rna interference
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RNA Interference. Team 1 [Chad, Brijesh, Shad, Niels]. 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 - PowerPoint PPT PresentationTRANSCRIPT
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From Gene To Bio Function, Fall 04 1
RNA Interference
Team 1[Chad, Brijesh, Shad, Niels]
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From Gene To Bio Function, Fall 04 2
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
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From Gene To Bio Function, Fall 04 3
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From Gene To Bio Function, Fall 04 4
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
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From Gene To Bio Function, Fall 04 5
Antisense
Ribozymes
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From Gene To Bio Function, Fall 04 6
Advances in RNAiFirst 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 21-
nucleotide 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).
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From Gene To Bio Function, Fall 04 7
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
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From Gene To Bio Function, Fall 04 8
QuickTime™ and aTIFF (Uncompressed) decompressor
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RNAi inaction
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From Gene To Bio Function, Fall 04 9
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From Gene To Bio Function, Fall 04 10
RNAi - the movie
QuickTime™ and aVideo decompressor
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From Gene To Bio Function, Fall 04 11
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
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From Gene To Bio Function, Fall 04 12
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
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From Gene To Bio Function, Fall 04 13
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
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From Gene To Bio Function, Fall 04 14
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.
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From Gene To Bio Function, Fall 04 15
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.
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Forward Genetics
• Look for rare individuals with unusual traits or phenotypes
• Then trace these traits to an underlying faulty allele or gene
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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-directed-mutagenesis or by gene knockout.
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From Gene To Bio Function, Fall 04 18
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
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From Gene To Bio Function, Fall 04 19
Knockout by RNA interference
• Relies on sequence specific interaction between siRNA and mRNA
• siRNA can be tailored to silence almost any gene
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From Gene To Bio Function, Fall 04 20
Method of gene silencingin 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
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From Gene To Bio Function, Fall 04 21
Example of success withgene 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
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Mammals
• Unfortunately, similar straight forward approaches for triggering silencing do not work in mammals.
• More advanced techniques are required.
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From Gene To Bio Function, Fall 04 23
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
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From Gene To Bio Function, Fall 04 24
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
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From Gene To Bio Function, Fall 04 25
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-of-function genetics in mammalian cells” available from pubmed
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From Gene To Bio Function, Fall 04 26
RNAi for Therapeutics
• Design of siRNA
• Specificity and Potency
• Safety profile
• Delivery Platforms and Issues
• Therapeutic examples
• How Promising is RNAi?
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From Gene To Bio Function, Fall 04 27
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
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From Gene To Bio Function, Fall 04 28
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
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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
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From Gene To Bio Function, Fall 04 30
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
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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
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From Gene To Bio Function, Fall 04 32
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
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From Gene To Bio Function, Fall 04 33
Examples, continued
• Huntington’s Disease– inhibit eGFP chimeras, reduced
aggregation
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From Gene To Bio Function, Fall 04 34
How Promising is RNAi ?
• RNAi might be used to silence dominant, gain-of-function mutations for example the neurodegenerative diseases such as:– ALS– Alzheimer’s-familial– Parkinson’s Disease-familial
• Documented gene sequence data lays a path for early-stage drug development
• Proteins/Small-molecules vs. siRNAs• $$$, economies of scale may help
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From Gene To Bio Function, Fall 04 35
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]