Efficacy of Gene Silencing as a Viable Clinical
Treatment Against West Nile Virus
by ______Senior Seminar Presentation
University of South Carolina Upstate
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
http://www.biosci.ohiou.edu/virology/WestNile/Virology.htm
1956
• Further studies in Egypt found that culex mosquito was primary vector for WNV transmission
http://www.entm.purdue.edu/publichealth/insects/mosquito.html
http://www.theodora.com/maps/new4/world_color.gif
19371951
1957
1956
1970s-1980s1990s
1998
1999 - 2005
• West Nile Virus first seen in Western Hemisphere – Queens, New York• West Nile Virus spread from eastern to western United States
http://pathmicro.med.sc.edu/mhunt/arbo.htm
http://www.microbeworld.org/news/west_nile/news_west_nile_01.aspx
Antiviral Treatment
• 2002 Epidemic raised concern about possible treatments
• Mechanism of gene silencing was proposed
• First seen in petunia plants (Que.Q et al. 1998)
Ancient Immune Defense
• Also seen in C elegans/nematode (Fire. A et al. 1998)
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Gene Silencing• Select probable target sequence on Viral genome
• Create vector and introduce into cells• Collect m-RNA to generate c-DNA
• Quantify
http://www.rzpd.de/products/rnai/rnai_mech
Gene Silencing Mechanism
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
In vitro
• The utility of siRNA transcripts produced by RNA polymerase I in down regulating viral gene expression and replication of negative an positive strand RNA viruses. McCown et al., 2003.
• Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus
replication. Ong et al., 2003.
Ong et al., 2003 McCown et al., 2006
Target gene: NS5 (8017-8035) 5’ Cap (312-332)
Cells: African green monkey kidney cells Human embryonic kidney cells
WNV strain: Israel 1950s New York 2000
Transfection: Lipid-based Lipid-based
Results: RT-PCR RT-PCR
Conclusion: (1) Reduction of protein expression and viral load
(2) Sequence specificity
Critique/Questions: (1) Post-infection reduction?
(2) Neuronal siRNA efficacy?
(3) In vivo viability?
(4) Duration of Rnai vs Viral replication rate
• Target gene for siRNA (Ong et al., 2003)
http://www.biosci.ohiou.edu/virology/WestNile/Virology.htm
• Target gene for siRNA (McCown et al., 2003)
http://www.biosci.ohiou.edu/virology/WestNile/Virology.htm
Ong et al., 2003
McCown et al., 2003
Figure 2: The effect of siRNAs targeting the WNV capsid and NS5 genes on WNV or DV RNA expression. 293T
cells were transfected with p-HH21, p-HH21 M-siRNA and p-HH21 WNV-CAP-siRNA. One day later, cells were
infected with WNV or DV and were harvested for WNV or DV RNA quantification by real-time fluorogenic RT-PCR.
(McCown et al. 2003)
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Post-infection• Actively replicating West Nile virus is
resistant to cytoplasmic delivery of siRNA. Geiss et al., 2003.
Geiss et al., 2003
Target gene: 5’ Cap (312-332)
Cells: Human Huh 7.5 hepatoma cells
WNV strain: New York 2000 (2)
Transfection: (1) Lipid-based (5’ Cap)
(2) TKO vs Electrophoresis (NS3)
Results: (1) Lipid-based (RT-PCR)
(2) TKO vs Electrophoresis (flow cytometry)
Conclusion: (1) Timing and mode of transfection affect efficacy of siRNA.
(2) Sequence specificity
(3) Inconsistency in results maybe reagent based.
Critique/Questions: (1) Mode of transfection compared different target genes.
(2) Neuronal siRNA efficacy?
(3) In vivo viability?
(4) Duration of RNAi vs. Viral replication rate?
• Target gene for siRNA (Geiss et al., 2005)
Geiss et al., 2005
Figure 3: A. Huh 7.5 cells were mock transfected or transfected with Cap or Cap Mut siRNA at the indicated times before and after WNV infection. Forty-eight hours after infection cells were harvested and WNV RNA levels were determined by quantitative real-time RT-PCR. The results are an average of three independent experiments and error bars indicate standard error of the mean.B. Induction of RNAi resistance by an attenuated lineage II WNV. 6337 denotes the target region of the lineage II specific siRNA. Huh 7.5 cells were transfected with Cap Mut, 6349, or 6337 siRNA at the indicated times prior to or after infection. Forty-eight hours after infection total RNA was collected and viral RNA was assessed.
Geiss et al., 2005A B
Figure 4:(A) siRNA treatment of Huh 7.5 cells were mock-transfected, transfected with TKO reagent complexed with 6337 or 6349 siRNAs, or electroporated with 6337 or 6349 siRNAs. Three days later, cells were processed for viral NS3 protein expression by flow cytometry using anti-NS3 antibody. Fold inhibition was determined using formula (% NS3 positive mock electroporated/ %NS3 positive siRNA electroporated). (B) RNA analysis of Huh 7.5 cells electroporated with siRNA. Cells were electroporated with 6349 or 7353 siRNAs. Three days later, total cellular RNA was collected and viral RNA was assessed. Fold inhibition was determined by dividing the amount of viral RNA in mock electroporated samples to the amount of viral RNA in siRNA electroporated samples.
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Neuronal Efficacy
• RNAi Functions in Cultured Mammalian Neurons. Krichevsky et al., 2003.
Krichevsky et al., 2003
Target gene: Green Fluorescent Protein (GFP)
Cells: Cerebral cortical and hippocampus cells of rat embryos
WNV strain: Not applicable
Transfection: Lipid-based (Lipofectamine 2000)
Results: Double Immunofluorescence, Microscopy and Image Analysis
Conclusion: (1) siRNA uptake in neurons less efficient than kidney cells.
(2) siRNA uptake has toxic effects possibly due to transfection reagents (support Geiss et al., 2005)
(3) Suggested use of cationic lipids (Crino et al., 1996).
Krichevsky et al., 2005
Figure 5: Effect of 21nt-siRNA targeting GFP expression (siGFP) in primary cortical neurons. Primary neurons were transfected with p-GFP and DsRed2 plasmids. For each transfected cell, green and red fluorescence were normalized to a background and plotted. For each cell the arctangent function (represents ration between red and green fluorescence) was calculated. The siGFP showed 42% reduction in GFP expression whereas sense-GFP and antisense-GFP showed no inhibition.
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Can siRNA be used to treat WNV?
yes
Post-infection
Ong et al. 2003
(1) Post-transfection viral load reduction
(2) Sequence Specificity
McCown et al. 2003
Neuronal efficacy
RNAi duration vs. Viral replication rate
In vivo viability
Geiss et al. 2003
(1) Timing/ mode of transfection affect siRNA uptake
(2) Inconsistency reagent based
(3) Sequence Specificity
Krichevsky et al. 2003
(1) Neuronal uptake less efficient
(2) Inconsistency reagent based
(3) Suggested use of cationic-lipid
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RECAP
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
RNAi activity vs. Viral replication rate
• Long-lasting RNAi activity in mammalian neurons. Omi et al., 2003.
• The mechanism of cell death during West Nile virus infection is dependent on initial infectious dose. Chu et al., 2003.
Omi et al., 2003 Target gene: GFP
Cells: Hippocampal neurons
WNV strain: Not applicable
Transfection: Lipid-based
Results: RT-PCR
Conclusion: (1) RNAi activity last up to 3 weeks in neurons, stable RISC# (Omi
et al., 2003).
(2) Burst phase for WNV 32h p.i (Chu et al., 2003)
Critique/Questions: (1) Investigation of alternate transfection reagents (cat-lip)?
(2) In vivo viability?
Not applicable
African green monkey kidney cells
Sarafend (Czech Republic 1997)
Not applicable
Tryphan blue-exclusion
Infection: Not applicable
WNV varying m.o.i
Chu et al., 2003
Omi et al., 2003
Figure 6: Persistence of RNAi activity in post-mitotic neurons. The La2 siRNA duplex (siLa2) and non-silencing control duplex (siCon) against the Photinus luciferase and Renilla luciferase were respectively transfected into mouse primary hippocampal neurons. RNAi activity was examined every week up to 3 weeks after RNAi induction. The expression levels were plotted in arbitrary luminescence units (a.u).
Chu et al., 2003
Figure 7: The effects of different infectious doses of WNV virus on Vero cells. Extracellular virus production( )and cell viability ( ) plotted against time (p.i). Vero cells were infected with WN virus at an m.o.i. of 0.1 (a), 1(b), 10 (c) and 100 (d). At the indicated time, cell supernatants were harvested and plaque assays were performed. The tryphan blue-exclusion method was used to determine cell viability throughout the study. Results from three independent experiments are plotted as the mean ± SE.
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
In vivo viability
• Use of RNA Interference to Prevent Lethal Murine West Nile Virus Infection. Bai et al., 2005.
• A single siRNA Suppresses Fatal Encephalitis Induced by two Different Flaviviruses. Kumar et al., 2006.
Bai et al., 2003 Kumar et al., 2006
Target gene: E gene(1053-1072),(1213-1232) E gene (1287-1305)
Organisms: 10wk female mice 4-6wk mice
WNV strain: Connecticut 1999 New York 1999
Transfection: Hydrodynamic lipid-based IC Lipid-based (JetSI/DOPE)
Results: Live counts Live counts
Conclusion: (1) Bai et al., 2003supported previous in vitro (McCown et al., 2003, Ong et al., 2003) and post-infectional studies (Geiss et al., 2005)
(2) Contrasted with Kumar et al., 2006.
(3) Hydrodynamic transfection not clinically viable.
Infection: Intraperitioneal Intracranial
• Target gene for siRNA (Bai et al., 2003and Kumar et al., 2006)
Bai et al., 2003
Figure 9: Survival curves of small interfering RNA (siRNA) - treated mice challenged with WNV. The siRNA W86, control siRNA, and siRNA W246 groups began with 31, 30, and 14 mice, respectively.
Kumar et al., 2003
Figure 9: siFvEprotects mice against lethal WNV-induced encephalitis. Mice (ten per group) were infected intercranially with WNV and 30 min or 6h later they were also injected with 3.2 nmoles of either control siLuc or siFvEcomplexed with JetSI/DOPE, and monitored for survival over time.
Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• Conclusions
Historical Background Mechanism of Gene Silencing
Can siRNA be used to treat WNV?
yes
Post-infection
Ong et al. 2003
(1) Post-transfection viral load reduction
(2) Sequence Specificity
McCown et al. 2003
Neuronal efficacy
RNAi duration vs. Viral replication rate
In vivo viability
Geiss et al. 2003
(1) Timing/ mode of transfection affect siRNA uptake
(2) Inconsistency reagent based
(3) Sequence Specificity
Krichevsky et al. 2003
Omi et al. 2003 Chu et al. 2003
(1) Neuronal uptake less efficient
(2) Inconsistency reagent based
(3) Suggested use of cationic-lipid
(1) RNAi last 3weeks, RISCs (Omi et al2003)
(2) Burst phase 14p.i (Chu et al.2003)
(3) Suggested alternate transfection reagent
Kumar et al. 2006
Bai et al. 2003
(1) Bai et al.2003 support in vitro studies.
(2) Contrasted with Kumar et al 2006 (Jet-SI/DOPE)
(3) Hydrodynamic injection not clinically
viable(Bai et al.2003)
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#3
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Outline• Introduction
• Studies
• In vitro
• Post-infection
• Neurons
RECAP
• Battle against time
• In vivo
GENERAL RECAP
• General Conclusions
Historical Background Mechanism of Gene Silencing
General Conclusions
• Notable progress towards clinical viability
• Better siRNA delivery systems needed.
• Combination of siRNAs gene targeting.
Questions?
http://www.csu.edu.au/faculty/health/biomed/subjects/molbol/DNA%20technology.htm
http://www.bcm.edu/mcfweb/index.cfm?PMID=3151
http://www.bcm.edu/mcfweb/index.cfm?PMID=3151
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