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Efficacy of Gene Silencing as a Viable Clinical Treatment Against West Nile Virus by ______ Senior Seminar Presentation University of South Carolina Upstate.

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Presentation on theme: "Efficacy of Gene Silencing as a Viable Clinical Treatment Against West Nile Virus by ______ Senior Seminar Presentation University of South Carolina Upstate."— Presentation transcript:

1 Efficacy of Gene Silencing as a Viable Clinical Treatment Against West Nile Virus by ______ Senior Seminar Presentation University of South Carolina Upstate

2 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

3 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

4

5 1956 Further studies in Egypt found that culex mosquito was primary vector for WNV transmission

6 s-1980s 1990s 1998

7 West Nile Virus first seen in Western Hemisphere – Queens, New York West Nile Virus spread from eastern to western United States

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10 Antiviral Treatment 2002 Epidemic raised concern about possible treatments Mechanism of gene silencing was proposed

11 First seen in petunia plants (Que.Q et al. 1998) Ancient Immune Defense

12 Also seen in C elegans/nematode (Fire. A et al. 1998)

13 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

14 Gene Silencing Select probable target sequence on Viral genome Create vector and introduce into cells Collect m-RNA to generate c-DNA Quantify

15 Gene Silencing Mechanism

16 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

17 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., Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication. Ong et al., 2003.

18 Ong et al., 2003 McCown et al., 2006 Target gene: NS5 ( ) 5’ Cap ( ) 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 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

19 Target gene for siRNA (Ong et al., 2003)

20 Target gene for siRNA (McCown et al., 2003)

21 Ong et al., 2003

22 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)

23 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

24 Post-infection Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA. Geiss et al., 2003.

25 Geiss et al., 2003 Target gene: 5’ Cap ( ) 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?

26 Target gene for siRNA (Geiss et al., 2005)

27 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 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.

28 Geiss et al., 2005 A 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.

29 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

30 Neuronal Efficacy RNAi Functions in Cultured Mammalian Neurons. Krichevsky et al., 2003.

31 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).

32 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.

33 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

34 Can siRNA be used to treat WNV? yes Post-infection Ong et al (1)Post-transfection viral load reduction (2) Sequence Specificity McCown et al Neuronal efficacy RNAi duration vs. Viral replication rate In vivo viability Geiss et al (1)Timing/ mode of transfection affect siRNA uptake (2)Inconsistency reagent based (3)Sequence Specificity Krichevsky et al (1)Neuronal uptake less efficient (2)Inconsistency reagent based (3)Suggested use of cationic-lipid #1 #2 #3 #4 RECAP

35 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

36 RNAi activity vs. Viral replication rate Long-lasting RNAi activity in mammalian neurons. Omi et al., The mechanism of cell death during West Nile virus infection is dependent on initial infectious dose. Chu et al., 2003.

37 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

38 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).

39 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.

40 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

41 In vivo viability Use of RNA Interference to Prevent Lethal Murine West Nile Virus Infection. Bai et al., A single siRNA Suppresses Fatal Encephalitis Induced by two Different Flaviviruses. Kumar et al., 2006.

42 Bai et al., 2003 Kumar et al., 2006 Target gene: E gene( ),( ) E gene ( ) Organisms: 10wk female mice 4-6wk mice WNV strain: Connecticut 1999 New York 1999 Transfection: Hydrodynamic l ipid-based IC Lipid-based (JetSI/DOPE) Results: 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., (3) Hydrodynamic transfection not clinically viable. Infection: Intraperitioneal Intracranial

43 Target gene for siRNA (Bai et al., 2003and Kumar et al., 2006)

44 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.

45 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.

46 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP Conclusions  Historical Background  Mechanism of Gene Silencing

47 Can siRNA be used to treat WNV? yes Post-infection Ong et al (1)Post-transfection viral load reduction (2) Sequence Specificity McCown et al Neuronal efficacy RNAi duration vs. Viral replication rate In vivo viability Geiss et al (1)Timing/ mode of transfection affect siRNA uptake (2)Inconsistency reagent based (3)Sequence Specificity Krichevsky et al Omi et al. 2003Chu et al (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 Bai et al (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) #1 #2 #3 #4

48 Outline Introduction Studies In vitro Post-infection Neurons RECAP Battle against time In vivo GENERAL RECAP General Conclusions  Historical Background  Mechanism of Gene Silencing

49 General Conclusions Notable progress towards clinical viability Better siRNA delivery systems needed. Combination of siRNAs gene targeting.

50 Questions?

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