Transcript The RNAi mechanism

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