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RNA Interference Team 1 [Chad, Brijesh, Shad, Niels] From Gene To Bio Function, Fall 04 1 Agenda • Introduction/History of RNAi [Chad] – What is RNAi? – Antisense and Ribozyme RNA – Experimental Breakthroughs • RNAi mechanism in detail [Brijesh] – Mammalian and non mammalian cells – microRNA • RNAi as a tool for Genetics [Shad] – Reverse Genetics – Knockout – Procedures • RNAi in Therapeutics [Niels] – Specificity and Potency – Delivery problems – Design of siRNA From Gene To Bio Function, Fall 04 2 From Gene To Bio Function, Fall 04 3 What is RNAi? • Post-transcriptional Gene Silencing (PTGS) • Double stranded RNA “interferes” with mRNA selectively and silences gene expression • Science magazine’s “breakthrough of the year” for 2002 From Gene To Bio Function, Fall 04 4 Ribozymes Antisense From Gene To Bio Function, Fall 04 5 Advances in RNAi First scientific observation in plants of what is known today as RNAi 1990 Napoli C, Lemieux C, and Jorgensen R. (1990) Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279-289 dsRNA shown to be capable of gene silencing in worms 1998 Guo S, and Kempheus KJ. (1995). Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81: 611-620. Discovery of RNA-induced silencing complex (RISC) 2000 Hammond, S.M.et all (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146-1150. siRNA of 21-25 base pair length shown to induce RNAi in mammals 2001 Elbashir, S. M., Haborth, J., Lendeckel, W., Yalcin, A., Weber, K., & Tuschl, T. Duplexes of 21nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (2001). RNAi shown to reduce the activity of viruses, such as HIV and Hepatitis C 2002 Novina C. D., Murray M. F., Dykxhoorn D., Beresford P. J., Riess J., Lee S.-K., Collman R. G., Lieberman J., Shankar P., & Sharp P. A. siRNA-directed inhibition of HIV-1 infection. Nature Med. 8(7), 681-686 (2002). Sarangi F., Harris-Brandts M., Beaulieu S., & Richardson C. D. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc. Natl. Acad. Sci. 100(5), 2783-2788 (2003). From Gene To Bio Function, Fall 04 6 RNAi in non-mammalian cells • Long strand of dsRNA introduced into cell • RNase III (aka Dicer) cuts up dsRNA into siRNA (21-23 bp) • siRNA forms RISC (RNA Induced Silencing Complex) • RISC binds to target mRNA and cleaves it in half • mRNA is degraded From Gene To Bio Function, Fall 04 7 RNAi in action QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. From Gene To Bio Function, Fall 04 8 From Gene To Bio Function, Fall 04 9 RNAi - the movie QuickTime™ and a Video decompressor are needed to see this picture. From Gene To Bio Function, Fall 04 10 RNAi in mammalian cells • Long dsRNA causes interferon response – Non-specific RNA degradation by PKR kinase – Likely evolved as virus protection – Does not occur in mouse embryonic stem cells • siRNA directly introduced into cells – Most effective are 21-nt with 2 nt 3’ overhangs • shRNA (short hairpin RNA) – Used for in-vivo production of siRNA – Inserted in DNA using expression vectors – More stable than siRNA From Gene To Bio Function, Fall 04 11 Power of RNAi silencing • Endogenous natural phenomenon – Observed in plants, nematodes – May occur in mammalian cells (esp. in developmental regulation) • Amplification – RISC can degrade many mRNA molecules – siRNA get replicated (by RdRP) – Few strands of dsRNA can silence gene expression • High specificity – Even single bp mismatch dramatically reduces silencing From Gene To Bio Function, Fall 04 12 miRNA vs. siRNA • microRNA is single-stranded RNA derived from introns and “junk” DNA • Over 150 identified – E.g.: lin-4, let-7 in developmental regulation – E.g.: lsy-6 controls neuronal asymmetry in C.Elegans • Behave in very similar manner to siRNA – pri-miRNA => pre-miRNA => miRNA – Dicer, RISC activity • Involved in gene regulation - developmental timing, tissue growth, apoptosis From Gene To Bio Function, Fall 04 13 RNAi as a tool for genetics • RNA interference is a powerful tool for studying the functions of specific genes • Uses Reverse Genetics methodology • Facilitates gene knockout • Rapidly developing new methods for successfully applying RNAi in different cell types. From Gene To Bio Function, Fall 04 14 Discovering the function of a gene • Forward genetics • Reverse genetics • In both forward and reverse genetics the goal is to deduce the function of a normal gene from the effects that follow from damaging or changing it. • However, except for this basic similarity, these methods differ. From Gene To Bio Function, Fall 04 15 Forward Genetics • Look for rare individuals with unusual traits or phenotypes • Then trace these traits to an underlying faulty allele or gene From Gene To Bio Function, Fall 04 16 Reverse Genetics • Procedure is opposite of how discoveries are made in classical or forward genetics. • Because of DNA Sequencing many genes are known before their function is understood. • In reverse genetics, researchers engineer a change or disruption and then observe the effect to determine the function of the gene. • Previously this was done by site-directedmutagenesis or by gene knockout. From Gene To Bio Function, Fall 04 17 RNAi for Reverse Genetics • RNA interference can be used to perform Reverse Genetics • The interference mechanism is applied to create a specific knockout effect • This does not require the mutation of the DNA of interest • RNAi has been used to systematically interfere with the expression of most genes in a genome From Gene To Bio Function, Fall 04 18 Knockout by RNA interference • Relies on sequence specific interaction between siRNA and mRNA • siRNA can be tailored to silence almost any gene From Gene To Bio Function, Fall 04 19 Method of gene silencing in C. elegans • Genes can be silenced in C. elegans by direct feeding of bacteria that express dsRNA • Or even by soaking the worms in dsRNA • The effect can also be transmitted to the next generation From Gene To Bio Function, Fall 04 20 Example of success with gene knockout • Julie Ahringer’s group at the University of Cambridge created a library of 16,000 cloned dsRNA which is about 86% of the C. elegans genome • By feeding these clones to worms, they have determined the function of 1722 genes, most of which were previously unknown From Gene To Bio Function, Fall 04 21 Mammals • Unfortunately, similar straight forward approaches for triggering silencing do not work in mammals. • More advanced techniques are required. From Gene To Bio Function, Fall 04 22 Cell Microarrays • First described by Ziauddin and Sabatini • Cells can be grown on a glass plate and take up DNA-lipid complexes deposited on the plate before the cells From Gene To Bio Function, Fall 04 23 RNAi Microarrays • Microdots of various dsRNA are printed onto a glass slide • A culture of cells is grown on the slide over the dsRNA deposits • The dsRNA is absorbed into the cells potentially causing a knockout • The effect of this knockout can then be observed • Performed in situ From Gene To Bio Function, Fall 04 24 Use of RNAi microarray • For example, grow tumor cells on slide • See which genes can be knocked out to effect tumor growth • Paper describing this: “RNA interference microarrays: High-throughput loss-offunction genetics in mammalian cells” available from pubmed From Gene To Bio Function, Fall 04 25 RNAi for Therapeutics • • • • • • Design of siRNA Specificity and Potency Safety profile Delivery Platforms and Issues Therapeutic examples How Promising is RNAi? From Gene To Bio Function, Fall 04 26 Design of siRNA • Double-stranded or dsRNA in ”short pieces” • 21-25 base pairs long • Chemically synthesized in the lab • Modified for stability • Companies like Ambion provide programs where you simply paste in your sequence and preferred end structure From Gene To Bio Function, Fall 04 27 Specificity and Potency • Target is specific mRNA • Block protein expression implicated in disease progression • Potency 1000-fold greater than antisense • 90% reduction in target mRNA levels with nanomolar or even picomolar amts of siRNA From Gene To Bio Function, Fall 04 28 Safety Profile • siRNA are recognized intracellularly and are free to disable mRNA • siRNA mimic a natural process thereby avoiding the toxicity associated with foreign molecules • Long term effects of triggering the RNAi pathway are unknown From Gene To Bio Function, Fall 04 29 Delivery Platforms • Current Delivery platforms: Lipid/Polymer formulations Viral delivery (e.g. Retroviruses) • • siRNAs in cationic lipids pass through cell membranes pDNA vectors, viruses can deliver genes encoding for siRNAs From Gene To Bio Function, Fall 04 30 Delivery Issues • Lipids work well in cultured cells , but who wants to inject them into their bloodstream? • Retroviruses, analogous to gene therapy, could change genome, cause cancer From Gene To Bio Function, Fall 04 31 Therapeutic Examples • AMD – Acuity Pharmaceuticals files IND for CanD for the regulation of VEGF in 08/2004 – Sirna Therapeutics files IND for Sima-027for the regulation of VEGF in 09/2004 • HIV – silence the expression of CD4 receptor – CCR5 may be more promising to allow normal immune response From Gene To Bio Function, Fall 04 32 Examples, continued • Huntington’s Disease – inhibit eGFP chimeras, reduced aggregation From Gene To Bio Function, Fall 04 33 How Promising is RNAi ? • RNAi might be used to silence dominant, gain-offunction mutations for example the neurodegenerative diseases such as: – ALS – Alzheimer’s-familial – Parkinson’s Disease-familial • Documented gene sequence data lays a path for earlystage drug development • Proteins/Small-molecules vs. siRNAs • $$$, economies of scale may help From Gene To Bio Function, Fall 04 34 References • www.ambion.com, www.alnylam.com, www.sirna.com • www.rnai.net • Intron derived microRNAs - fine tuning of gene functions [Ying, Lin 04] • RNA interference microarrays: High-throughput loss-of-function genetics in mammalian cells • RNAi Therapeutics: How likely, how soon? [Robinson 04] From Gene To Bio Function, Fall 04 35