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Gene Delivery Course: MSMVM 3465, Lecture 7, Feb 18, 2009.

This lecture discusses the host immune response to gene delivery and explores strategies to overcome immune responses for successful gene delivery. Topics covered include innate immune response to viruses, viral components and their sensors, immune evasion strategies of viruses, host immune responses to gene delivery, and the use of modified vectors and immune suppressors. The lecture also delves into the kinetics of immune response after virus infection and the signaling pathways involved. Additionally, the lecture highlights the role of interferons and their signaling pathways in gene induction and viral evasion. Students are assigned a paper critique to further explore relevant topics in TLR, RLR, NLR, IFN, ISG, and viral vector research.

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Gene Delivery Course: MSMVM 3465, Lecture 7, Feb 18, 2009.

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  1. Gene Delivery Course: MSMVM 3465, Lecture 7, Feb 18, 2009. Who Am I? Saumendra N. Sarkar, Ph.D. (Saumen) University of Pittsburgh Cancer Institute, HCC 1.8 5117 Centre Avenue, Pittsburgh, PA 15213  Phone: (412) 623-7720, Fax: (412) 623-7715 E-mail: saumen@pitt.edu Web: http://labs.mmg.pitt.edu/sarkar/ Course Material: http://labs.mmg.pitt.edu/sarkar/Learning.htm What Am I going to talk about? And Why? • Overcoming the host immune response for gene delivery success. • Host responses to virus infection • Innate immune response to virus • Viral components and their Sensors • TLR, RLR, NLRs • IFN • ISGs • Innate Immune evasion strategies of viruses and how it can provide us insights to improve gene delivery

  2. Host immune responses to gene delivery. Adenoviral and Adeno-associated Vectors: (Ad and AAV) Gene Therapy and Innate Immunity - Problems & Solutions • Induction of type I IFN by Ad vectors – Capsids • PBMCs induce IL-6, IL-1b, IL-8, TNFaafter UV-inactivated Ad vector infection. • Ad vector DNA induces inflammation both TLR dependent or independent pathways. • UnmethylatedCpG DNA – TLR9. • Cytoplasmic DNA – DAI, Inflammosome. • Proinflammatory Cytokines – Side effects • Vector Clearance – Unsuccessful Gene transfer • Modified Vectors • Immune Suppressors

  3. Kinetics of immune response after virus infection Biron & Sen (2007) Fields Virology

  4. Virus - dsRNA – Interferon – anti-viral state

  5. Interferon bEnhansosome Escalante et al, Mol. Cell 26, 703-16

  6. Transcription Factors: NF-kB, STAT and IRF

  7. Toll-like Receptors

  8. TLR Structures Jin et al, Immunity 29, 182-91

  9. NLRs, RLRs Inflammosomes Meylan et al, Nature 442, 39-44

  10. Cellular Sensors of DNA and DNA viruses • DNA transfection induces IFNb independent of TLRs (Stetson, 2005) • Cytoplasmic DNA sensor – DAI/ZBP1 (Takaoka, 2007) • Transient Expression allows DNA dependent IRF activation, siRNA. • DAI interaction with DNA • NALP3/ASC Inflammosome (Muruve, 2008) • Adenoviral particles, capsid, DNA induced IL-1 secretion in a NALP3, ASC dependent manner. • No IRF activation. • AIM2, new protein which interacts with ASC (Hornung, 2009) • Binding, localization with ASC • Direct DNA binding • IRF activation?

  11. PRR Signaling Pathways

  12. TLR Signaling Pathways: Gene Induction

  13. RLR Signaling Pathways: Gene Induction Takeuchi and Akira Curr. Opin. Immun. 20, 17-22

  14. NLR Signaling Pathways: IL-1 Production & Gene Induction Meylan et al, Nature 442, 39-44

  15. Interferons

  16. Interferons Type I (a1, a2.., b, d, e, t, k..) Type II (g)

  17. IFN Signaling

  18. Protein Kinase RNA activated (PKR) Enzyme: Ser/Thr kinase, Activator dsRNA, PACT (cofactor) Substrate: eIF2a, PKR, other substrates in vitro. Function:Translation inhibition, other PKR Structure PKR Activation

  19. INTERFERON 2’-5’ OAS ATP dsRNA Inactive RNase L Active RNase L RNA Degradation 2’-5’ OligoadenylateSynthetases (OAS)

  20. ATP, dsRNA pppA(pA)2 Large, P100 pppApA pppA(pA)29 ATP, dsRNA Medium: P69, P71 ATP, dsRNA pppA(pA)5 pppApA Small: E16, E18, 9-2 pppApA Human OAS Isozymes

  21. Structure of OAS1 Hartmann, Sarkar et al. (2003) Mol Cell. 19, 1173

  22. Other ISGs ISG56 Family: Human ISG 56, 54, 60, aka IFIT Interaction with eIF3, translation inhibition ADAR: RNA-Specific Adenosine Deaminase Mx/GBP: small GTP binding proteins ISG15: Similar to Ubiquitin A || U I || U I || C

  23. Strategies to evade host response Finlay et al, Cell 124, 767

  24. Examples of Viral Evasion TLR/RLR IFN ISG Pox V IFNARs IFNGRs TNFR Flu NS1 Paramyxo V, W HCV NS3/4A Adeno VAI RNA Mu V Nipah W Adeno VAI RNA EBV EBER RNA HIV TAR RNA Vaccinia E3L Adeno E1A EBNA1 HHV8 K9 HPV E6 Multifunctional viral proteins with multiple targets!! Paramyxo V and W, Flu NS1, Adeno E1A, SV40 T……

  25. Viral inhibitors of IRF3

  26. Assignment • Pick a paper (25 % Credit) • Relevant Topic: TLR, RLR, NLR, IFN, ISG, Viral Evasion, Viral Vector. • Recent (2007 – 2008) • Quality • Write a critique (75 % Credit) • Overall Summary • Background • Description of Problem/Hypothesis • Experimental Approach • Discussion • Unanswered Questions/Future direction • References Template available online: http://labs.mmg.pitt.edu/sarkar/MSMVM 3465.htm E-mail the paper PDF and your critique to saumen@pitt.edu by Feb 28, 2009

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