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RNA interference


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RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.

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RNA interference

  1. 1. Isfahan University of Medical Science, School of Pharmacy Department of Clinical BiochemistryJune 25, 2012 1 Total slides : 51
  2. 2. RNA interferenceMechanism, Applications & Delivery Methods Presented by: A.N. Emami Razavi
  3. 3. O u t lin e s Introduction RNA silencing Definition of RNA interference Discovery of RNAi Mechanism of RNA interference Generation of small interfering RNA Small interfering RNA delivery methods Applications of RNA interference Therapeutic applications Other applications Conclusion
  4. 4. In t r o d u c t io n RN A i (R N A In t e r f e r e n c e )
  5. 5. RNA silencingSeveral terms are used to described RNA silencing; usually there are three phenotypically different but mechanistically similar phenomena:3. Cosuppression or post-trascriptional gene silencing (PTGS) in plants5. Quelling in fungi7. RNA interference in animal kingdom
  6. 6. Quelling:The silencing in fungal system Quelling came to light during attempts to boost the production of an orange pigment made by the gene al1 of the fungus Neurospora crassa N. crassa (al1+) transformed with plasmid+al1 few transformants show albino phenotype al1-quelled strain had similar level of unspliced al1 mRNA to wilde-type. Native al1 mRNA was highly reduced indicating that quelling and NOT the rate of transcription affected the level of mature mRNA in a homology- dependent manner
  7. 7. RNAi:Silencing in Cenorhabditis elegans dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function.
  8. 8. Definition RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons.
  9. 9. Need for interference Defense Mechanism Defense against Infection by viruses, etc As a defense mechanism to protect against transposons and other insertional elements Genome Wide Regulation RNAi plays a role in regulating development and genome maintenance. 30% of human genome regulated
  10. 10. PTGS in plants:The discovery of Jorgensen and Napoli in1990 They were trying to make petunias more purple Overexpression of petunia gene Entered homologous RNA Expected:more pigments Observed:white sectorsCosuppression:• Loss of mRNAs of both endo-and transgene
  11. 11. Andrew Fire Craig C. Mellow
  12. 12. Cenorhabditis elegansRNAi can be induced in C. elegans in three simple ways: Injection of dsRNA into the worm gonads Soaking the worms in dsRNA solution Feeding the worms engineered bacteria producing dsRNA
  13. 13. DiscoveryInject worms with dsRNAcorresponding to a gene(important for muscle function)involved in wiggling (unc-22)
  14. 14. DiscoveryInject worms with dsRNAcorresponding to a gene(important for muscle function)involved in wiggling (unc-22)Conclusion: dsRNA triggers potent and specific gene silencing
  15. 15. RNAi vs AntisenseEffects of mex-3 RNA interference on levels of the endogenous mRNA.• Negative control showing lack of staining in the absence of the hybridization probe.• Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining).• Embryo from parent injected with purified mex-3 antisense RNA.• Embryo from a parent injected with dsRNA corresponding to mex-3. Injected antisense or dsRNA into C. elegans dsRNA was more effective than ssRNA (antisense) Effective even in tiny amounts Inactivation was due to degradation of target mRNA
  16. 16. What is the secret of this little worms success?Simple organismSimilarity to other complicated animals It has neurons,muscles, gut, …Simple life cycle Rapid growth Produce about 300 progeny in 6 days Eat bacteriaSuitable for genetic investigation
  17. 17. What is the secret of this little wormssuccess?
  18. 18. Nobel prize winners in the C. elegans field Sidney Brenner John Sulston Robert Horvitz Andrew FireCraig Mello
  19. 19. Happy ending These results were thoroughly reproducible RNAi was found to work in many species RNAi became a powerful method that is changing the face of biology MicroRNAs were discovered (the next topic) Andy and Craig win the 2006 Nobel Prize in Physiology or Medicine!
  20. 20. What happened after this paper?
  21. 21. RNAi was found to work in many diverse species Fungi Trypanosomes Insects ZebrafishMice
  22. 22. M e c h a n is m o f R N Ai RN A i (R N A In t e r f e r e n c e )
  23. 23. RNAi Overview During RNAi Double-stranded RNAs cut into short double-stranded RNAs, s(small) i(interfering) RNAs, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi
  24. 24. The Players In Interference RNA siRNA: dsRNA 21-22 nt. miRNA: ssRNA 19-25nt. Encoded by non protein coding genome RISC: RNA induced Silencing Complex, that cleaves mRNA Enzymes Dicer : produces 20-21 nt cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre miRNA; which is later processed by Dicer
  25. 25. siRNAs Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post- transcriptional gene silencing RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation A single base pair difference between the siRNA template and the target mRNA is enough to block the process. Each strand of siRNA has: a. 5’-phosphate termini b. 3’-hydroxyl termini c. 2/3-nucleotide 3’ overhangs
  26. 26. miRNA Originate from capped & polyadenylated full length precursors (pri-miRNA) Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA)
  27. 27. Difference between miRNA and siRNA Function of both species is regulation of gene expression. Difference is in where they originate. siRNA originates with dsRNA. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. miRNA originates with ssRNA that forms a hairpin secondary structure. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target. And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS) RITS
  28. 28. Dicer RNase III-like dsRNA-specific ribonuclease • Enzyme involved in the initiation of RNAi. • It is able to digest dsRNA into uniformly sized small RNAs (siRNA) Dicer family proteins are ATP- dependent nucleases. Rnase III enzyme acts as a dimer Loss of dicer→loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer is a conserved protein)
  29. 29. Dicer’s domainsDicer is a ribonuclease (Rnase III family) with 4 distinct domains: domains 1 4 2 2 31. Amino-terminal helicase domain2. Dual Rnase III motifs in the carboxy terminal segment3. dsRNA binding domain4. PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it is thought to be important for protein-protein interaction
  30. 30. RISC RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA Unwinding of double- stranded siRNA by ATP independent helicase. The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand.
  31. 31. Mechanism of RNA interference • dsRNA are chopped into short interfering RNAs (siRNA) by Dicer. siRNA Dicer 2. The siRNA-Dicer complex recruits additional components to form an RNA-Induced Silencing Complex (RISC). The siRNA unwinds. RISC 3. The unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target mRNA. 4. The target mRNA is effectively cleaved and subsequently degraded – resulting in gene silencing. silencing
  32. 32. Exogenous dsRNA is detected and bound by aneffector protein, known as RDE-4 in C. elegans andR2D2 in Drosophila, that stimulate dicer activity.Thisprotein only binds long dsRNAs.These RNA-binding proteins then facilitate transfer ofcleaved siRNA to the RISC complex.
  33. 33. Mechanism of RNA interference
  34. 34. Mechanism of RNA interference
  35. 35. Amplification of siRNA
  36. 36. Illustration of miRNA processing
  37. 37. Another View
  38. 38. Summary of Players Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) Drosha and Dicer are RNase III enzymes Pasha is a dsRNA binding protein Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran and GTP Argonautes are RNase H enzymes
  39. 39. G e n e r a t io n o f s iR N A RN A i (R N A In t e r f e r e n c e )
  40. 40. Generation of small interference RNA
  41. 41. siRNA Expression Vectors
  42. 42. siRNA design 21-23nt 2-nt 3 overhangs ( UU overhangs ) G/C content: 30-50%. No basepair mismatch Synthesised siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). BLAST : eliminate any target sequences with significant homology to other coding sequences.
  43. 43. s iR N A d e l iv e r y method s RN A i (R N A In t e r f e r e n c e )
  44. 44. Effective methods for the delivery of small RNA to allow asufficient silencing effect in the target organ(s) and/or cells areyet to be developed.In particular, toxicity and side effects of RNAi must be wellcharacterized and limited.Therefore, careful design and selection of target sequence andquantification of the effect on the expression of target protein andmRNA are essential for success of gene interfering approaches.
  45. 45. High-pressure injection “High-pressure injection” was the first strategy to demonstrate injection successful delivery of siRNA in vivo. A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs mainly the liver, kidney and to a lesser degree the lung. Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight).
  46. 46. Electroporation• Electroporation of small RNA directly into target tissues and organs has also been developed to successfully silence gene function.
  47. 47. Problem Delivery of siRNA to tissue is a problem both because: The material must reach the target organ And must also enter the cytoplasm of target cells. RNA cannot penetrate cellular membranes, so systemic delivery of siRNA is unlikely to be successful. RNA is quickly degraded by RNAse activity in serum and even siRNA chemically modified to be more stable has a half-life of only a few hours at most.
  48. 48. Solution For these reasons, other mechanisms to deliver siRNA to target cells has been devised. These methods include: Viral delivery The use of liposomes or nanoparticles Bacterial delivery Chemical modification of siRNA to improve stability
  49. 49. Viral delivery Viral delivery has been used extensively in gene therapy to deliver DNA to target cells. There are 5 main classes of viruses used in the delivery of nucleotides to cells: Retrovirus Adenovirus Lentivirus Baculovirus Adeno-associated-virus (AAV).
  50. 50. Retroviruses Retroviruses were one of the first vectors used to transduct cells with plasmids expressing hairpin-RNA constructs. Despite the relative ease of use in vitro, use of the retrovirus in vivo has safety concerns and significant limitations. Retroviruses integrate their DNA into the host.s genomic DNA, bringing with it, the risk of mutagenesis and carcinogenesis. carcinogenesis Another problem is that retroviral transduction is limited to actively dividing cells, which means that the majority of mammalian cells will not receive the siRNA.
  51. 51. Adenovirus Adenoviral vectors are commonly used in gene therapy trials. Since the adenovirus does not integrate DNA into the host.s genome, the effects are short-lived, usually lost after several cell divisions. For this reason, the adenovirus is used when a short duration of action is sufficient or desirable, such as tumor-targeting therapy. Despite the lowered risk of insertional mutagenesis, the adenovirus is associated with significant dose-dependent liver toxicity that can severely limit therapy. Another major disadvantage of adenoviral vectors is the dependence on specific surface receptors on the target cell which are often absent, rendering transduction impossible in many cases.
  52. 52. Lentivirus Lentiviral vectors are a subclass of retroviruses that lack the risk of insertional mutagenesis and are able to transducer primary and non-dividing cells. Several studies have demonstrated the use of lentiviral vectors to deliver RNAi to target cells.
  53. 53. Baculoviruses Baculoviruses are insect viruses that can carry large quantities of genetic information. This may allow their use for combined RNAi therapy and gene therapy. Safety concerns are less prominent with these vectors since the virus is unable to replicate or express proteins in mammalian cells.
  54. 54. Adeno-associated viruses (AAV) Adeno-associated viruses (AAV) are another possible vector for siRNA. These viruses do not appear to be pathogenic and can transducer non-dividing cells.
  55. 55. Liposomes and nanoparticles Liposomes and nanoparticles have been known as an alternative to viral delivery systems. Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys. Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types. Liposomes such as Lipofectamine, cationic DOTAP, neutral DOPC have been used to carry siRNA into cells. Nanoparticles such as the cationic polymer, polyethyleneimine (PEI) have also been used to successfully deliver siRNA to target cells.
  56. 56. Bacterial delivery Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a process known as transkingdom RNA interference (tkRNAi). Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom). The bacteria can also be engineered to carry shRNA encoding DNA plasmids. The advantages of this system include: Safety Ability to control the vector using antibiotics
  57. 57. Chemical modification Finally, chemical modification of siRNA has been used to improve stability and prevent degradation by serum RNAase. Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA. One of the most common modifications is the use of locked nucleic acid residues (LNA). A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect.
  58. 58. Side effects of gene silencing by smallRNA molecules Unspecific silencing Caused by the failure to identify similar sequences with only few nt difference in other genes. Activation of intracellular PKR and immune pathways that are linked to toll-like receptor activation. PKR ( protein kinase R) is activated by dsRNA longer than 30 nt, which subsequently induces the production of cytokines of the IFN family. These IFNs ultimately promote inflammatory responses.
  59. 59. Side effects of gene silencing… High pressure injection” and electroporation can cause significant injection damage to the integrity of the normal tissues and organs and thus preclude the utilisation in a clinical set-up. Liposomes/cationic encapsulated siRNA may also be toxic to the host and may cause severe host immune responses. Other emerging strategies includes chemical modification of siRNA molecules and encapsulated with different molecules are still in their infancy and need to be thoroughly investigated before used in therapeutic applications.
  60. 60. Ap p l ic a t io n s o f R N Ai RN A i (R N A In t e r f e r e n c e )
  61. 61. Therapeutic uses of RNAi Hematology (blood) Oncology (cancer) Stem cell biology Infectious diseases
  62. 62. Hematology (blood) Hematologic disorders result from Loss of gene function Mutant gene function Absent gene function RNAi May be used to create models of disease processes Could help to develop pharmacologic and genetic therapeutic targets
  63. 63. Oncology (cancer)Targeting of oncogenes Dominant mutant oncogenes, amplified oncogenes, viral oncogenesDefine role of signaling molecules in tumor-creationImprovement efficacy of chemotherapy andradiotherapyTumor regression through creation of potentially newmode of chemotherapy
  64. 64. Stem cell biology Mouse research Knock out tumor-suppression gene in mouse embryonic stem cell Observe tumor phenotype Positive correlation between extent of Trp 53 (suppression gene) inhibition and severity of disease
  65. 65. Infectious Diseases Virus targeting RNAi – inhibit cellular and viral factors of disease RNA transcriptase is RNAi target Inhibition of replicationMain goal Render cells resistant to infectious organisms
  66. 66. Hepatitis C Infects ~200 million people worldwide Often fatal 2002, Anton McCaffrey and Mark Kay at Stanford University Injected "naked" RNA strands into the tail veins of mice RNAi treatment controlled the virus in mice
  67. 67. Silencing genes in HIVAIM: Silence the main structural protein in the virus, p24, and the human protein CD4. Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infections.
  68. 68. Respiratory infections RSV, infects almost every child by the age of two 2005, Sailen Barik University of South Alabama Short strands of "naked" RNA Controlled the virus in mice Clinical trials are ongoing
  69. 69. Macular degeneration Macular degeneration is the leading cause of adult blindness Excess VEGF which leads to sprouting of excess blood vessels behind the retina & obscuring vision. The new RNAi drugs shut down genes that produce VEGF. The drug can be injected directly into the eye First clinical trial: 24 patients, launched in 2004. Two months after being injected with the drug, 6 of the patients had significantly clearer vision Other patients vision had at least stabilized More extensive trials are ongoing
  70. 70. Huntington’s diseaseIdeal candidate for RNAi therapyDisease caused by protein, thataffects more than 30,000 peoplein the U.S. alone.We would want to shut down theexpression of the gene coding forthe abberant protein2004, Beverly Davidson andcolleagues at the University ofIowaDavidson treated mice withHuntingtons
  71. 71. Other uses of RNAi Studying cell division Testing Hypotheses of Gene Function Target Validation Pathway Analysis • Gene Redundancy Functional Screening
  72. 72. Studying cell division using RNAi Genes involved in cell division identified by using RNAi RNA interference (RNAi) used to assign functions of genes involved in C. elegans cell division 133 genes identified. Only 11 previously identified
  73. 73. Testing Hypotheses of Gene Function Array analysis and other methods for identifying differentially expressed genes have created an enormous database of genes and associated phenotypes. In many cases, scientists make predictions about gene function based on expression patterns in different samples. Other predictions of mammalian gene function are developed using homology searches with genes whose functions are known in model organisms like Drosophila, C. elegans, and S. cerevisiae. In many cases, testing the accuracy of these predictions can be accomplished using siRNAs.
  74. 74. Testing Hypotheses of Gene Function:……… still workingAl-Khalili et al treated myotubes with serum and showed that increased glucose uptake correlated with increased cell-surface content of glucose transporter (GLUT1). To confirm that glucose transport depends on GLUT1 expression, cells were treated with GLUT1 siRNA and were shown to have reduced levels of serum- stimulated glucose transport.Chen and Barritt used siRNAs to study the transient receptor potential canonical 1 (TRPC1) gene. The TRPC1 gene was thought to encode a non-selective cation channel activated by depletion of cellular storage and/or an intracellular messenger. When liver cells were treated with the TRPC1 siRNA, they exhibited increased cell volume and decreased inflow of Ca2+, Mn2+.
  75. 75. Target Validation In its simplest form, drug development follows the path of target identification → target validation → therapeutic compound development → compound testing in model systems → clinical trials. siRNA are easy to use and highly specific, they provide the ultimate tool for validation studies. Reducing the expression of a potential therapeutic target and determining if the desired phenotype results provides confidence that an inhibitor of the same target gene should have therapeutic value.
  76. 76. Target Validation:… an interesting example Filleur et al showed that the antiangiogenic molecule thrombospondin-1 (TSP-1) could reduce vascularization and delay tumor onset. Over time, tumor cells producing active TSP1 began to form exponentially growing tumors. These tumors were composed of cells secreting unusually high amounts of the angiogenic stimulator, vascular endothelial growth factor (VEGF), which were sufficient to overcome the inhibitory TSP1. Treating tumor cells with a combination of TSP1 and a VEGF- specific siRNA caused a striking reduction in cell proliferation. This result suggested that using a combination of TSP1 and an anti-VEGF compound would slow or eliminate tumor growth
  77. 77. Pathway Analysis Reducing the expression of a single gene has implications on the expression and activities of genes that are in the same pathway(s). Treating cells with an siRNA targeting a given gene and then monitoring the expression of other genes using a microarray will make it possible to identify genes that are associated with the target gene. Furthermore, a specific pathway can be dissected by treating cells sequentially with siRNAs targeting the various genes in the pathway and assaying which genes are affected. This will make it possible to assign a position in the pathway for each gene.
  78. 78. Gene Redundancy In many cases, eliminating the expression of a single gene in higher eukaryotes can be tolerated even if that gene product functions in a critical pathway. This is because many critical cell functions are accomplished by more than one gene product. When one gene product is eliminated, the redundant gene product compensates to allow the cell or animal to survive. Identifying redundant genes could be achieved by co-transfecting siRNAs and assaying for a given phenotype. Evaluating each of the candidate genes alone to ensure that they only cause the cell cycle defect when reduced in combination with the target gene would help pinpoint the most likely redundant gene
  79. 79. Functional Screening Libraries of siRNAs targeting broad collections of genes will enable screening experiments to tie genes to cellular function. To date, libraries with more than a couple of hundred siRNAs have been limited to a few large research organizations. Recognizing the benefits of siRNA libraries, Ambion is preparing a collection of more than 1800 siRNAs targeting the known human kinases. There have been no published reports on the application of siRNA libraries in screening experiments, but screens in Drosophila and C. elegans using dsRNA libraries exemplify the opportunities.
  80. 80. Biotechnology & Agriculture RNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzymes production in other parts of the plant, where gossypol is important in preventing damage from plant pests.
  81. 81. Biotechnology & Agriculture… Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants. Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidants.
  82. 82. C o n c l u s io n RN A i (R N A In t e r f e r e n c e )
  83. 83. D NA RN AP r o t e in
  84. 84. A RND NA n ei ot Pr
  85. 85. Alzheimer’sAsthmaArthritisCancerHIVHepatitis C & BALSMuscular dystrophyCystic fibrosisSmallpoxSARSMacular degenerationInfluenza
  86. 86. RN A i
  87. 87. RNA interference characteristics dsRNA needs to be directed against an exon, not an intron in order to be effective Homology of the dsRNA and the target gene/mRNA is required Targeted mRNA is lost (degraded) after RNAi The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism) ssRNA does not work as well as dsRNA
  88. 88. Advantage of RNAi Downregulation of gene expression simplifies "knockout" analysis. Easier than use of antisense oligonucleotides. siRNA more effective and sensitive at lower concentration. Cost effective High Specifity middle region 9-14 are most sensitive With siRNA, the researcher can simultaneously perform experiments in any cell type of interest Can be labelled Ease of transfection by use of vector
  89. 89. Importance of RNAi Powerful for analyzing unknown genes in sequenced genomes. ⇒ efforts are being undertaken to target every human gene via siRNAs Faster identification of gene function Gene therapy: down-regulation of certain genes/ mutated alleles Cancer treatments knock-out of genes required for cell proliferation knock-out of genes encoding key structural proteins Agriculture
  90. 90. Th a n k y o u A n y q u e s t io n ?