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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#2

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#4

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