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RNA interference: new tool, ancient immune system

Bergen lecture – April 21st 2005

Torgeir Holen, CMBN

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Aims of this talkPart I: To give a short introduction to the newly discovered immune system RNA interference (RNAi) in various speciesPart II: To look at some practical limitations to knocking down mRNA gene expression by RNAi & siRNA, and some recent solutions...Part III: siRNA is already old hat – what’s going on now...? short RNA : DNA/chromatin interactions...

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Part I: Gene silencing exist in many multicellular organisms

PetuniasTransgenic Petunias

roundworm C. elegans

fungus Neurospora

Transgenic potatoes resist virus infection

fruitfly Drosophila

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Gene silencing go back to the earliest epochs of life on Earth This ancient immune

system seems conserved over 1.5 billion years

...thus we can learn about different aspects of gene and RNA silencing wherever it is easiest... plants... roundworms... flies...

human cell lines...

Wang, Kumar & Hedges, 1999

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A brief history of RNA silencing

1998: Andrew Fire and colleagues show that double-stranded RNA induce gene silencing in C. elegans

• 1999: Hamilton & Baulcombe find small RNA where genes are silenced in plants

• 2001: Thomas Tuschl and colleagues show that synthetic small RNA introduced into human cells can silence human genes

• 2000: Greg Hannon and colleagues isolate Dicer, the siRNA producing RNase-III enzyme in Drosophila

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Plasmid produced siRNA

•Brummelkamp, 2002; Paddision, 2002; Miyahishi, 2002; Paul, 2002; Sui, 2002; Lee, 2002; Jacque, 2002; Yu, 2002; Yang, 2002

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The PRK/interferon response immune-system virus-response system in

mammalian cells activated by long dsRNA mechanism: shuts down all translation by by

phosphorylating eIF2-alpha (see Williams, Oncogene, 1999)

the siRNA break-through of Thomas Tuschl consisted of the PKR/interferon response not being activated (Elbashir et al, Nature, 2001; also shown by Natasha Caplen, PNAS, 2001)

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1. siRNA exhibit position effects – some target positions superior to others

2. siRNA activity fades out – in our case 3-4 days after transfection

3. siRNA activity can be blocked by competition with less active siRNA

4. Some siRNA have great tolerance for chemical modifications

5. Some siRNA tolerate mutations

6. Main problem of RNAi-field today: delivery, in vitro & in vivo

Part II: Limits to siRNA activity - some other limitations to these great, but very new, tools

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pTRE pTRELUC (37-2255)... ...

TF (47-951)

Fusion Reporter Construct

Finding good target positions:

TF-luciferase-reporter assay- Holen et al (Nucleic Acids Research, 2002).

- Tissue Factor (TF), is the principal coagulation trigger and has been implicated in cardiac diseases. TF is also involved in angiogenesis and metastasis of cancer.

pTRE pTRELUC (37-2255)... ...

Fen1/Aqp4/GS

Fen1, Aqp4 and GlnS Constructs

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Why the position effects?

hTF478i

HaCaT

Cos-1

HeLa

293

PSK314ihTF167ihTF372i

hTF562ihTF478i

PSK314ihTF167ihTF372i

hTF562i

PSK314ihTF167ihTF372i

hTF562ihTF478i

PSK314i

hTF167i

hTF372i

hTF562ihTF478i

PSK546iPSK566iPSK739i

20 40 60 10080 120 140

hTF929i

hTF77i

hTF256i

hTF459i

Normalised TF-LUC/RLUC

mRNA AGGUGGCCGGCGCUUCAGGCACUACAAAUACUGUGGC

hTF158i AGGUGGCCGGCGCUUCAGGTT

hTF161i UGGCCGGCGCUUCAGGCACTT

hTF164i CCGGCGCUUCAGGCACUACTT

hTF167i GCGCUUCAGGCACUACAAATT

hTF170i CUUCAGGCACUACAAAUACTT

hTF173i CAGGCACUACAAAUACUGUTT

hTF176i GCACUACAAAUACUGUGGCTT

Nor

mal

ised

TF-

LUC

/RLU

C

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Knock-downs must be verified...- position effect consistent also in Northern assays, protein assays and coagulation assays…

TF

GAPDH

moc

k

hTF3

72i

hTF1

67i

hTF5

62i

PS

K31

4i

PS

K54

6i

PS

K56

6i

PS

K73

9i

hTF4

78i

Northerns

moc

k

TF

GAPDH

hTF1

58i

hTF1

61i

hTF1

64i

hTF1

67i

hTF1

70i

hTF1

73i

hTF1

76i N

orm

alis

ed T

F ex

pres

sion

Legend: mRNA (filled bars), procoagulant activity (dotted bars) and TF protein (hatched bars)

Protein & Coagulation

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Some recent computational solutions to position effects:

Khvorova, Cell, 2003: 5’-end of active antisense unstable...

Ui-Tei et al, Nucleic Acids Research, 2004: (i) A/U at the 5' end of the antisense strand (ii) G/C at the 5' end of the sense strand (iii) at least five A/U residues in the 5' terminal one-third of the

antisense strand (iv) the absence of any GC stretch of more than 9 nt in length.

siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells

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RNAi silencing sets in slowly…

Nor

mal

ised

TF/

GA

PD

H m

RN

A

Measurements at 4 h, 8 h, 24 h, 48 h

Nor

mal

ised

TF

expr

essi

on…and fades out even more slowly, but steadily…

…why?

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Tolerance for chemical modifications

5’-g*c-gcuucaggcacuaca-a-a-u*a-3’ 3’-g*c-c-g-cgaaguccgugaugu-u*u-5’ 5’-g-c-gcuucaggcacuaca-a-a*u*a-3’ 3’-g*c*c-g-cgaaguccgugaugu-u-u-5’

5’-GcgcuucaggcacuacaaauA-3’ 3’-GccgcgaaguccgugauguuU-5’ 5’-gcgcuucaggcacuacaaaUA-3’ 3’-GCcgcgaaguccgugauguuu-5’

5’-GcgcuucaggcacuacaaauA-3’ 3’-GccgcgaaguccgugauguuU-5’ 5’-gcgcuucaggcacuacaaaUA-3’ 3’-GCcgcgaaguccgugauguuu-5’

5’-g*c*gcuucaggcacuaca-a-a*u*a-3’ 3’-g*c*c-g-cgaaguccgugaugu*u*u-5’ 5’-g*c*gcuucaggcacuaca*a*a*u*a-3’ 3’-g*c*c*g*cgaaguccgugaugu*u*u-5’

5’-GCgcuucaggcacuacaaaUA-3’ 3’-GCcgcgaaguccgugauguUU-5’ 5’-GCgcuucaggcacuacaAAUA-3’ 3’-GCCGcgaaguccgugauguUU-5’

P1+1

P0+2

M1+1

M0+2

A1+1

A0+2

P2+2

P2+4

M2+2

M2+4

Nor

mal

ised

TF/

GA

PD

H m

RN

A

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Inhibition of cholesterol synthesis by siRNA: A recent study by Alnylam Inc

"...Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. "

Soutschek et al, Nature, 2004

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An siRNA mutation studyN

orm

alis

ed T

F/G

AP

DH

mR

NA

**** * ** * *5'-GCGCUUCAGGCACUACAAAUA GCCGCGAAGUCCGUGAUGUUU-5'

Amarzguioui M, Holen T, Babaie E, Prydz H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 2003 Jan 15;31(2):589-95.

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Other genes possibly targeted by laminB2CLSTN25'-AAGAGGAGGAAGAAGCCGAGG-3' |||||||||| ||||||||| 3'-UUCUCCUCCUCCUUCGGCUCA-5'

HS6ST35'-AAGAGGAGGAGGAAGACGAGC-3' ||||||||||||||| |||| 3'-UUCUCCUCCUCCUUCGGCUCA-5'

NM_0158975'-AAGAGGAGGAGGAAGACGAGG-3' ||||||||||||||| |||| 3'-UUCUCCUCCUCCUUCGGCUCA-5'

NM_0153555'-ACUCGGCCUCCUCCUCCUCCU-3' ||||||| ||||||||||| |3'-UGAGCCGAAGGAGGAGGAGAA-5'

ATBF15'-AAGAGGAGGAGGAAGACGAGG-3' ||||||||||||||| |||| 3'-UUCUCCUCCUCCUUCGGCUCA-5'

SPTB5'-AAGAGGAGGAGGAAACAGAGU-3' |||||||||||||| | ||||3'-UUCUCCUCCUCCUUCGGCUCA-5'

The dangers of siRNA in functional genomics:“…the two other lamins, B1 and B2, are now identified as essential proteins” (Harborth et al, JCS, 2001)

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0

20

40

60

80

100

120

140

as-16

7

hTF16

7i

as-P

SK411

as-16

7

hTF16

7i

as-P

SK411

as-16

7

hTF16

7i

as-P

SK411

as-16

7

hTF16

7i

as-P

SK411

The delivery problem - comparing different transfection agents Lipofectamine2000, Lipofectamine, Oligofectamine and RNAifect…

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Indirect delivery in vivo- Tissue Factor in cancer metastasis

set-up: metastatic B16 cells transfected with siRNA against TF, then injected into tail-vein of mice

tumors will then colonize lungs of mouse

mTF

GAP

mTF223i hTF167imock

D

hTF167i

mTF223i

Day 10 Day 15

mTF223i

mTF321i

hTF167i

Day 20

Mohammed Amarzguioui, Qian Peng, Torgeir Holen, Vlada Vasovic, Eshrat Babaie, Jahn M. Nesland & Hans Prydz

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RNAi delivery by virus Lentiviruses delivery especially interesting for

delivery to neuronal cells. An et al, Human Gene Theraphy, 2003 Stewart et al, RNA, 2003 Paddison PJ, Silva JM, Conklin DS, Schlabach M, Li

M, Aruleba S, Balija V, O'Shaughnessy A, Gnoj L, Scobie K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, Hannon GJ. A resource for large-scale RNA-interference-based screens in mammals.Nature. 2004 Mar 25;428(6981):427-31.

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Transgenic RNAi: mice expressing siRNA

Carmell, Rosenquist et al, Nature Structural & Molecular Biology, 2003

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Regulatable knock-downs

From Matsukura et al (Nucleic Acids Research, 2003)

What of it? Can’t the Cre/LoxP system or other conditional knock-outs do the same?

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1) RNA silencing, more than siRNA2) DNA & chromatin regulation: still

unsolved...3) Three cases of RNA-DNA silencing link

a) dsRNA-induced DNA methylation in plants b) transgene DNA silencing in Drosophilac) centromere silencing in budding yeast (S.

pombe)d) X-chromatin silencing in humans...e) recently: chromatin silencing induced by short

RNA in human cells

Part III: The possible link between RNA and DNA silencing in higher organisms

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• microRNA comes from microGenes, transcripted as ~70 nt hairpins, and processed (as is shRNA) to short ~21-mer RNAs...

• latest research hotspot: heterochromatic siRNA might affect centromeres in budding yeast (S.pombe) (Reinhart, 2002; Volpe, 2002)

More small RNA exist• stRNA (short temporal RNA, also called micro-RNA,

miRNA) inhibit mRNA translation (Lee & Ambros, 1993; Reinhart, 2000; Lee, 2001; Lau, 2001; Lagos-Quintana, 2001; Llave, 2002; Lim, 2003)

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PTSVd viroids (Wassenegger, Cell, 1994)

• PTSVd can lead to methylation of a target down to 30 bp of DNA (Pelissier & Wassenegger, RNA, 2000)

Double-stranded RNA cause silencing in plants

Lehninger, 1993

HOW???

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X-chromosome inactivation centre (Xic)

Xic (450 kb region) can be transferred to chromosome 12, resulting in gene silencing, hypoacetylation, histone methylation (His3mLys9), delayed DNA replacation and RNA coating... (Lee & Jaenisch, Nature, 1997)

Lee, Cell, 2000

Lewin, 2000

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Is the proposed mechanism right?

Nobody knows... yet

...and does it exist in higher organisms?

Summary: What is the mechanism?

Allshire, Science, 2002

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Two recent papers find chromatin modifications in mammalian cells

Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science. 2004 Aug 27;305(5688):1289-92. Epub 2004 Aug 05.

Kawasaki & Taira. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature. 2004 Sep 9;431(7005):211-7. Epub 2004 Aug 15.