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Discovery and Brief Description
of RNAi
RNAi, also known as RNA silencing
or post-transcriptional gene silencing is a
mechanism of inactivating specific target
gene expressions by reducing their rates
of transcription, stability of target mR-
NAs, or the translational process of those
mRNAs, thereby promoting degradation
of the related RNAs.
1
Critical event lead-
ing up to the discovery of RNAi in mam-
mals is investigated by Guo and Kemphues
2
who successfully inhibit gene expression
of nematode C.elegans using sense or an-
tisense single strand RNA (ssRNA). Later,
Fire et.al
3
discovered that double-stranded
RNA (dsRNA) is more potent than sense or
antisense ssRNA in silencing gene expres-
sion of the same species; this mechanism
was named RNA interference.
4
During inhibition of a specific gene
product, dsRNA plays a major role as an
intermediate to the triggerring process of
silencing phenomena.
1,4,5
Many research
performed to reveal how dsRNA trigger the
gene silencing. It is reported that dsRNA
could cause homologous genes silencing in
transgenic plants which subsequently resist
viral infection (so-called cosuppression).
6-8
Therefore, RNA silencing can be triggered
by viruses or transposons that generate
dsRNA during their replication or as
an alternative by introducing synthetic
dsRNA.
4,9,10
In brief, RNAi machinery uses
dsRNA as a guide to target and degrade
specific cellular or viral RNAs.
11
Scientists have discovered ways to con-
trol RNAi in manipulating gene expression
of various biological systems, including vi-
ruses. Many investigations have proven the
enormous therapeutic potential of RNAi in
preventing the establishment
12
, reducing
replicative activity
13
, or even promoting
viral clearance
4,5
including: human im-
munodeficiency virus type 1 ( HIV-1)
12-16
,
hepatitis virus (B, C)
17-19
, human papil-
lomavirus (HPV)
20
, Rous sarcoma virus
13
,
poliovirus
21
, respiratory syncytial virus
22
,
and also influenza virus
4
(this dicussion
focused on RNAi as therapeutic agents for
HIV-1 and hepatitis virus). It is essential
to discuss the recent progress of RNAi in
response to their molecular mechanism of
viral gene silencing, major obstacles, and
future directions toward their optimum
application.
Molecular Principles of Gene
Silencing
The molecular pathway underlying the
work of RNAi is relatively simple. Firstly,
dsRNAs are introduced into cells either
with the use of plasmid and virus vector-
based cassette or by using non-viral de-
livery strategies such as integration with a
cholesterol-lipoprotein complex (common
in targeting liver infected by hepatitis vi-
rus) or encapsulated in stable nucleic acid
lipid particles (SNALPs)
24
. dsRNA can be
produced via bidirectional transcription,
transcription of an inverted repeat (hairpin
sequence), or physically introduced into
feeding dsRNA-expressing bacteria.
1,25
After intracellular entry, long dsR-
NAs are then cleaved into small fragments
called short interfering RNAs (siRNAs) by
the action of a 218-kDa dsRNA-specific
endonuclease (RNase type III) known as
Dicer.
1,4,5,9,11
The resultant siRNAs are 21 to
25 nucleotides in length, double-stranded,
and have 3' overhangs of 2 nucleotides.
1,4,5
These siRNAs in turn are incorporated into
a complex of nuclease (Argonaute sub-
units; in humans only Ago2 possesses an
active catalytic domain for cleavage activ-
ity) known as RNA-induced silencing com-
plex (RISC) by the help of dsRNA-binding
protein R2D2.
25
siRNAs unwind in ATP-
dependent manner to activate the RISC.
11
The unwound antisense siRNA act as a
guide to direct RISC toward homologous
target RNA ( i.e. viral mRNAs or genomic
RNA itself) which later undergo endonu-
cleolytic cleavage by Slicer enzyme along
with the role of Dicer.
4,5
Cleavage of target RNA begins at a
single site 10 nucleotides upstream of the
5'-most residue of the siRNA-target RNA
duplex.
1,4
A perfect degradation of target
RNA can be achieved if RNAi-mRNA
complex (RISC) is highly organized in a
sequence-specific pattern, thus promot-
ing natural endogenous degradation by
Slicer. However, in case RISC possesses
any mismatch in nucleotide sequences
(several nucleotides may not have equal
base pair), the target mRNA would still
exist but the tRNA in ribosome will be
unable to translate the codon sequences
into expected amino acids, thus aborting
the translational process.
1,12
These two
mechanisms obstruct the protein synthe-
sis in a gene construction, hence silencing
its final end-product.
Prospect of Nucleic - Acid Based
Immune System RNAi as Potent
Antiviral Agents
Andreas Soejitno
1
, Prichilia Sarah Permadi
1
, Desak Made Wihandani
2
1
undergraduate 4
th
semester Faculty of Medicine, udayana university, Denpasar, Indonesia
2
Department of Biochemistry and Biomolecular, Faculty of Medicine, udayana university, Denpasar, Indonesia
Interferon respponse and
nonspecific gene silencing
dsRNA
(homologous to target)
ATP
ATP
OH
Cytoplasm
Cell
membrane
polyA
polyA
polyA
Target mRNA
Target gene
Nucleus
Expressed
siRNA
ADP + Pi
ADP + Pi
Active RISC
siRNA
RISC
dsRNA
processing
Assembly
of RISC
siRNA
unwinding
Recruitment of RISC
to target mRNA
Target mRNA
cleavage
Sequence-specific
gene silencing
Delivery
Stability
Specific targeting to disease tissue
Activation of interferon response
Saturation of RISC
Persistence of silencing effect
Acute liver
Failure
Fas and
caspase
Hepatitis
Cancer
Allele-specific
oncogene
silence
Multidrug
resistance
Applications
Infection disease
HIV-1 salvage therapy
Influenzaviruses
Hepatitis B and C
viruses
West Nileviruses
Papillomaviruses
Herpesviruses
Insertional activation
Saturation of RISC
Specific expression in
desease tissue
Activation of interferon
response
Obstacles
Obstacles
Mechanism of RNAi as a Potent
Antiviral Agent
Many infectious diseases still can not
be eradicated safely, completely, and effi-
ciently; especially certain viral pathogens
such as HIV-1 and hepatitis B, C (HBV,
HCV) ; HIV-1 has a high rate of mutation
and complexity (also related with toxicity)
to HAART regimen, while acute/subacute
liver failure and hepatocellular carcinoma
induced by HBV and HCV are still unable
to be resolved completely by conventional
therapy. Yet, RNAi offers a promising
therapeutic application as antiviral since
the RNA targets are exogenous and can be
inhibited without affecting cellular func-
tion.
4
Several molecular pathways in disrupt-
ing HIV-1 infection have been elucidated.
To date, RNAi can be used to silence the
expression of CCR5 and CXCR4 corecep-
tor that is involved in the entry process of
HIV-1 to host cell. Martinez et.al (2002)
26
transfected siRNAs-specific for CCR5 and
CXCR4 gene expression in HIV-1+ cells and
found siRNA that target those chemokine
receptors could effectively inhibit surface
protein expression and their function as
HIV-coreceptors. The inhibitory effect
of RNAi does not overlap (i.e. specific to
certain coreceptor) with the blockage per-
centage of CXCR4 and CCR5 reaching
63% and 48%, respectively. Although not
curing, this finding is worthwhile because
individuals with ± 50% decrease in CCR5
surface expression have lower plasma viral
load and a substantially prolonged course
of disease.
27
RNAi through virus-specific
RNA-inducing silencing complex (vRISC)
could also be used to target deletion of 32-
bp homozygote gene located in CCR5 gene
(CCR532).
28
If successfully performed,
this deletion would cause zero expres-
sion of CCR5 coreceptor in CD4
+
T cells,
thereby inducing high resistance to HIV-1
infection. Furthermore, a reduction in vi-
ral replication rate was observed by the de-
crease of p24 intracellular antigen in HIV-
1-infected cells.
27
RNAi inhibits the expression of DC-
SIGN, a specific dendritic cell which in-
ternalizes HIV-1 and introduces HIV-1
to CD4
+
T lymphocytes via CD80-CD86
interaction-activating MAPK pathway in
the lymph nodes as viral replicating sites.
RNAi targets DC-SIGN's mRNA for gene
encoding CD40, CD80 and CD86 expres-
sions, therefore inhibiting the p38 MAPK
Figure 1. Mechanism of Gene Silencing by RNA interference. (ADP = adenosine
diphosphate, Pi = inorganic phosphate, P = phosphate, OH = hydroxyl) (cited with
permission from Stevenson).
4
Figure 2. Inhibitory mechanism of RNAi to CCR5 coreceptor expression in CD4
+
T cells.
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Discovery and Brief Description
of RNAi
RNAi, also known as RNA silencing
or post-transcriptional gene silencing is a
mechanism of inactivating specific target
gene expressions by reducing their rates
of transcription, stability of target mR-
NAs, or the translational process of those
mRNAs, thereby promoting degradation
of the related RNAs.
1
Critical event lead-
ing up to the discovery of RNAi in mam-
mals is investigated by Guo and Kemphues
2
who successfully inhibit gene expression
of nematode C.elegans using sense or an-
tisense single strand RNA (ssRNA). Later,
Fire et.al
3
discovered that double-stranded
RNA (dsRNA) is more potent than sense or
antisense ssRNA in silencing gene expres-
sion of the same species; this mechanism
was named RNA interference.
4
During inhibition of a specific gene
product, dsRNA plays a major role as an
intermediate to the triggerring process of
silencing phenomena.
1,4,5
Many research
performed to reveal how dsRNA trigger the
gene silencing. It is reported that dsRNA
could cause homologous genes silencing in
transgenic plants which subsequently resist
viral infection (so-called cosuppression).
6-8
Therefore, RNA silencing can be triggered
by viruses or transposons that generate
dsRNA during their replication or as
an alternative by introducing synthetic
dsRNA.
4,9,10
In brief, RNAi machinery uses
dsRNA as a guide to target and degrade
specific cellular or viral RNAs.
11
Scientists have discovered ways to con-
trol RNAi in manipulating gene expression
of various biological systems, including vi-
ruses. Many investigations have proven the
enormous therapeutic potential of RNAi in
preventing the establishment
12
, reducing
replicative activity
13
, or even promoting
viral clearance
4,5
including: human im-
munodeficiency virus type 1 ( HIV-1)
12-16
,
hepatitis virus (B, C)
17-19
, human papil-
lomavirus (HPV)
20
, Rous sarcoma virus
13
,
poliovirus
21
, respiratory syncytial virus
22
,
and also influenza virus
4
(this dicussion
focused on RNAi as therapeutic agents for
HIV-1 and hepatitis virus). It is essential
to discuss the recent progress of RNAi in
response to their molecular mechanism of
viral gene silencing, major obstacles, and
future directions toward their optimum
application.
Molecular Principles of Gene
Silencing
The molecular pathway underlying the
work of RNAi is relatively simple. Firstly,
dsRNAs are introduced into cells either
with the use of plasmid and virus vector-
based cassette or by using non-viral de-
livery strategies such as integration with a
cholesterol-lipoprotein complex (common
in targeting liver infected by hepatitis vi-
rus) or encapsulated in stable nucleic acid
lipid particles (SNALPs)
24
. dsRNA can be
produced via bidirectional transcription,
transcription of an inverted repeat (hairpin
sequence), or physically introduced into
feeding dsRNA-expressing bacteria.
1,25
After intracellular entry, long dsR-
NAs are then cleaved into small fragments
called short interfering RNAs (siRNAs) by
the action of a 218-kDa dsRNA-specific
endonuclease (RNase type III) known as
Dicer.
1,4,5,9,11
The resultant siRNAs are 21 to
25 nucleotides in length, double-stranded,
and have 3' overhangs of 2 nucleotides.
1,4,5
These siRNAs in turn are incorporated into
a complex of nuclease (Argonaute sub-
units; in humans only Ago2 possesses an
active catalytic domain for cleavage activ-
ity) known as RNA-induced silencing com-
plex (RISC) by the help of dsRNA-binding
protein R2D2.
25
siRNAs unwind in ATP-
dependent manner to activate the RISC.
11
The unwound antisense siRNA act as a
guide to direct RISC toward homologous
target RNA ( i.e. viral mRNAs or genomic
RNA itself) which later undergo endonu-
cleolytic cleavage by Slicer enzyme along
with the role of Dicer.
4,5
Cleavage of target RNA begins at a
single site 10 nucleotides upstream of the
5'-most residue of the siRNA-target RNA
duplex.
1,4
A perfect degradation of target
RNA can be achieved if RNAi-mRNA
complex (RISC) is highly organized in a
sequence-specific pattern, thus promot-
ing natural endogenous degradation by
Slicer. However, in case RISC possesses
any mismatch in nucleotide sequences
(several nucleotides may not have equal
base pair), the target mRNA would still
exist but the tRNA in ribosome will be
unable to translate the codon sequences
into expected amino acids, thus aborting
the translational process.
1,12
These two
mechanisms obstruct the protein synthe-
sis in a gene construction, hence silencing
its final end-product.
Prospect of Nucleic - Acid Based
Immune System RNAi as Potent
Antiviral Agents
Andreas Soejitno
1
, Prichilia Sarah Permadi
1
, Desak Made Wihandani
2
1
undergraduate 4
th
semester Faculty of Medicine, udayana university, Denpasar, Indonesia
2
Department of Biochemistry and Biomolecular, Faculty of Medicine, udayana university, Denpasar, Indonesia
Interferon respponse and
nonspecific gene silencing
dsRNA
(homologous to target)
ATP
ATP
OH
Cytoplasm
Cell
membrane
polyA
polyA
polyA
Target mRNA
Target gene
Nucleus
Expressed
siRNA
ADP + Pi
ADP + Pi
Active RISC
siRNA
RISC
dsRNA
processing
Assembly
of RISC
siRNA
unwinding
Recruitment of RISC
to target mRNA
Target mRNA
cleavage
Sequence-specific
gene silencing
Delivery
Stability
Specific targeting to disease tissue
Activation of interferon response
Saturation of RISC
Persistence of silencing effect
Acute liver
Failure
Fas and
caspase
Hepatitis
Cancer
Allele-specific
oncogene
silence
Multidrug
resistance
Applications
Infection disease
HIV-1 salvage therapy
Influenzaviruses
Hepatitis B and C
viruses
West Nileviruses
Papillomaviruses
Herpesviruses
Insertional activation
Saturation of RISC
Specific expression in
desease tissue
Activation of interferon
response
Obstacles
Obstacles
Mechanism of RNAi as a Potent
Antiviral Agent
Many infectious diseases still can not
be eradicated safely, completely, and effi-
ciently; especially certain viral pathogens
such as HIV-1 and hepatitis B, C (HBV,
HCV) ; HIV-1 has a high rate of mutation
and complexity (also related with toxicity)
to HAART regimen, while acute/subacute
liver failure and hepatocellular carcinoma
induced by HBV and HCV are still unable
to be resolved completely by conventional
therapy. Yet, RNAi offers a promising
therapeutic application as antiviral since
the RNA targets are exogenous and can be
inhibited without affecting cellular func-
tion.
4
Several molecular pathways in disrupt-
ing HIV-1 infection have been elucidated.
To date, RNAi can be used to silence the
expression of CCR5 and CXCR4 corecep-
tor that is involved in the entry process of
HIV-1 to host cell. Martinez et.al (2002)
26
transfected siRNAs-specific for CCR5 and
CXCR4 gene expression in HIV-1+ cells and
found siRNA that target those chemokine
receptors could effectively inhibit surface
protein expression and their function as
HIV-coreceptors. The inhibitory effect
of RNAi does not overlap (i.e. specific to
certain coreceptor) with the blockage per-
centage of CXCR4 and CCR5 reaching
63% and 48%, respectively. Although not
curing, this finding is worthwhile because
individuals with ± 50% decrease in CCR5
surface expression have lower plasma viral
load and a substantially prolonged course
of disease.
27
RNAi through virus-specific
RNA-inducing silencing complex (vRISC)
could also be used to target deletion of 32-
bp homozygote gene located in CCR5 gene
(CCR532).
28
If successfully performed,
this deletion would cause zero expres-
sion of CCR5 coreceptor in CD4
+
T cells,
thereby inducing high resistance to HIV-1
infection. Furthermore, a reduction in vi-
ral replication rate was observed by the de-
crease of p24 intracellular antigen in HIV-
1-infected cells.
27
RNAi inhibits the expression of DC-
SIGN, a specific dendritic cell which in-
ternalizes HIV-1 and introduces HIV-1
to CD4
+
T lymphocytes via CD80-CD86
interaction-activating MAPK pathway in
the lymph nodes as viral replicating sites.
RNAi targets DC-SIGN's mRNA for gene
encoding CD40, CD80 and CD86 expres-
sions, therefore inhibiting the p38 MAPK
Figure 1. Mechanism of Gene Silencing by RNA interference. (ADP = adenosine
diphosphate, Pi = inorganic phosphate, P = phosphate, OH = hydroxyl) (cited with
permission from Stevenson).
4
Figure 2. Inhibitory mechanism of RNAi to CCR5 coreceptor expression in CD4
+
T cells.
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signalling pathway which in turn, reduces
co-stimulatory molecules expression and
viral replication.
29,30
Another HIV-1 inhibitory mechanism
is through NF- inactivation, the tran-
scription factor family which consists of
five structural proteins (c-Rel/p65, RelA,
RelB, p50/p105 and p52/p100) and plays
major role in host immune responses. NF-
inactivation via silencing of interference
sites on p65 subunit will abort the binding
process to HIV-1 LTR proximal promoter
via hindrance to gp120 in signaling to
p56
lck
tyrosine kinase, which is required by
the virus to begin the transcriptional pro-
cess, after internalization in the host's cell
cytoplasm.
30-32
HBV and HCV can be inhibited by
the application of RNAi in a similar fash-
ion to HIV-1. HBV possesses the life cycle
and genome structure which are both
susceptible to specific siRNAs. HBV has a
open reading frames (ORFs) and mRNAs
within the genome can be targeted by siR-
NAs. This includes the 3.5 kb pregenomic
RNA (serves as the template for HBV DNA
replication, also encodes the viral core and
polymerase protein), 2.4 kb and 2.1 kb
mRNA (encodes the viral envelope pro-
teins), and 0.7 kb mRNA (encodes the viral
X protein).
From the clinical perspective, decrease
in HBV replication by siRNA has been con-
firmed. A reduction of HBV DNA-RNA and
HBsAg-HBcAg as many as 7792% and 85-
99% is achieved when siRNAs target C, S, P,
and X genes by using plasmid vector in the
Huh7 or HepG2.2.15 cells.
18
Whereas a re-
ductive stage of more than 90% reduction of
HBV DNA-RNA and up to 100% reduction
of HBsAg-HBcAg can be obtained when the
plasmid vector was substituted with adeno-
virus.
19
The most effective sequence, which
targeted a region of the surface and overlap-
property is distinct from anti-HBV nucleo-
side or nucleoside analogues, which act on
the viral DNA polymerase to have their
therapeutic effect. Efficacy of surface ORF-
targeted siRNAs was confirmed in other
studies
36,37
and improved in viral replica-
tion decrement by repeated siRNA trans-
fection of cells in culture was also report-
ed.
38
In addition, RNAi also able to reduce
the incidence of acute/subacute liver fail-
ure by preventing apoptosis of hepatocytes
through inhibiton of cell death receptors
expression.
4
The siRNAs targeted to Fas
RNA to the liver of mice were shown suc-
cessfully inhibit Fas expression and protect
mice from hepatitis.
39
Similarly, siRNAs
that target CASP RNA (encoding caspase
8) can prevent acule liver failure induced
by Fas activation.
40
Major Obstacles and Future
Directions to Therapy
The studies of the effect of RNA silenc-
ing on viral replication in mammalian cells
pose several barriers which can be divided
into two groups: philosophical and techni-
cal. Philosophical barriers comprise critical
questions regarding intrinsic therapeutic
characterization of RNAi that is not totally
elucidated by recent studies. For instance,
can RNAi target the incoming viral RNA
when in transit to the nucleus while it is
still associated with nucleocapsid proteins?
This is crucial since certain viral in-
fections can be blocked during this phase
to prevent further dissemination, while
viruses genomes are often protected by a
proteinaceous structure (dsRNA viruses),
nucleoproteins and matrix layers (negative-
stranded RNA viruses), or by association
with cellular membranes during replica-
tion (positive-stranded RNA viruses).
5
Another important question is about
the duration and amount of RNAi should
be administered to exert optimal effects.
The ability of siRNA-transfected cells to
resist virus infection was maintained for
several days.
12,21
However, it could either
indicate that the interference ability per-
sists for a few days or as a caused of the
slow-released siRNAs characteristic.
5
Sec-
ondly, the amplification system (a catalytic
activity in which small amount of dsRNAs
could exert plenty of siRNAs)
2,4
has been
proven to be RdRP-dependent, whereas re-
cent investigation failed to identify RdRP
in humans.
1
The delivery mechanism probably be-
comes the biggest technical problem. To
date, dsRNAs can be delivered to cells by
compact genome with a partially double
stranded DNA of approximately 3200 bases
in length and contains four open reading
frames (ORFs) that encode precore/core,
polymerase, surface and HBVx (HBx)
proteins.
17
The core and polymerase genes
are essential for viral DNA replication and
encodes the viral capsid protein, known
as hepatitis B virus core antigen (HBcAg).
While HBV life cycle is characterized by
the synthesis of a 3 kb partially double-
stranded, relaxed-circular DNA (rcDNA)
genome which has important roles in the
entry, uncoating, and delivery of the viral
genome into the cell nucleus. HBV viral
ping polymerase ORF, inhibited HBV sur-
face antigen secretion by 94% in transfected
cultured cells, and 85% in vivo in the murine
hydrodynamic tail vein injection (MHI)
model. Inhibitory effects were observed
in normal (C57BL/6) as well as immuno-
compromised mice, which indicate that the
expressed shRNAs have a direct effect that
is not dependent on an antigen-dependent
immune response.
33,34
siRNA duplex that targeted sequence
nucleotides 9-27 from the surface ORF
initiation codon was found to be particu-
larly effective against HBV without a re-
quirement for HBV DNA synthesis.
35
This
Figure 3. A schematic diagram depicting the location of potentially siRNAs targeting
in association with viral open reading frames and viral mRNAs within the HBV
genome.
17
PreS 1
PreS 2
Surface protein
HBV DNA
X protein
Pre core
Core protein
Core protein
Polymerase-reverse transcriptase
Transcription
Polydenylation signal
siRNA
0.7kb mRNA
2.1kb mRNA
siRNA 1
2
3
siRNA 5
6
7
2.4kb mRNA
3.5kb mRNA
vectors or as artificial siRNAs.
9,23
However,
there are concerns regarding the hazards
that could be arise when inserting foreign
vector sequences into chromosomal DNA,
such as insertional activation or inactiva-
tion of cellular genes.
4
Furthermore, in-
travenous administration requires siRNAs
that is resistant to nucleases.
These problems perhaps could be re-
solved by the use of synthetic siRNAs and
conjugated carrier such as cholesterol con-
jugates or SNALPs.
23
In addition to the
potential harm of using integrated vectors
to genome, insertion of dsRNA more than
Tomari Y, Zamore PD. Perspective: machines for RNAi. Gen Dev 2005; 19:
1.
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Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in
2.
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Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and
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Stevenson M. Therapeutic Potential of RNA Interference. N Engl J Med 2004;
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Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that
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Sen GL, Blau HM. A brief history of RNAi: the silence of the genes. FASEB J.
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2006; 20: 1293-99.
Jaque J-M, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA
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Hu W, Myers C, Kilzer J, Pfaff S, Bushman F. Inhibition of retroviral pathogen-
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Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small Inter-
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Nakai H, Pandey K
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21.
ral immunity in human cells. Nature 2002; 418: 4304.
Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-
22.
specific double-stranded short interfering RNA and its application in the
reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol.
2001; 1: 34.
Grim D, Kay MA. RNAi and gene therapy: A mutual attraction. Hematology
23.
2007; 2007: 473-81.
Billy E, Brondani V, Zhang H, Muller U, Filipowicz W. Specific interference with
24.
gene expression induced by long, double-stranded RNA in mouse embryonal
teratocarcinoma cell lines. Proc. Natl. Acad. Sci. 2001; 98: 1442833.
Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T. Single-stranded
25.
antisense siRNAs guide target RNA cleavage in RNAi. Cell 2002; 110: 563-74.
Martinez MA, Gutie´rrez A, Armand-Ugo M, et al. Suppression of chemokine
26.
receptor expression by RNAinterference allows for inhibition of HIV-1 replica-
tion. Lippincott Williams &Wilkins 2002.
An DS, Donahue RE, Kamata M. Stable reduction of CCR5 by RNAi through
27.
hematopoietic stem cell transplant in non-human primates.
Proc. Natl. Acad.
Sci. 2007; 104(32): 1311015.
Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1 infection
28.
and progression to AIDS by a deletionallele of the CKR5 structural gene. Sci-
ence 1996; 273: 1856-62.
Madhavan PN, Reynolds JL, Supriya D, et al. RNAi-directed inhibition of
29.
DC-SIGN by dendritic ells: prospects for HIV-1 therapy. AAPS Journal 2005;
E572-8.
Hiscott J , Kwon H , Genin P. Hostile takeovers: viral appropriation of the NF-
30.
kappaB pathway. J Clin Invest 2001; 107: 143-51.
Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily
31.
conserved mediators of immune responses. Annu. Rev. Immunol. 1998; 16:
22560.
Kwon H. Inducible expression of I
32.
B repressor mutants interferes with
NF-B activity and HIV-1 replication in Jurkat T cells. J. Biol. Chem. 1998; 273:
743140.
Ren X, Luo G, Xie Z, Zhou L, Kong X, Xu A. Inhibition of multiple gene expres-
33.
sion and virus replication of HBV by stable RNA interference in 2.2.15 cells. J
Hepatol 2006; 44: 663-70.
Ren XR, Zhou LJ, Luo GB, Lin B, Xu A. Inhibition of hepatitis B virus replication
34.
in 2.2.15 cells by expressed shRNA. J Viral Hepat 2005; 12: 236-42.
Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small
35.
interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 2003;
8: 769-76.
Klein C, Bock CT, Wedemeyer H et al. Inhibition of hepatitis B virus replication
36.
in vivo by nucleoside analogues and siRNA. Gastroenterology 2003; 125: 9-18.
Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable
37.
HBV-producing cell line. Hepatology 2003; 38: 842-850.
Hamasaki K, Nakao K, Matsumoto K, Ichikawa T, Ishikawa H, Eguchi K. Short
38.
interfering RNA-directed inhibition of hepatitis B virus replication. FEBS Lett
2003; 543: 51-54.
Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice
39.
from fulminant hepatitis. Nat Med 2003; 9: 347-51.
Zender L, Hutker S, Liedtke C, et al. Caspase 8 small interfering RNA prevents
40.
acute liver failure in mice. Proc. Natl. Acad. Sci. 2003; 100: 7797-802.
Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the
41.
interferon system by short-interfering RNAs. Nat Cell Biol 2003; 5: 834-9.
500 bp can trigger the activation of inter-
ferons, despite there is no evidence that
this activation could interfere the extent of
RNA silencing.
41
Lastly, it is important to consider the
viral escape possibility after RNAi therapy.
This is crucial since mismatch potential
(the presence of a single or multiple un-
complement base siRNA with target RNA)
of RNAi machinery is not well-tolerated.
5
The tolerance of RNAi machinery to mis-
matches is critical to ensure that the ability
of the virus to escape inhibition is blunted.
Therefore, it is recommended to target
multiple viral genes by RNAi to reduce the
chances of a virus escaping RNAi repres-
sion through spontaneous mutation.
4
Conclusions
RNAi is a potent antiviral agent which
is compatible to nearly most of labile patho-
gens that has not been able to be eradicated
yet. Given the need for therapeutic ma-
chinery that is able to maintain pace with
the high mutation rate of viruses such as
HIV, it is wise to expect RNAi-based thera-
peutic potential nearly in the future con-
temporary medicine. n
References
342
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I
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j
A
u
A
n
P
u
S
T
A
K
A
|
a g u s t u s 2 0 0 9
343
T
I
n
j
A
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signalling pathway which in turn, reduces
co-stimulatory molecules expression and
viral replication.
29,30
Another HIV-1 inhibitory mechanism
is through NF- inactivation, the tran-
scription factor family which consists of
five structural proteins (c-Rel/p65, RelA,
RelB, p50/p105 and p52/p100) and plays
major role in host immune responses. NF-
inactivation via silencing of interference
sites on p65 subunit will abort the binding
process to HIV-1 LTR proximal promoter
via hindrance to gp120 in signaling to
p56
lck
tyrosine kinase, which is required by
the virus to begin the transcriptional pro-
cess, after internalization in the host's cell
cytoplasm.
30-32
HBV and HCV can be inhibited by
the application of RNAi in a similar fash-
ion to HIV-1. HBV possesses the life cycle
and genome structure which are both
susceptible to specific siRNAs. HBV has a
open reading frames (ORFs) and mRNAs
within the genome can be targeted by siR-
NAs. This includes the 3.5 kb pregenomic
RNA (serves as the template for HBV DNA
replication, also encodes the viral core and
polymerase protein), 2.4 kb and 2.1 kb
mRNA (encodes the viral envelope pro-
teins), and 0.7 kb mRNA (encodes the viral
X protein).
From the clinical perspective, decrease
in HBV replication by siRNA has been con-
firmed. A reduction of HBV DNA-RNA and
HBsAg-HBcAg as many as 7792% and 85-
99% is achieved when siRNAs target C, S, P,
and X genes by using plasmid vector in the
Huh7 or HepG2.2.15 cells.
18
Whereas a re-
ductive stage of more than 90% reduction of
HBV DNA-RNA and up to 100% reduction
of HBsAg-HBcAg can be obtained when the
plasmid vector was substituted with adeno-
virus.
19
The most effective sequence, which
targeted a region of the surface and overlap-
property is distinct from anti-HBV nucleo-
side or nucleoside analogues, which act on
the viral DNA polymerase to have their
therapeutic effect. Efficacy of surface ORF-
targeted siRNAs was confirmed in other
studies
36,37
and improved in viral replica-
tion decrement by repeated siRNA trans-
fection of cells in culture was also report-
ed.
38
In addition, RNAi also able to reduce
the incidence of acute/subacute liver fail-
ure by preventing apoptosis of hepatocytes
through inhibiton of cell death receptors
expression.
4
The siRNAs targeted to Fas
RNA to the liver of mice were shown suc-
cessfully inhibit Fas expression and protect
mice from hepatitis.
39
Similarly, siRNAs
that target CASP RNA (encoding caspase
8) can prevent acule liver failure induced
by Fas activation.
40
Major Obstacles and Future
Directions to Therapy
The studies of the effect of RNA silenc-
ing on viral replication in mammalian cells
pose several barriers which can be divided
into two groups: philosophical and techni-
cal. Philosophical barriers comprise critical
questions regarding intrinsic therapeutic
characterization of RNAi that is not totally
elucidated by recent studies. For instance,
can RNAi target the incoming viral RNA
when in transit to the nucleus while it is
still associated with nucleocapsid proteins?
This is crucial since certain viral in-
fections can be blocked during this phase
to prevent further dissemination, while
viruses genomes are often protected by a
proteinaceous structure (dsRNA viruses),
nucleoproteins and matrix layers (negative-
stranded RNA viruses), or by association
with cellular membranes during replica-
tion (positive-stranded RNA viruses).
5
Another important question is about
the duration and amount of RNAi should
be administered to exert optimal effects.
The ability of siRNA-transfected cells to
resist virus infection was maintained for
several days.
12,21
However, it could either
indicate that the interference ability per-
sists for a few days or as a caused of the
slow-released siRNAs characteristic.
5
Sec-
ondly, the amplification system (a catalytic
activity in which small amount of dsRNAs
could exert plenty of siRNAs)
2,4
has been
proven to be RdRP-dependent, whereas re-
cent investigation failed to identify RdRP
in humans.
1
The delivery mechanism probably be-
comes the biggest technical problem. To
date, dsRNAs can be delivered to cells by
compact genome with a partially double
stranded DNA of approximately 3200 bases
in length and contains four open reading
frames (ORFs) that encode precore/core,
polymerase, surface and HBVx (HBx)
proteins.
17
The core and polymerase genes
are essential for viral DNA replication and
encodes the viral capsid protein, known
as hepatitis B virus core antigen (HBcAg).
While HBV life cycle is characterized by
the synthesis of a 3 kb partially double-
stranded, relaxed-circular DNA (rcDNA)
genome which has important roles in the
entry, uncoating, and delivery of the viral
genome into the cell nucleus. HBV viral
ping polymerase ORF, inhibited HBV sur-
face antigen secretion by 94% in transfected
cultured cells, and 85% in vivo in the murine
hydrodynamic tail vein injection (MHI)
model. Inhibitory effects were observed
in normal (C57BL/6) as well as immuno-
compromised mice, which indicate that the
expressed shRNAs have a direct effect that
is not dependent on an antigen-dependent
immune response.
33,34
siRNA duplex that targeted sequence
nucleotides 9-27 from the surface ORF
initiation codon was found to be particu-
larly effective against HBV without a re-
quirement for HBV DNA synthesis.
35
This
Figure 3. A schematic diagram depicting the location of potentially siRNAs targeting
in association with viral open reading frames and viral mRNAs within the HBV
genome.
17
PreS 1
PreS 2
Surface protein
HBV DNA
X protein
Pre core
Core protein
Core protein
Polymerase-reverse transcriptase
Transcription
Polydenylation signal
siRNA
0.7kb mRNA
2.1kb mRNA
siRNA 1
2
3
siRNA 5
6
7
2.4kb mRNA
3.5kb mRNA
vectors or as artificial siRNAs.
9,23
However,
there are concerns regarding the hazards
that could be arise when inserting foreign
vector sequences into chromosomal DNA,
such as insertional activation or inactiva-
tion of cellular genes.
4
Furthermore, in-
travenous administration requires siRNAs
that is resistant to nucleases.
These problems perhaps could be re-
solved by the use of synthetic siRNAs and
conjugated carrier such as cholesterol con-
jugates or SNALPs.
23
In addition to the
potential harm of using integrated vectors
to genome, insertion of dsRNA more than
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21.
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22.
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2001; 1: 34.
Grim D, Kay MA. RNAi and gene therapy: A mutual attraction. Hematology
23.
2007; 2007: 473-81.
Billy E, Brondani V, Zhang H, Muller U, Filipowicz W. Specific interference with
24.
gene expression induced by long, double-stranded RNA in mouse embryonal
teratocarcinoma cell lines. Proc. Natl. Acad. Sci. 2001; 98: 1442833.
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25.
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26.
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tion. Lippincott Williams &Wilkins 2002.
An DS, Donahue RE, Kamata M. Stable reduction of CCR5 by RNAi through
27.
hematopoietic stem cell transplant in non-human primates.
Proc. Natl. Acad.
Sci. 2007; 104(32): 1311015.
Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1 infection
28.
and progression to AIDS by a deletionallele of the CKR5 structural gene. Sci-
ence 1996; 273: 1856-62.
Madhavan PN, Reynolds JL, Supriya D, et al. RNAi-directed inhibition of
29.
DC-SIGN by dendritic ells: prospects for HIV-1 therapy. AAPS Journal 2005;
E572-8.
Hiscott J , Kwon H , Genin P. Hostile takeovers: viral appropriation of the NF-
30.
kappaB pathway. J Clin Invest 2001; 107: 143-51.
Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily
31.
conserved mediators of immune responses. Annu. Rev. Immunol. 1998; 16:
22560.
Kwon H. Inducible expression of I
32.
B repressor mutants interferes with
NF-B activity and HIV-1 replication in Jurkat T cells. J. Biol. Chem. 1998; 273:
743140.
Ren X, Luo G, Xie Z, Zhou L, Kong X, Xu A. Inhibition of multiple gene expres-
33.
sion and virus replication of HBV by stable RNA interference in 2.2.15 cells. J
Hepatol 2006; 44: 663-70.
Ren XR, Zhou LJ, Luo GB, Lin B, Xu A. Inhibition of hepatitis B virus replication
34.
in 2.2.15 cells by expressed shRNA. J Viral Hepat 2005; 12: 236-42.
Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E. Small
35.
interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 2003;
8: 769-76.
Klein C, Bock CT, Wedemeyer H et al. Inhibition of hepatitis B virus replication
36.
in vivo by nucleoside analogues and siRNA. Gastroenterology 2003; 125: 9-18.
Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable
37.
HBV-producing cell line. Hepatology 2003; 38: 842-850.
Hamasaki K, Nakao K, Matsumoto K, Ichikawa T, Ishikawa H, Eguchi K. Short
38.
interfering RNA-directed inhibition of hepatitis B virus replication. FEBS Lett
2003; 543: 51-54.
Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice
39.
from fulminant hepatitis. Nat Med 2003; 9: 347-51.
Zender L, Hutker S, Liedtke C, et al. Caspase 8 small interfering RNA prevents
40.
acute liver failure in mice. Proc. Natl. Acad. Sci. 2003; 100: 7797-802.
Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. Activation of the
41.
interferon system by short-interfering RNAs. Nat Cell Biol 2003; 5: 834-9.
500 bp can trigger the activation of inter-
ferons, despite there is no evidence that
this activation could interfere the extent of
RNA silencing.
41
Lastly, it is important to consider the
viral escape possibility after RNAi therapy.
This is crucial since mismatch potential
(the presence of a single or multiple un-
complement base siRNA with target RNA)
of RNAi machinery is not well-tolerated.
5
The tolerance of RNAi machinery to mis-
matches is critical to ensure that the ability
of the virus to escape inhibition is blunted.
Therefore, it is recommended to target
multiple viral genes by RNAi to reduce the
chances of a virus escaping RNAi repres-
sion through spontaneous mutation.
4
Conclusions
RNAi is a potent antiviral agent which
is compatible to nearly most of labile patho-
gens that has not been able to be eradicated
yet. Given the need for therapeutic ma-
chinery that is able to maintain pace with
the high mutation rate of viruses such as
HIV, it is wise to expect RNAi-based thera-
peutic potential nearly in the future con-
temporary medicine. n
References