Long Term Anti-HIV RNAi Therapy

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Long Term Anti-HIV RNAi
Therapy For AIDS
Dr. Rajesh Kumar

Ph.D (Dairy Microbiology)
Molecular Biology Unit
Dairy Microbiology Division
N.D.R.I
rajesh.mbu@gmail.com
Get yours now!
INTRODUCTION:
HIV/AIDS : major public health problem worldwide.
 No effective vaccines are currently available.

Although combinatorial therapies such as HAART have
proven to be effective, but do not afford a complete
cure.
Previous approach involved use of transdominant
proteins, decoys and ribozymes shown initial promise only.
 Intracellular immunization by gene therapy strategies
offers a promising approach.
Get yours now!
Cont….
 RNA interference is a kind of secret weapon inside us.
 Silencing genes in HIV is straight forward
:- HIT THE VIRUS where it counts.
Synthetic siRNAs can specifically and effectively inhibit

HIV-I gene expression.
 shRNAs
:- Powerful tools for long term HIV gene therapy.
Get yours now!
HIV/AIDS Challenges
• Continuing spread in Africa, Caribbean

• Emerging epidemics
• Possible resurgence in high income
countries
• Anticipated escalation of new infections
40 million individuals infected with HIV in
world presently.
28 million infections are preventable.
16000 infections occur worldwide each day.
Get yours now!
Global Summary of the HIV/AIDS Epidemic
December 2002
Number of people living with HIV/AIDS
Total 42 million
Adults 38.6 million
Women 19.2 million
Children under 15 years 3.2 million
People newly infected with HIV in 2002
Total 5 million
Adults 4.2 million
Women 2 million
Children under 15 years 800 000
AIDS deaths in 2002
Total 3.1 million

Adults 2.5 million
Women 1.2 million
3.5 to 4.5 million in India Children under 15 years 610 000
Get yours now!
Adults and Children Estimated to Be Living with
HIV/AIDS as of End 2002
Eastern Europe
and Central Asia
Western Europe
North America 570,000 1.2 million
980,000
East Asia and Pacific
1.2 million
North Africa
Caribbean and Middle East South and
440,000 550,000 South-
South-East Asia
6 million
Sub-
Sub-Saharan
Africa
Latin America 29.4 million
1.5 million
Australia
and New Zealand
15,000
Total: 42 million
Get yours now!
Molecular and Biological Features of HIV:-
Member of the lentivirus family of animal retroviruses.
HIV variants:
a) X4 T cell tropic.
b) R5 macrophage tropic.
c) dual tropic.
HIV virion consists of two identical strands of RNA (9.2 kb)

packaged with in a core of viral proteins.
Get yours now!
Complex Structure of HIV
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HIV-I genome:
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Entry of HIV virion :- CD4 binding which leads to CCR5 / CXCR4
binding and eventual fusion peptide exposure
CXCR4
or
Get yours now!
HIV Life Cycle
Reverse Transcriptase (RT) Protease
Inhibitors Inhibitors
–AZT (Zidovudine)
Entry/fusion inhibitiors –Nevaripine
Integration Assembly
Reverse Transcription Translation &
Binding & Uncoating Transcription Release
Entry
Genomic DS
CD4 CCR5
viral
RNA DNA
mRNA
X X X
Get yours now!
Cytoplasm Nucleus
“Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your Cytoplasm
Rationale for Gene Therapy for AIDS
• The benefits of HAART

• The limitations of HAART:-
 Cost—$10,000 to $20,000 per year
 Reservoir of latently HIV-infected cells persists despite

suppression of replication to undetectable levels
 Toxicity
 Emergence of drug-resistant HIV strains
 Side effects.
Get yours now!
Vaccine against AIDS:
 There is currently no vaccine to prevent HIV infection.
 Only HIV-negative individuals can volunteer for a preventive
HIV vaccine trial.
 You cannot become infected with HIV from the vaccines being
tested.
 All populations must be involved in HIV vaccine research.
 Obstacles in the development of a vaccine:-
 Complex structure and life cycle
 Genetic potential of the virus for great antigenic
variability.
International AIDS Vaccine Initiative (IAVI).
Get yours now!
Approaches to Anti -HIV Gene Therapy
Anti-HIV
The inhibition strategies can be divided into two
groups:-
a) The RNA-based strategies :
 RNA Decoys(sense RNA)

Ribozymes
Antisense RNA
siRNA
RNA aptamers
miRNA
Get yours now!
b) Protein-based strategies including :-
 Transdominant negative proteins (TNPs)
 Chimeric proteins (fusion proteins)
 Nucleases.
 Anti-infective cellular proteins.
 Intracellular single-chain antibodies.
 Monoclonal antibodies (Mabs).
Get yours now!
Intracellular Ab
& Transdominant
RNA Decoys Proteins
Integration Assembly
Binding & Uncoating Reverse Transcription Translation &
Entry Transcription Release
CD4 CCR5
Genomic DS viral
RNA DNA
mRNA Rev
Tat
siRNA
Antisense RNA
Ribozymes
Cytoplasm Nucleus Cytoplasm
Get yours now!
Double stranded RNA mediated RNA interference
 Simple & rapid method of silencing gene expression.
 Gene silencing is a consequence of degradation of

RNA into short RNAs that activate ribonuclease
target homologous mRNA.
 It is a two step mechanism:-
a) First step involves degradation of ds RNA

into siRNAs (21-22bp) by Dicer.
b) siRNAs join RISC which acts on cognate

mRNA & degrades it.
Get yours now!
Silencing of Gene Expression by Small Interfering
RNA (siRNA)
Get yours now!
Strategies using siRNA to Inhibit HIV-I
Replication
Expression of small interfering RNAs targeted

against HIV-1 rev transcripts in human cells.
(Lee NS et al, may 2002.)
 Mammalian Pol III promoter system capable of expressing

functional ds siRNAs following transfection into human cells.
 In 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA

and the siRNA-producing constructs, 4 logs of inhibition of
expression from the HIV-1 DNA was achieved.
Get yours now!
Potent and Specific Inhibition of Human
Immunodeficiency Virus Type 1 Replication
by RNA Interference.
(Glen A. Coburn & Bryan R. Cullen, sep 2002.)
 siRNA duplexes targeted against the essential

Tat and Rev regulatory proteins encoded by HIV-1
can specifically block Tat and Rev expression and
function.
 Observations demonstrates thst RNA can

effectively block virus replication in human cells.
Get yours now!
Suppression of chemokine receptor expression by
RNA interference allows for inhibition of HIV-1
replication.
(Martinez MA et al, 2003)
 Inhibitory effect directed to CXCR4 was detected in

48 h after transfection of CXCR+U87-CD4+ cells.
 Expression of CXCR4 & CCR5 was blocked in 63 &

48% of positive cells by corresponding siRNA.
 siRNA directed to CXCR4 did not suppress CCR5

expression or vice-versa.
Get yours now!
Inhibiting HIV-1 infection in human T cells by
lentiviral-mediated delivery of small interfering
RNA against CCR5.
(Qin XF et al,2003)
 Lentivirus based vector designed to introduce siRNAs

against the HIV-I Coreceptor CCR5.
 Expression of a potent CCR5-siRNA resulted into

upto 10 fold inhibition of CCR5 expression on cell
surface.
 Lentiviral vector mediated delivery of siRNA proven

to be effective.
Get yours now!
Targeting CCR5 with siRNAs: using recombinant SV40-
derived vectors to protect macrophages and microglia
from R5-tropic HIV. (Cordelier P et al,2003)
Recombinant Tag-deleted SV-40 vectors used to transduce

unselected CCR5-bearing cell lines.
 SV-40 were designed to express two different anti-CCR5

siRNAs.
These siRNAs largely protected CCR5+ cell lines from R5-tropic

HIV.
Therefore, strategies to target CCR5 using rSV40-delivered,

VA promoter-driven siRNAs may be useful therapeutic options for
treating HIV infection.
Get yours now!
Potent suppression of HIV type 1 infection
by a short hairpin anti-CXCR4 siRNA.
(Anderson J et al, 2003)
 A stem-loop hair structured anti-CXCR siRNA was designed.
 FACS analysis showed marked downregulation of CXCR4.
 siRNA transfected cells exhibited marked viarl resistance.
 Delivery of these siRNA into hematopoietic stem cells via

lentiviral vectors may have potential gene therapeutic
applications.
Get yours now!
Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4
and CCR5 confer HIV-1 resistance. (Anderson J et al, 2003)
 Previous studies was based on utility of monospecific siRNAs in suppression

of HIV-I infection.
 High mutation rate considerable challenges in design of fully effective

constructs.
Bispecific siRNA constructs was designed targeted against CXCR4 or CCR5.
Transfected Magi-CXCR4 and CCR5 cells showed significant downregulation

of their respective coreceptors.
 Results demonstrated the practical utility of short hairpin siRNA

bispecific constructs.
 it is now possible to introduce promising multivalent siRNA constructs into

specific
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Human Immunodeficiency Virus Type 1 Escapes from
RNAi Mediated Inhibition.
(Atze T. Das et al, 2004.)
 Short-term assays suggested that RNAi may be a powerful new
method.
 However, RNAi has not yet been shown to protect cells against
HIV-1 in long-term virus replication assays.
 In this study siRNA directed against viral Nef gene confers

resistance to HIV-1.
 siRNA-Nef resistant virus containing substitution or deletion in

Nef gene emerged.
 Like HAART, antiviral approaches involving RNAi should be used in

a combined fashion.
Get yours now!
Inhibition of HIV-1 fusion with small interfering
RNAs targeting the chemokine coreceptor CXCR4.
(Zohu N et al, Dec 2004.)
Immunofluorescence microscopy demonstrated downregulation of

CXCR4 with specific siRNAs.
Transfections with siRNAs targeting CXCR4 mRNA shown to

inhibit HIV-I envelope fusion.
The specificity of this effect was demonstrated by the inhibition

of fusion by CXCR4-tropic and dual-tropic envelope glycoproteins
from HIV-1 on CXCR4+ indicator cells.
Targeting cellular cofactor, rather than the HIV-I specific

mRNAs holds promise.
Get yours now!
HIV-1 resistance conferred by siRNA cosuppression of
CXCR4 and CCR5 coreceptors by a bispecific
lentiviral vector.
(Anderson J, Akkina R, Jan 2005)
 Monospecific siRNAs targeting individual coreceptors are

inadequate against R5 & X4 viral strains.
 Bispecific constructs with dual specificity are required.
 Lentiviral vectors incorporating CXCR4 & CCR5 siRNAs of short

hairpin design were constructed.
 High efficient transduction with a concomitant down regulation was

achieved with lentiviral vectors.
Get yours now!
Cont…
 siRNA expressing transduced cells demonstrated

marked viral resistance against X4 & R5 tropic
HIV-I.
 HIV-I resistance was also observed in transduced

PBMCs.
 CXCR4 & CCR5 could be simultaneously targeted for

downregulation by a single combinatorial lentiviral
vector.
Get yours now!
HIV-1 can escape from RNA interference by evolving
an alternative structure in its RNA genome.
(Ellen M. Westerhout et al, Feb 2005)
 HIV-1 replication can be efficiently inhibited by intracellular

expression of an siRNA.
 Recently long term inhibitionof HIV-I replication in human T cells

stably expressing siRNA-Nef was demonstrated.
 However, HIV-1 escape variants emerged after prolonged

culturing.
 These siRNA resistant viruses contain nucleotide substitution or

deletion in or near the target sequence.
Get yours now!
Cont….
 Out of 9 escape variants, two virus were found to escape through

mutations that induce an alternative secondary structure of target
RNA.
 Results demonstrates that occlusion of an siRNA target sequence

by RNA secondary structure reduces RNAi efficiency.
 These results highlight the extreme versatility of HIV-I and its

evolutionary Capacity to escape from RNAi mediated antiviral
therapy.
Get yours now!
Computational Design of Antiviral RNA Interference Strategies
That Resist Human Immunodeficiency Virus Escape.
(Joshua N. Leonard and David V. Schaffer, Feb 2005.)
 Recently developed strategies based upon RNAi appear highly promising and
offer alternative approaches.
 RNAi has emerged as a robust and highly evolutionary conserved mechanism

for down regulating gene expression.
 Despite this the clinical application of RNAi faces a number of challenges:-

 Certain type of siRNA can induce considerable off-target changes in
gene expression.
 Exquisite sequence specificity of RNAi makes the problem of viral
escape.
 Optimization is required in the design of effective antiviral RNAi therapies.
 Computational model of HIV-I replication used to elucidate design

principles for applying RNAi against HIV-I in a manner that delays
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Contd…..
A novel type of computer model:
 MASS simulation that incorporates molecular level details
of HIV-I reproduction and susceptibility to RNAi.
 This system could be used to study more fundamental aspects of

viral evolution.
 Increasing no of RNAi targets even while keeping siRNA level

constant dramatically mproves RNAi efficiency.
 This approach can yield insights into highly complex process by

which replicating entities evolve.
Get yours now!
Lentiviral siRNAs targeting multiple highly conserved RNA
sequences of HIV-I. (Chang LJ, Liu X, HeJ, Jul 2005)
 High mutation rate of HIV makes it difficult for any therapy to

sustain prolonged effect.
 To explore novel therapy, lentiviral siRNA vectors targeting

multiple highly conserved regions in HIV-I genome was devloped &
tested.
 siRNA expression cassette was cloned into self-inactivating

insulator vector.
 Some siRNA targeting sites were also present in the helper

construct of vector system.
 siRNA targeting gag, pol and vpu but not nef efficiently
inhibited replication of pNL4-3(subtype B), p89.6 (subtype B)
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Cont….
 Transduction of a long term chronically infected human lymphoma

cell line with lentiviral siRNAs resulted in stable inhibition of
HIV-I replication.
 Northeren analysis showed that both genomic & subgenomic viral

RNA species were downregulated.
 Inhibition of viral RNA persisted after prolonged passage.
 Using these vectors reduced replication kinetics of HIV-I in

human PBLs was demonstrated.
 Lentiviral siRNAs targeting multiple conserved HIV-I sequences

holds significant promise for the treatment of HIV-I infections.
Get yours now!
CXCR4 and CCR5 shRNA transgenic CD34+ cell derived
macrophages are functionally normal and resist HIV-1 infection.
(Joseph
(Joseph Anderson
Anderson and
and Ramesh
Ramesh Akkina,
Akkina, Aug
Aug 2005)
2005)
 RNAi can effectively down regulate the expression of either

viral or cellular RNA.
 The potency of shRNAs in targeted gene silencing qualifies

them as powerful tools for long term HIV gene therapy.
 A no of reports have shown, delivery of siRNAs by transfection

of presynthesized siRNAs into cultured cells can effectively
inhibit HIV-I replication.
 Due to transient nature of transfected nucleic acids, the

antiviral effects are only temporary.
 For long range therapy prolonged maintenance & expression of

siRNA coding transgenes in a susceptible target cell is
necessary.
Get yours now!
Cont….
 In this regard lentiviral vectors have proven to highly effective

in high efficiency gene transduction & sustained gene expression.
 Targeting HIV-I genes alone will not be sufficient due to high

possibility of generating escape mutants.
 More sustained efficacy of antiviral effects may be obtained

by targeting host cellular genes critical for viral entry and/or
replication.
 CXCR4 and CCR5 play critical roles as coreceptors for viral

entry.
CXCR4  T cell tropic X4 HIV-I.
CCR5  Macrophage tropic R5 HIV-I.
Their sustained knock down may prove to be more efficacious

for long range siRNA therapy.
Get yours now!
Cont…
 Considering both coreceptors will be imp in developing effective

therapeutics.
 Individuals carrying naturally occurring 32-bp deletion in the

CCR5 gene results function thus conferring significant resistance
to HIV infection.
 Homozygous or heterozygous individuals with this mutation

remain physiologically normal.
 Based on this rationale, recent work with synthetic siRNAs

demonstrated that down regulating either CXCR4 or CCR5 will
protect cells from X4 or R5 HIV-I strains, respectively, at the
level of viral entry.
 Down regulating CCR5 alone in the face of an HIV-1 infection

is insufficient.
Get yours now!
Cont…
 Therefore synthetic bispecific combinatorial constructs as well as

a bispecific lentiviral vector targeting both CXCR4 and CCR5
showed efficacy in inhibiting HIV-1 infections in cell culture lines.
 In translating these findings into a stem cell gene therapy

setting, this bispecific lentiviral vector was used to generate
shRNA expressing transgenic macrophages.
 Macrophages, along with T cells, are major cell targets of HIV

infections.
 Programming these cells to express shRNAs targeted CXCR4 and

CCR5, could confer resistance to HIV infection.
 shRNAs can have possible off target effects thus transgenic

macrophages also need to be assessed for proper functionality.
Get yours now!
CXCR4 and CCR5 bispecific siRNA lentiviral vector XHR
 Transfer vector pHIV-7-GFP was designed to contain an anti-

CXCR4 shRNA cassette under the control of the Pol-III U6
promoter and an anti-CCR5 shRNA cassette under the control of
the Pol-III H1 promoter.
anti-CXCR4------targets--- CXCR4 transcript at 3-23 nt.

anti-CCR5--------targets-- CCR5 transcript at 13-31 nt.
 Two cis-acting elements used to enhance the performance of

vector, namely:-
a) central DNA flap.
b) WPRE.
 This vector also contains an EGFP reporter gene downstream from

shRNA cassettes.
Get yours now!
Bispecific
Bispecific lentiviral
lentiviral vector
vector (XHR)
(XHR) encoding
encoding anti-CXCR4
anti-CXCR4 and
and
CCR5
CCR5 shRNAs.
shRNAs.
Get yours now!
Results:-
(I) Lentiviral vector transduction of CD34+ cells with

CXCR4 and CCR5 shRNAs and derivation of mature
macrophages.
 CD34+ hematopoietic progenitor cells transduced with either

control GFP or XHR vectors sorted for EGFP and driven towards a
myeloid lineage in semi-solid methyl cellulose cytokine media to
generate transgenic macrophage.
 No significant differences were found in the levels of

macrophages obtained when compared between the control GFP
vector and XHR vector transduced cells or control non-transduced
CD34+ cells.
 The morphology of the transgenic macrophages also appeared

normal (data not shown).
Get yours now!
(II) Down regulation of HIV-1 coreceptors
CXCR4 and CCR5 in transgenic macrophages.
 In XHR transduced cells FACS analysis showed an 82%

decrease in CXCR4 expression.
 GFP-alone control vector transduced cells and non-

transduced cells displayed normal levels of CXCR4
expression (94%).
 Similar analysis for CCR5 expression (75% decrease in

transduced macrophages)
 Thus, stably transduced macrophages exhibited

significant down regulation of both the coreceptors
CXCR4 and CCR5 due to shRNA targeting.
Get yours now!
FACS Analysis
Fig II.  Down regulation of the coreceptors CXCR4 and CCR5

in XHR transgenic macrophages.
Get yours now!
(III) XHR transgenic macrophages resist HIV-I
challenge.
 Transduced macrophages were challenged with X4-

tropic (NL4-3) and R5-tropic (BaL-1) strains of
HIV-1.
 Antigen ELISA to detect viral p24 in culture

supernatants were performed on various days post-
infection.
 Over a 2-log reduction in viral yield was seen in XHR

transduced macrophages compared to control cells.
 Thus stable coreceptor down regulation by siRNAs

resulted in marked protection of transgenic -
macrophages against viral challenge.
Get yours now!
1-log reduction in
viral yield.
2-log reduction in
viral yield.
Fig III.  HIV-1 resistance of XHR transgenic macrophages
Get yours now!
(IV) Transgenic macrophages display characteristic
phenotypic cell surface markers.
 Off target effects of some siRNAs may disrupt the phenotypic

properties of macrophages.
 Therefore, transgenic macrophages were subjected to phenotypic

analyses to assess their characteristic cell surface markers by
FACS.
 Levels of the monocyte/macrophage marker CD14 in XHR

macrophages were found to be similar to GFP-alone transduced
or nontransduced cells (98% and 97% respectively).
 Similarly the levels of CD4 were found at comparable levels for

XHR and GFP-alone transduced macrophages at 95% and 93%
respectively.
Get yours now!
Fig IV.  Transgenic macrophages display normal cell
surface markers.
Get yours now!
(V) Transgenic macrophages are functionally
normal.
 Some shRNAs could have possible off-target global effects

leading to disruption of normal cellular functions.
 Functional assays on transgenic macrophages to evaluate this

possibilty were performed.
 Macrophages are typically antigen presenting cells which are

phagocytic in nature as well.
 To determine if XHR transgenic macrophages retained the

phagocytic function, they were presented with fluorescently
labeled E. coli (Bioparticles).
 Foreign cell uptake was measured by FACS.
 No significant difference in phagocytic capacity were found.

Get yours now!
Contd…
 Based on fluorecscence levels, XHR macrophage phagocytosis
was quantified at 68.2% compared to non transduced and
GFP-alone cells at 63.5% and 61.5%, respectively.
 Transduced Magi-CXCR4 cells, serving as non-phagocytic cell

controls did not display any phagocytic activity.
 Macrophages secrete and respond to a number of important

cytokines that include IL-1 and TNF-á.
 To determine functional capacity, siRNA transgenic

macrophages were stimulated with LPS.
 No significant differences were seen in levels of IL-1 and

TNF-á cytokine secretion among the transgenic and control
cell types.
 These finding showed that siRNA transgenic macrophages are

phenotypically and functionally normal.
Get yours now!
0% 63.5% 61.5% 68.2%
Fig V (a)  Phagocytosis of fluorescently labeled E.coli

by CD34+ derived macrophages
Get yours now!
LPS stimulation
Fig V (b)  XHR transgenic macrophages secrete

normal levels of the cytokines IL-1 and TNFá.
Get yours now!
CONCLUSION
 siRNAs are recognized as powerful reagents to reduce the

expression of specific genes.
 To use siRNAs as reagents to protect cells against viral

infection, effective methods for introducing siRNAs into
primary cells are required.
 Stable simultaneous knock down of both the coreceptors

CCR5 and CXCR4 is necessary to prevent HIV-1 infection at
the entry level by both R5 and X4, as well as dual tropic
viral strains.
 Present studies have demonstrated that a bispecific

lentiviral vector could be used to stably deliver shRNAs
targeted to both CCR5 and CXCR4 coreceptors into CD34+
hematopoietic progenitor cells and derive transgenic
macrophages. Get yours now!
Contd…..
 Stable down regulation of both the coreceptors was
achieved in transgenic macrophages.
 The siRNA expressing macrophages were also found to

be phenotypically and functionally normal.
 It is now possible to construct gene therapeutic

lentiviral vectors incorporating multiple siRNAs targeted to
cellular molecules that aid in HIV-I infection.
 Use of these vectors in a stem cell setting shows great

promise for sustained HIV/AIDS gene therapy.
Get yours now!
CD4+ T cell
CD4+CD8+
CD8+ T cell
lymphoid
stem cell
pre-B cell B cell plasma cell
dendritic cell
Pluripotent
Stem Cell
CFU-M monocyte
multilineage
myeloid CFU-G/M
stem cell CFU-G
(CFU-S) PMN
erythrocyte
BFU-E
E/mega
CFU-E
CFU-Mega megakaryocyte
Get yours now!
Get yours now!

Long Term Anti-HIV RNAi Therapy

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Welcome

Dr. Rajesh Kumar

 No effective vaccines are currently available.

 RNA interference is a kind of secret weapon inside us.

 Silencing genes in HIV is straight forward

:- HIT THE VIRUS where it counts.

Synthetic siRNAs can specifically and effectively inhibit

:- Powerful tools for long term HIV gene therapy.

• Continuing spread in Africa, Caribbean

People newly infected with HIV in 2002

AIDS deaths in 2002

Total 3.1 million

Member of the lentivirus family of animal retroviruses.

HIV virion consists of two identical strands of RNA (9.2 kb)

• The benefits of HAART

 Reservoir of latently HIV-infected cells persists despite

 Emergence of drug-resistant HIV strains

International AIDS Vaccine Initiative (IAVI).

a) The RNA-based strategies :

 RNA Decoys(sense RNA)

 Transdominant negative proteins (TNPs)

 Chimeric proteins (fusion proteins)

 Anti-infective cellular proteins.

 Intracellular single-chain antibodies.

 Monoclonal antibodies (Mabs).

 Simple & rapid method of silencing gene expression.

 Gene silencing is a consequence of degradation of

 It is a two step mechanism:-

a) First step involves degradation of ds RNA

b) siRNAs join RISC which acts on cognate

Expression of small interfering RNAs targeted

 Mammalian Pol III promoter system capable of expressing

 In 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA

 siRNA duplexes targeted against the essential

 Observations demonstrates thst RNA can

 Inhibitory effect directed to CXCR4 was detected in

 Expression of CXCR4 & CCR5 was blocked in 63 &

 siRNA directed to CXCR4 did not suppress CCR5

 Lentivirus based vector designed to introduce siRNAs

 Expression of a potent CCR5-siRNA resulted into

 Lentiviral vector mediated delivery of siRNA proven

Recombinant Tag-deleted SV-40 vectors used to transduce

 SV-40 were designed to express two different anti-CCR5

These siRNAs largely protected CCR5+ cell lines from R5-tropic

Therefore, strategies to target CCR5 using rSV40-delivered,

 A stem-loop hair structured anti-CXCR siRNA was designed.

 FACS analysis showed marked downregulation of CXCR4.

 siRNA transfected cells exhibited marked viarl resistance.

 Delivery of these siRNA into hematopoietic stem cells via

 Previous studies was based on utility of monospecific siRNAs in suppression

 High mutation rate considerable challenges in design of fully effective

Transfected Magi-CXCR4 and CCR5 cells showed significant downregulation

 Results demonstrated the practical utility of short hairpin siRNA

 it is now possible to introduce promising multivalent siRNA constructs into

 In this study siRNA directed against viral Nef gene confers

 siRNA-Nef resistant virus containing substitution or deletion in

 Like HAART, antiviral approaches involving RNAi should be used in

Immunofluorescence microscopy demonstrated downregulation of

Transfections with siRNAs targeting CXCR4 mRNA shown to

The specificity of this effect was demonstrated by the inhibition

Targeting cellular cofactor, rather than the HIV-I specific

 Monospecific siRNAs targeting individual coreceptors are

 Bispecific constructs with dual specificity are required.

 Lentiviral vectors incorporating CXCR4 & CCR5 siRNAs of short