Transcript Gene Delivery Course - University of Pittsburgh

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: [email protected]
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
o
Viral components and their Sensors
o
TLR, RLR, NLRs
o
IFN
o
ISGs
• Innate Immune evasion strategies of viruses and how it can provide us insights
to improve gene delivery
Host immune responses to gene delivery.
Adenoviral and Adeno-associated Vectors: (Ad and AAV)
• Induction of type I IFN by Ad vectors – Capsids
o PBMCs induce IL-6, IL-1b, IL-8, TNFa after UVinactivated Ad vector infection.
• Ad vector DNA induces inflammation both TLR dependent
or independent pathways.
o Unmethylated CpG DNA – TLR9.
o Cytoplasmic DNA – DAI, Inflammosome.
Gene Therapy and Innate Immunity - Problems & Solutions
• Proinflammatory Cytokines – Side effects
• Vector Clearance – Unsuccessful Gene transfer
• Modified Vectors
• Immune Suppressors
Kinetics of immune response after virus infection
Biron & Sen (2007) Fields Virology
Virus - dsRNA – Interferon – anti-viral state
Interferon b Enhansosome
Escalante et al, Mol. Cell 26, 703-16
Transcription Factors: NF-kB, STAT and IRF
NF-kB
IRF
STAT
Family
P50, p52, p65 (RelA),
RelB, c-Rel
IRF1 to IRF9
STAT1 to STAT6; 5A & 5B
Activation
IkB Degradation, pS, pT
pS, pT, Ub
pY, pS, pT
Activators
Everything!!
TLR, RLR
IFN, Hormone
Repressor
IkB
Not Clear
SOCS
Kinases
IKK family to IkB
TBK1, IKKe
JAK
Genes
Cytokines, Inflammation,
Growth Promoting
IFN, Innate Immunity,
Growth Inhibitory
ISGs, Growth
Promoting/Inhibitory.
Toll-like Receptors
TLR Structures
Jin et al, Immunity 29, 182-91
NLRs, RLRs Inflammosomes
Meylan et al, Nature 442, 39-44
Cellular Sensors of DNA and DNA viruses
• DNA transfection induces IFNb independent of TLRs (Stetson, 2005)
• Cytoplasmic DNA sensor – DAI/ZBP1 (Takaoka, 2007)
o
o
Transient Expression allows DNA dependent IRF
activation, siRNA.
DAI interaction with DNA
• NALP3/ASC Inflammosome (Muruve, 2008)
o
o
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)
o
Binding, localization with ASC
o
Direct DNA binding
o
IRF activation?
PRR Signaling Pathways
TLR Signaling Pathways: Gene Induction
RLR Signaling Pathways: Gene Induction
Takeuchi and Akira Curr. Opin. Immun. 20, 17-22
NLR Signaling Pathways: IL-1 Production & Gene Induction
Meylan et al, Nature 442, 39-44
Interferons
Interferons
Type I (a1, a2.., b, d, e, t, k..)
Size
Type II (g)
~ 18 kDa
21 kDa
Cell Source
Most Nucleated cells
Activated T Cells, NK Cells
Target Cells
All
Monocytic Cells and others
Mainly Antiviral
Antiviral and Antitumor
IFNAR
IFNGR
Effect
Receptor
IFN Signaling
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
2’-5’ Oligoadenylate Synthetases (OAS)
INTERFERON
2’-5’ OAS
ATP
dsRNA
O
O
O
P
O 3
N
O
5
3
N
N
2
N
N
O
O
O
P
O
O
N
O
5
3
N
N
2
N
O
O
Inactive RNase L
Active RNase L
RNA Degradation
N
Human OAS Isozymes
ATP, dsRNA
pppA(pA)2
Large, P100
pppA(pA)29
ATP, dsRNA
Medium: P69, P71
ATP, dsRNA
pppA(pA)5
Small: E16, E18,
9-2
pppApA
pppApA
pppApA
Structure of OAS1
Hartmann, Sarkar et al. (2003) Mol Cell. 19, 1173
Other ISGs
ISG56 Family:
Human ISG 56, 54, 60, aka IFIT
Interaction with eIF3, translation inhibition
ADAR:
RNA-Specific Adenosine Deaminase
A
||
U
Mx/GBP: small GTP binding proteins
ISG15: Similar to Ubiquitin
I
||
U
I
||
C
Strategies to evade host response
Finlay et al, Cell 124, 767
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 E1A
EBNA1
HHV8 K9
HPV E6
Adeno VAI RNA
EBV EBER RNA
HIV TAR RNA
Vaccinia E3L
Multifunctional viral proteins with multiple targets!!
Paramyxo V and W, Flu NS1, Adeno E1A, SV40 T……
Viral inhibitors of IRF3
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)
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–
–
–
–
–
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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 [email protected] by Feb 28, 2009