From Gene To Bio Function, Fall 04 1

RNA Interference

Team 1[Chad, Brijesh, Shad, Niels]

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

From Gene To Bio Function, Fall 04 3

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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Antisense

Ribozymes

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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).

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

From Gene To Bio Function, Fall 04 8

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RNAi inaction

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RNAi - the movie

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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 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

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

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.

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.

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

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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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

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

From Gene To Bio Function, Fall 04 22

Mammals

• Unfortunately, similar straight forward approaches for triggering silencing do not work in mammals.

• More advanced techniques are required.

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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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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 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

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?

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

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

From Gene To Bio Function, Fall 04 29

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 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

From Gene To Bio Function, Fall 04 31

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 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

From Gene To Bio Function, Fall 04 33

Examples, continued

• Huntington’s Disease– inhibit eGFP chimeras, reduced

aggregation

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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

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]