1. From Gene To Bio Function, Fall 041 RNA Interference Team 1 [Chad, Brijesh, Shad, Niels]

2. From Gene To Bio Function, Fall 042 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

3. From Gene To Bio Function, Fall 043

4. 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

5. From Gene To Bio Function, Fall 045 Antisense Ribozymes

6. From Gene To Bio Function, Fall 046 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 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).

7. From Gene To Bio Function, Fall 047 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

8. From Gene To Bio Function, Fall 048 RNAi in action

9. From Gene To Bio Function, Fall 049

10. 10 RNAi - the movie

11. From Gene To Bio Function, Fall 0411 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

12. From Gene To Bio Function, Fall 0412 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

13. From Gene To Bio Function, Fall 0413 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

14. From Gene To Bio Function, Fall 0414 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.

15. From Gene To Bio Function, Fall 0415 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.

16. From Gene To Bio Function, Fall 0416 Forward Genetics Look for rare individuals with unusual traits or phenotypes Then trace these traits to an underlying faulty allele or gene

17. From Gene To Bio Function, Fall 0417 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.

18. From Gene To Bio Function, Fall 0418 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

19. From Gene To Bio Function, Fall 0419 Knockout by RNA interference Relies on sequence specific interaction between siRNA and mRNA siRNA can be tailored to silence almost any gene

20. From Gene To Bio Function, Fall 0420 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

21. From Gene To Bio Function, Fall 0421 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

22. From Gene To Bio Function, Fall 0422 Mammals Unfortunately, similar straight forward approaches for triggering silencing do not work in mammals. More advanced techniques are required.

23. From Gene To Bio Function, Fall 0423 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

24. From Gene To Bio Function, Fall 0424 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

25. From Gene To Bio Function, Fall 0425 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

26. From Gene To Bio Function, Fall 0426 RNAi for Therapeutics Design of siRNA Specificity and Potency Safety profile Delivery Platforms and Issues Therapeutic examples How Promising is RNAi?

27. From Gene To Bio Function, Fall 0427 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

28. From Gene To Bio Function, Fall 0428 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

29. From Gene To Bio Function, Fall 0429 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

30. From Gene To Bio Function, Fall 0430 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

31. From Gene To Bio Function, Fall 0431 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

32. From Gene To Bio Function, Fall 0432 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

33. From Gene To Bio Function, Fall 0433 Examples, continued Huntington’s Disease –inhibit eGFP chimeras, reduced aggregation

34. From Gene To Bio Function, Fall 0434 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

35. From Gene To Bio Function, Fall 0435 References www.ambion.com, www.alnylam.com, www.sirna.comwww.ambion.comwww.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]