RNA interference (RNAi):A therapeutic strategy for aquaculture

RNA interference (RNAi):A therapeutic strategy for aquaculture

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  RNA interference (RNAi): Atherapeutic strategy for aquaculture Presented By – MD. IDRISH RAJA KHAN M.F.Sc. (AAH – 01/15) COF, CAU (I), TRIPURA Master’s seminar onMaster’s seminar on
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  INTRODUCTIONINTRODUCTION • RNAi, anatural mechanism for post-transcriptional silencing of homologous genes by dsRNA resulting in inhibition of translation (Fire et al., 1998). • In 1998, Andrew Fire and Craig Mello were the first to reveal the induction of RNAi in the nematode worm Caenorhabditis elegans. • Later on for the year 2006 they were awarded by Nobel Prize in the field of Physiology or Medicine. • RNAi, a natural mechanism for post-transcriptional silencing of homologous genes by dsRNA resulting in inhibition of translation (Fire et al., 1998). • In 1998, Andrew Fire and Craig Mello were the first to reveal the induction of RNAi in the nematode worm Caenorhabditis elegans. • Later on for the year 2006 they were awarded by Nobel Prize in the field of Physiology or Medicine.
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  How RNAi Works?How RNAi Works ? Two-step mechanism for RNAi: A. INITIATION STEP 1. The entry of long dSRNA, triggers the RNAi pathway of cells. This results in the activation of the enzyme Dicer. 2. Dicer cleaves the dsRNA into short, 20-21 basepairs long, fragments, called small interfering RNA (siRNA). These small molecules have a characteristic 2 nucleotide 3’ overhang. Two-step mechanism for RNAi: A. INITIATION STEP 1. The entry of long dSRNA, triggers the RNAi pathway of cells. This results in the activation of the enzyme Dicer. 2. Dicer cleaves the dsRNA into short, 20-21 basepairs long, fragments, called small interfering RNA (siRNA). These small molecules have a characteristic 2 nucleotide 3’ overhang.
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  B. EFFECTOR STAGE 1.Short RNA duplexes are incorporated into a multimeric protein complex, known as RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) protein as one of its main components. 2. RISC binds and unwinds the siRNAs into single- stranded molecules. The sense strand is released and the antisense strand remains bound to RISC, serving as guide to select fully complementary mRNA (seed) substrates for degradation. B. EFFECTOR STAGE 1. Short RNA duplexes are incorporated into a multimeric protein complex, known as RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) protein as one of its main components. 2. RISC binds and unwinds the siRNAs into single- stranded molecules. The sense strand is released and the antisense strand remains bound to RISC, serving as guide to select fully complementary mRNA (seed) substrates for degradation.
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  Fig. RNA interferencemechanism Source – www.nature.com
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  DEVELOPING THERAPEUTIC RNAiDEVELOPINGTHERAPEUTIC RNAi There are several stages in developing a RNAi based therapy for viral diseases – 1. Target gene 2. Target sequence 3. Effective design 4. Therapeutic molecules - siRNA, miRNA 5. Delivery mode Immersion, feeding, hydrodynamic i.v. injection, electroporation, cholesterol conjugates, cationic delivery systems, nano delivery systems etc. There are several stages in developing a RNAi based therapy for viral diseases – 1. Target gene 2. Target sequence 3. Effective design 4. Therapeutic molecules - siRNA, miRNA 5. Delivery mode Immersion, feeding, hydrodynamic i.v. injection, electroporation, cholesterol conjugates, cationic delivery systems, nano delivery systems etc.
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  SOME SUCCESSFULL IMPLEMENTATIONOF RNAi IN AQUACULTURE SOME SUCCESSFULL IMPLEMENTATION OF RNAi IN AQUACULTURE Shrimps Gene construct Protection Studies against Gene References Penaeus monodon siRNA WSSV Vp28, vp15 Westenberg et al. (2005) Litopenaeus vannamei siRNA WSSV DNA pol, Rr2, ORF252, vp28 Robalino et al. (2005) P. monodon siRNA YHV Protease Yodmuang et al. (2006) L. vannamei siRNA TSV DNA pol, Rr2, Wu et al. (2007) P. chinensis siRNA WSSV vp28, vp281, Protein kinase Kim et al. (2007) Metapenaeus japonicus siRNA WSSV vp28 Xu et al. (2007)
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  A case studyof RNAi useA case study of RNAi use • Place - Netherland • Organism – Shrimp (P. monodon) • Disease – WSD • Causative agent – WSSV • Gene construct – siRNA (vp28, vp15, gfp) • Delivery – Intramuscular injection (40µl of 100µM siRNA duplexes with buffer) • Result – About 40 – 45% reduction in the mortality where as control is having about 55 - 60%. • Place - Netherland • Organism – Shrimp (P. monodon) • Disease – WSD • Causative agent – WSSV • Gene construct – siRNA (vp28, vp15, gfp) • Delivery – Intramuscular injection (40µl of 100µM siRNA duplexes with buffer) • Result – About 40 – 45% reduction in the mortality where as control is having about 55 - 60%.
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  APPLICATIONAPPLICATION • Antiviral mechanism •‘Neo-females’ production • Secures genome stability by keeping mobile elements silent • Offers a new experimental tool to repress genes specifically • GMO • Investigate the functional role of specific genes • Shelf life and bio-fortification • Elimination of allergens and toxic substances • Antiviral mechanism • ‘Neo-females’ production • Secures genome stability by keeping mobile elements silent • Offers a new experimental tool to repress genes specifically • GMO • Investigate the functional role of specific genes • Shelf life and bio-fortification • Elimination of allergens and toxic substances
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  LIMITATIONSLIMITATIONS • Most importantlyeffective delivery in vivo • Off-target effects • Triggering • Innate immune responses • Excessive levels of exogenous RNAs can saturate one or more components of the endogenous RNAi pathways, causing potential hazard • Some viruses to escape RNAi mediated • Most importantly effective delivery in vivo • Off-target effects • Triggering • Innate immune responses • Excessive levels of exogenous RNAs can saturate one or more components of the endogenous RNAi pathways, causing potential hazard • Some viruses to escape RNAi mediated
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  CONCLUSIONCONCLUSION • One ofthe most lucrative methodology of the modern era but still has lot to understand in terms of practical utility. • Further study needs to be done in the field of aquaculture practices. • One of the most lucrative methodology of the modern era but still has lot to understand in terms of practical utility. • Further study needs to be done in the field of aquaculture practices.
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  •Fire, A., Xu,S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391: 806–811. •Kim, C. S., Kosuke, Z., Nam, Y. K., Kim, S. K. and Kim, K. H., 2007. Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double stranded RNA. Fish Shellfish Immunol., 23:242–246. •Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S. and Rossi, J. J., 2004. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol., 23: 222–226. •Robalino, J., Browdy, C. L., Prior, S., Metz ,A., Parnel, P. and Warr, G., 2004. Induction of antiviral immunity by double stranded RNA in a marine invertebrate. J Virol., 78: 10442– 10448. •Wu, Y., Lu, L., Yang, L., Weng, S. and Chan, j., 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence specific siRNA. Aquaculture, 271: 21–30. •Westenberg, M., Heinhuis, B., Zuidema, D. and Vlak, J. M., 2005. siRNA injection induces sequence independent protection in Penaeus monodon against white spot syndrome virus (WSSV). Virus Res., 114: 133–139. •Xu, J., Han, F. and Zhang, X., 2007. Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA. Antiviral Res., 73: 126–131. •Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W. and Panyim, S., 2006. YHVprotease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality. Biochem Biophys Res. Commun., 341: 351–356. •Http://www.nature.com/nrg/rnai.html [Accessed on 9 December 2016] •Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391: 806–811. •Kim, C. S., Kosuke, Z., Nam, Y. K., Kim, S. K. and Kim, K. H., 2007. Protection of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV) challenge by double stranded RNA. Fish Shellfish Immunol., 23:242–246. •Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S. and Rossi, J. J., 2004. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol., 23: 222–226. •Robalino, J., Browdy, C. L., Prior, S., Metz ,A., Parnel, P. and Warr, G., 2004. Induction of antiviral immunity by double stranded RNA in a marine invertebrate. J Virol., 78: 10442– 10448. •Wu, Y., Lu, L., Yang, L., Weng, S. and Chan, j., 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequence specific siRNA. Aquaculture, 271: 21–30. •Westenberg, M., Heinhuis, B., Zuidema, D. and Vlak, J. M., 2005. siRNA injection induces sequence independent protection in Penaeus monodon against white spot syndrome virus (WSSV). Virus Res., 114: 133–139. •Xu, J., Han, F. and Zhang, X., 2007. Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA. Antiviral Res., 73: 126–131. •Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W. and Panyim, S., 2006. YHVprotease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality. Biochem Biophys Res. Commun., 341: 351–356. •Http://www.nature.com/nrg/rnai.html [Accessed on 9 December 2016]
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  Heros of CRISPR GeorgeChurchJennifer Doudna Feng ZhangEmmanuelle Charpentier Thank you for attention But big THANK YOU TO..