Transcript Slide 1

Emergence and Applications of RNA Interference
Omar Memon, Vandana Sekhar, Varnika Roy, Yizhou Yin, Alison Heffer
University of Maryland, College Park
APPLICATIONS
HISTORY
More than a decade ago, a surprising observation was made in petunias. While
trying to deepen the purple color of these flowers, Rich Jorgensen and colleagues
introduced a pigment-producing gene under the control of a powerful promoter.
Instead of the expected deep purple color, many of the flowers appeared
variegated or even white. This phenomenon was considered to be posttranscriptional gene silencing (PTGS), since the expression of both the introduced
gene and the homologous endogenous gene was suppressed.
Years later, experiments in Caenorhabditis elegans by Andrew Fire and Craig
Mello revealed that injection of either “sense” or “anti-sense” mRNA molecules
encoding muscle protein, led to no behavioral changes in the worms. But when
they injected sense and antisense RNA together, they observed that the worms
displayed peculiar, twitching movements. Similar movements were seen in worms
that completely lacked a functioning gene for the muscle protein.
CRITIQUES
Functional Genomics
RNAi Advantages
Different from classical forward genetics, RNAi is a very powerful technique to investigate gene function in
the reverse genetics way. Because of its convenience, high efficiency and economy, it is ideal for analyzing
the functions of large numbers of genes and whole genome-wide screens. Based on the completion of
sequencing of several organisms and the development of techniques such as cell microarrays, highthroughput RNAi screen is an invaluable tool for functional genomics in a wide range of different species.
Use of RNAi in genome-wide screening
Step 1 Choose organisms
or cell lines
Step 2 Choose RNAi reagents: Long
dsRNA, synthetic siRNA, plasmid or
viruses based shRNA
Step 3 Screening with some
specific paradigm and format
Finding potential therapy
targets
 Easy study of gene
function
 Elucidating cellular
mechanisms
 Analyze many genes at
once; RNAi libraries
 Knock-downs can show a
variety of phenotypes
 More specific than most
other therapies
 Can be used to target
many cell and tissue types
 Efficiently transfers the
gene to its target

Functional
genomics
Therapeutics
N.benthamiana C.elegans D.melagonaster
Arabidopsis
Mouse
Human
Step 4 Read out and analyze results,
microarray can be imaged or stained
Large-scale RNAi screens
have been done:
Fire and Mello tested the hypothesis that injection of sense and antisense RNA
molecules resulted in the formation of double-stranded RNA (dsRNA). In every
experiment, injection of double-stranded RNA carrying a genetic code led to
silencing of the gene containing that particular code. From this, they deduced
that dsRNA can silence genes and that this RNA interference is specific for the
gene whose code matches that of the injected RNA molecule, and that RNA
interference can spread between cells and even be inherited.
•About 90% genes on
C.elegans chromosome III for
several basic cellular
processes,
•Screen on C.elegans
chromosome I for embryonic
lethal genes,
Fire and Mello published their findings in the journal Nature on February 19, 1998.
Their discovery clarified many confusing and contradictory experimental
observations and revealed a natural mechanism for controlling the flow of genetic
information. This research awarded them a Nobel prize and heralded the start of a
new research field.
•Functional screen for RNAi
itself in C.elegans
Meanwhile, high-throughput
screens and RNAi libraries
have proved to be very useful
to therapeutic research
MECHANISM
1. Introduction of ds RNA in the
cell by viral infection or by
artificial means using vectors
based short hairpin RNA
(shRNA)
ds RNA virus
1
shRNA
2
3. Duplexes of siRNA of 21-24
nucleotides length formed by
Dicer
DICER
3
4
miRNA
ATP
ADP + Pi
5
RISC
ds DNA
6
ATP
ADP + Pi
RISC
activation
7
Target
mRNA
8
Degraded
target
mRNA
2. Recognition and processing of
long dsRNA by Dicer, an RNase
III enzyme
4. miRNA are naturally
synthesized long ds RNA in the
nucleus, which are processed
by Drosha enzyme into small
pre-miRNA and exported to
cytoplasm.
Disease Therapy
Wide therapeutic applications of siRNA are the new sensation in the biotechnology drug world. Major
traditional drug targets have been proteins (enzymes and receptors), which are targeted at the post
translational level. But siRNA drug selectively silences a disease causing gene, at the post transcriptional
level itself. Side-effects are decreased by targeting a disease inducing gene in which genetic
polymorphisms distinguish it from the RNA of wild type alleles.
Unlike the antisense approach, dsRNA employs a normal cellular process thus it is more specific and
allows a cell-cell spreading of the gene silencing effect. The knockdown of the target gene by RNAi is
heritable and stable.
Taking RNAi from Bench to Bedside- First Trial Treating Age Related Macular Degeneration
DELIVERY OF DRUG
Local intravitreal injection
of siRNA (100-800µg) per
eye diluted in phosphate
buffer saline)
7. Recruitment of RISC along with
antisense strand to target
mRNA
8. Cleavage of target mRNA by an
unidentified RNase (Slicer)
within RISC. Degrades mRNA
at sites not bound by siRNA
siRNA
duplex
RISC
activation
VEGF target
recognized
PHENOTYPIC EFFECT
The biggest problem with
the use of RNAi is its
successful delivery to the
target.
RNAi must be
stable in a cell for
prolonged activity without
getting degraded. Nonspecific interactions can
occur because, though
siRNA can be designed to
target a specific sequence,
a difference in one or two
base-pairs is sufficient to
cause off-target binding.
Table 2. Different delivery methods of RNAi and the
advantages and disadvantages of each
Knock-downs do not
completely inhibit gene
expression or activity
 Number of genes targeted
at one time limited; RNAi
overload
 RNAi doesn’t always work

Difficult to deliver siRNA to
the target
 Side effect of activating the
interferon response (IR)
 siRNA stability is a
concern for effective
therapy
 Concern that the siRNA
might interfere with natural
RNAi mechanisms in the
cell
 May bind non-specifically
to some tissues

Delivery
system
The research conducted
over past few years has
shown
the
promising
potential of RNAi.
This
powerful genetic tool has
been used to a certain
level of success in both
proteomics
and
drug
therapy. Though both will
continue to be active fields
of scientific research, the
drawbacks must also be
considered (Table 1).
Table 1. Advantages and disadvantages of
using RNAi in two applications
Major
characteristics
Promising results in
cancer therapy through
in vitro cell studies

Related to retroviruses;
eliminate some
disadvantages
 Effective in targeting
genes in the brains of
Alzheimer’s patients


Retrovirus

Viral
Lentivirus
Vector based on adenoassociated viruses
 Can target tumors
 For transient
expression
 Balances stability of
siRNA without
influencing RNAi
mechanisms
 Modifications: locked
nucleic acids (LNA),
phosphothioates (PS),
2’ modifications to
ribose
 siRNA packaged into an
envelope with a signal
for target cells

Adenovirus
Chemically
modified
siRNA
Nonviral
Liposomes

Naked siRNA
Advantages
siRNA is injected
directly into the
organism

Very efficient
Genes are passed on
during mitosis
Applies to non-dividing
cells
 Virus can be specific for
recognizing one type of
tissue
 Good for carrying larger
genes
 Small risk of host
genome integration
since replication occurs
outside of the nucleus
Stability of siRNA is
increased
 More specific targeting




Easy to obtain
Targets many different
types of organs
Accomplished with
relatively little work
Disadvantages
Gene is integrated into
the genome; risk of
mutagenesis
 Only for dividing cells
 Can only target specific
areas; no systemic
applications
 Risk of mutagenesis

Since the DNA is
outside of the nucleus,
it is less stable and can
be lost after many cell
divisions
 No in vivo studies on
many modified RNAs
 5’ modifications might
interfere with silencing
 Bulky modifications
may hinder RNA
unwinding

Non-specific targeting
Liposome electric
charge on may interfere
with tissue uptake
 Encounters RNAses in
serum
 Delivery to non-specific
sites


SUMMARY
RNAi is a powerful and attractive genetic approach because of the diversity of its
applications. The potential uses currently in progress include the identification of
specific gene functions in living systems and creation of genome wide screens.
Development of antiviral and anticancer therapies are broadening the horizons of
the therapeutic arena.
Another value of RNAi screens is in combining it with other functional genomic
assays enabling mapping of biochemical pathways. Impact of RNAi is also being
extended to the field of agriculture for example by increasing disease resistance in
plants.
Many potential obstacles in the path of RNAi therapeutics can be overcome, but
further insight into the non-coding functions of RNA in vivo will provide better
understanding of mechanisms underlying RNAi. Future applications of RNAi
technology will revolutionize genetic, genomic and proteomic aspects of biology
and will take the field of medicine into new scientific realms.
Target
cleaved
SIRNA-027 Target = VEGFR-1
REFERENCES
5. Incorporation of both
synthetic siRNA or
endogenously expressed
miRNA into RNA-induced
silencing complex(RISC)
6. Unwinding of duplex siRNA by
a helicase in RISC and removal
of passenger strand (RISC
activation)
IN VIVO MECHANISM
RNAi Disadvantages
Ocular angiogenesis
Reduced
Dose dependent
Improvement of Vision
Common RNAi Targeted Diseases
ONCOGENESIS
•siRNA drugs directly target
cancer promoting genes
•Chemotherapeutic avoidance of
tumors is decreased by targeting
clusterin (antiapoptotic gene).
•Ex:-Imatinib drug for Philadelphia
chromosome target BCR-ABL fusion
protein causes chronic myelogenous
leukemia
NEURODEGENERATIVE
DISEASES
•RNAi is an important process
in normal neuronal function
•Its manipulation is important
for treating many untreatable
neurological disorders
• Ex:-Mouse models for Alzheimer's
disease, DYT1 dystonia, and
polyglutamine disease in progress
VIRAL DISEASES
•Targets are viral and host
genes that are essential for
entry of the virus
•Hepatitis B and C, Influenza
and HIV are common targets
•Ex:- Silencing of the HIV
chemokine receptor (CCR5) by
RNAi therapy is under trial by
Benetic and City of Hope company
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