. ^ Martinez, LA; Naguibneva, I; Lehrmann, H; Vervisch, A; Tchénio, T; Lozano, G; Harel-Bellan, A (Nov 12, 2002). "Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways.". Proceedings of the National Academy of Sciences of the United States of America 99 (23): 14849–54. doi:10.1073/pnas.222406899. PMID 12403821. 
  16. ^ Lapteva, N; Yang, AG; Sanders, DE; Strube, RW; Chen, SY (January 2005). "CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo.". Cancer gene therapy 12 (1): 84–9. doi:10.1038/sj.cgt.7700770. PMID 15472715. 
  17. ^ a b July, LV; Beraldi, E; So, A; Fazli, L; Evans, K; English, JC; Gleave, ME (March 2004). "Nucleotide-based therapies targeting clusterin chemosensitize human lung adenocarcinoma cells both in vitro and in vivo.". Molecular cancer therapeutics 3 (3): 223–32. PMID 15026542. 
  18. ^ a b Ning, S; Fuessel, S; Kotzsch, M; Kraemer, K; Kappler, M; Schmidt, U; Taubert, H; Wirth, MP; Meye, A (October 2004). "siRNA-mediated down-regulation of survivin inhibits bladder cancer cell growth.". International journal of oncology 25 (4): 1065–71. PMID 15375557. 
  19. ^ Verma, UN; Surabhi, RM; Schmaltieg, A; Becerra, C; Gaynor, RB (April 2003). "Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells.". Clinical cancer research : an official journal of the American Association for Cancer Research 9 (4): 1291–300. PMID 12684397. 
  20. ^ Dave, RS; Pomerantz, RJ (December 2004). "Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains.". Journal of Virology 78 (24): 13687–96. doi:10.1128/JVI.78.24.13687-13696.2004. PMID 15564478. 
  21. ^ Wilson, JA; Jayasena, S; Khvorova, A; Sabatinos, S; Rodrigue-Gervais, IG; Arya, S; Sarangi, F; Harris-Brandts, M; Beaulieu, S; Richardson, CD (Mar 4, 2003). "RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells.". Proceedings of the National Academy of Sciences of the United States of America 100 (5): 2783–8. doi:10.1073/pnas.252758799. PMID 12594341. 
  22. ^ a b Qin, XF; An, DS; Chen, IS; Baltimore, D (Jan 7, 2003). "Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.". Proceedings of the National Academy of Sciences of the United States of America 100 (1): 183–8. doi:10.1073/pnas.232688199. PMID 12518064. 
  23. ^ Li, MJ; Bauer, G; Michienzi, A; Yee, JK; Lee, NS; Kim, J; Li, S; Castanotto, D; Zaia, J; Rossi, JJ (August 2003). "Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs.". Molecular therapy : the journal of the American Society of Gene Therapy 8 (2): 196–206. doi:10.1016/s1525-0016(03)00165-5. PMID 12907142. 
  24. ^ Giladi, H; Ketzinel-Gilad, M; Rivkin, L; Felig, Y; Nussbaum, O; Galun, E (November 2003). "Small interfering RNA inhibits hepatitis B virus replication in mice.". Molecular therapy : the journal of the American Society of Gene Therapy 8 (5): 769–76. doi:10.1016/s1525-0016(03)00244-2. PMID 14599810. 
  25. ^ Randall, G; Grakoui, A; Rice, CM (Jan 7, 2003). "Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs.". Proceedings of the National Academy of Sciences of the United States of America 100 (1): 235–40. doi:10.1073/pnas.0235524100. PMID 12518066. 
  26. ^ Randall, G; Rice, CM (Jun 1, 2004). "Interfering with hepatitis C virus RNA replication.". Virus research 102 (1): 19–25. doi:10.1016/j.virusres.2004.01.011. PMID 15068876. 
  27. ^ Butz, K; Ristriani, T; Hengstermann, A; Denk, C; Scheffner, M; Hoppe-Seyler, F (Sep 4, 2003). "siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.". Oncogene 22 (38): 5938–45. doi:10.1038/sj.onc.1206894. PMID 12955072. 
  28. ^ McCown, M; Diamond, MS; Pekosz, A (Sep 1, 2003). "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.". Virology 313 (2): 514–24. doi:10.1016/s0042-6822(03)00341-6. PMID 12954218. 
  29. ^ a b Fan, Qiang; Wei, Chao; Xia, Ming; Jiang, Xi (January 2013). "Inhibition of Tulane virus replication in vitro with RNA interference". Journal of Medical Virology 85 (1): 179–186. doi:10.1002/jmv.23340. PMID 23154881. 
  30. ^ "Norovirus Overview". Center for Disease Control and Prevention. 
  31. ^ a b c d Lieberman, J; Song, E; Lee, SK; Shankar, P (September 2003). "Interfering with disease: opportunities and roadblocks to harnessing RNA interference.". Trends in molecular medicine 9 (9): 397–403. doi:10.1016/s1471-4914(03)00143-6. PMID 13129706. 
  32. ^ a b c Leung, RK; Whittaker, PA (August 2005). "RNA interference: from gene silencing to gene-specific therapeutics.". Pharmacology & therapeutics 107 (2): 222–39. doi:10.1016/j.pharmthera.2005.03.004. PMID 15908010. 
  33. ^ a b Sørensen, DR; Sioud, M (2010). "Systemic delivery of synthetic siRNAs.". Methods in molecular biology (Clifton, N.J.) 629: 87–91. doi:10.1007/978-1-60761-657-3_6. PMID 20387144. 
  34. ^ Zaas, DW; Duncan, MJ; Li, G; Wright, JR; Abraham, SN (Feb 11, 2005). "Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2.". The Journal of Biological Chemistry 280 (6): 4864–72. doi:10.1074/jbc.M411702200. PMID 15545264. 
  35. ^ Popescu, FD; Popescu, F (September 2007). "A review of antisense therapeutic interventions for molecular biological targets in asthma.". Biologics : targets & therapy 1 (3): 271–83. PMID 19707336. 
  36. ^ Pistelli, R; Lange, P; Miller, DL (May 2003). "Determinants of prognosis of COPD in the elderly: mucus hypersecretion, infections, cardiovascular comorbidity.". The European respiratory journal. Supplement 40: 10s–14s. doi:10.1183/09031936.03.00403403. PMID 12762568. 
  37. ^ Shao, MX; Nakanaga, T; Nadel, JA (August 2004). "Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells.". American journal of physiology. Lung cellular and molecular physiology 287 (2): L420–7. doi:10.1152/ajplung.00019.2004. PMID 15121636. 
  38. ^ Rennard, SI (November 1999). "Inflammation and repair processes in chronic obstructive pulmonary disease.". American Journal of Respiratory and Critical Care Medicine 160 (5 Pt 2): S12–6. doi:10.1164/ajrccm.160.supplement_1.5. PMID 10556162. 
  39. ^ Sacco, O; Silvestri, M; Sabatini, F; Sale, R; Defilippi, AC; Rossi, GA (2004). "Epithelial cells and fibroblasts: structural repair and remodelling in the airways.". Paediatric respiratory reviews. 5 Suppl A: S35–40. doi:10.1016/s1526-0542(04)90008-5. PMID 14980241. 
  40. ^ "Pulmonary Fibrosis". Mayo Clinic. Retrieved 13 December 2013. 
  41. ^ Gurujeyalakshmi, G; Giri, SN (Sep–Oct 1995). "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.". Experimental lung research 21 (5): 791–808. doi:10.3109/01902149509050842. PMID 8556994. 
  42. ^ a b c d e f g h i j k l m n "Gene Silencing". HOPES - Huntington's Outreach Project for Education, at Stanford. Stanford University. Retrieved 13 December 2013. 
  43. ^ a b Mantha, N; Das, SK; Das, NG (September 2012). "RNAi-based therapies for Huntington's disease: delivery challenges and opportunities.". Therapeutic delivery 3 (9): 1061–76. doi:10.4155/tde.12.80. PMID 23035592. 
  44. ^ a b Harper, SQ (August 2009). "Progress and challenges in RNA interference therapy for Huntington disease.". Archives of neurology 66 (8): 933–8. doi:10.1001/archneurol.2009.180. PMID 19667213. 
  45. ^ "What is ALS?". The ALS Association. 
  46. ^ a b c Geng, CM; Ding, HL (February 2008). "Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing allele.". Acta pharmacologica Sinica 29 (2): 211–6. doi:10.1111/j.1745-7254.2008.00740.x. PMID 18215350. 
  47. ^ Boulis, Nicholas. "Gene Therapy for Motor Neuron Disease". Society for Neuroscience. Retrieved 13 December 2013. 
  48. ^ Ding, H; Schwarz, DS; Keene, A; Affar el, B; Fenton, L; Xia, X; Shi, Y; Zamore, PD; Xu, Z (August 2003). "Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.". Aging cell 2 (4): 209–17. doi:10.1046/j.1474-9728.2003.00054.x. PMID 12934714. 

External links[edit]