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Workshop W02
Viral Vectors in Neuroscience Research
American Society for Neurochemistry
March 21, 2011
Saint Louis, MO
B. Joy Snider, MD, PhD
Malú Tansey, PhD
Mark Sands, PhD
Mingjie Li, MD, PhD
VIRAL VECTORS WORKSHOP SCHEDULE OF EVENTS
4:30 pm WO2-01 B. Joy Snider, Washington University School of
Medicine, Department of Neurology
Overview of Viral Vectors in Neuroscience Research- What they are and how you can use them
4:55 pm WO2-02 Malú Tansey, Emory University School of Medicine,
Department of Physiology
Viral vector tools to investigate the role of neuroinflammation in models of neurodegeneration
5:25 pm WO2-03 Mark Sands, Washington University School of Medicine,
Department of Medicine
CNS-Directed Gene Therapy for Inherited Metabolic Diseases 6:00 pm PANEL DISCUSSION,Q&A
Drs. Snider, Tansey, Sands and Dr. Mingjie Li, Coordinator, Hope Center Viral Vectors Core, Washington University School of Medicine, Department of Neurology
CONTACT INFORMATION FOR HOPE CENTER VIRAL VECTORS CORE
Website: http://hopecenter.wustl.edu/cores/viralVectors/ Core Coordinator: Dr. Mingjie Li, [email protected] 314-747-1123 Core Director: Dr. B. Joy Snider, [email protected] 314-747-2107
SPECIAL THANKS TO BECKMAN COULTER FOR SPONSORING THIS WORKSHOP!
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
American Society for NeurochemistryMarch 21, 2011Saint Louis MO
Workshop W02Viral Vectors in Neuroscience Research
Saint Louis, MO
B. Joy Snider, MD, PhDMalú Tansey, PhDMark Sands, PhD
Mingjie Li, MD, PhD
American Society for NeurochemistryMarch 21, 2011Saint Louis, MO
Overview of Viral Vectors in NeuroscienceResearch:
What they are and how youcan use them
Saint Louis, MO
B. Joy Snider, MD, PhD
Disclosures
• Snider Research Support– National Institute of Health (P30, PPG)– The Knight ADRC– Clinical therapeutic drug trials (Eli Lilly, Jannsen)
• Dr Snider is the faculty director of the Hope Center Viral• Dr. Snider is the faculty director of the Hope Center Viral Vectors Core
• Support for this workshop was generously provided by Beckman Coulter, Inc.
VIRAL VECTORS• Why use viral vectors?
– In cell culture models: transfection with plasmid DNA is easier and more cost-effective in many cell lines, but primary neurons and some neuronal cell lines are difficult to transfect at high efficiency.
– In animal models: Viral vectors offer an alternative to the more expensive, time and labor-intensive approach of generating transgenic/recombinant mice and for most applications are more efficient than gene gun, transfection and electroporation.
• If your lab doesn’t have the expertise to make these vectors, they are also available through commercial vendors and University-based cores.
TYPES OF VIRAL VECTORS
VECTOR TYPE Cloning Capacity
Customize Easy to make?
OTHER
HSV‐1 Large up to 150 kB
Yes No
Adenovirus Medium ~ 36 Yes OK ToxicityAdenovirus Medium 36 kB max
Yes OK Toxicity
Alphaviruses Max 6.5 kb No OK Short‐lived expression
Lentivirus (LV) ~ 7 kB Yes OK Local expression in vivo
Adeno Associated Virus (AAV)
~ 5 kB Yes No Nontoxic; very useful in vivo
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
LENTIVIRAL VECTORS
• Cloning capacity (promoter + insert) ~ 8 kb• Many different promoters, shRNA, miRNA
and regulated expression availableVSV G d t i lt i i• VSV-G pseudotyping results in expression in many cell types
• Other pseudotypes are possible• Time required for expression varies
between cell types• Less expensive to make than AAV
Lentiviral basics• LV is a retroviral vector based on HIV-1
– Retroviruses have an RNA genome.– Upon infection, genome is converted to DNA by reverse
transcriptase and then integrated into the host genome as a “provirus”
– Integration is “semi” random, occuring preferentially at transcriptionally active sitestranscriptionally active sites
• Safety features of the third generation “SIN” lentiviral vectors– Many genes essential for replication deleted– LTRs mutated to make virus “self-inactivating”– Viral genes are divided among 2 plasmids, with capsid
protein provided on a third plasmid (“four vector system”)
Advantages of Lentiviral vectors:• Can transduce both dividing and non-dividing cells• Sustained long term expression of transduced genes
and less transcriptional gene silencing• Large cloning capacity (7 kb promoter + insert)• Easy purification (from media) means viral preps can
be made quickly, e.g. within 2 weeks• Vesicular stomatitis virus glycoprotein (VSV-G)
pseudotyping provides broad tropism and high titer• Can express multiple genes in a single vector
– Pol II promoters – typically 1-2 genes of interest either via separate promoters or IRES
– Pol III promoters for shRNA- up to 4 shRNAs in a single vector
• Relatively low toxicity in most cell types (e.g., less than adenovirus)
Downside of Lentiviral vectors
• These are HIV-based vectors, they are BL2 • Titers lower than AAV• May have some toxicity• Limited diffusion/volume transduction in vivo• May be more difficult to target specific cell types,
ti l l if ll t ifi t tparticularly if cell-type specific promoters are not available (e.g., in gene silencing applications)
• Cloning capacity may not be large enough for really big genes or large promoters
• Integration site concerns- potential for oncogenesis
Two Approaches for Targeting LV Transduction • Promoter selection
– Nonspecific, robust expression in most cells:» CMV (cytomegalovirus)» PGK (phosphoglycerate kinase)» MND (myeloproliferative sarcoma virus enhancer) –
longer-term expression?» CAG (hybrid of CMV/beta-actin)» EF1alpha (elongation factor 1alpha)» EF1alpha (elongation factor 1alpha)
– Cell-type specific» MBP (myelin basic protein)- oligodendrocytes» GFAP (glial fibrillary acidic protein) –astrocytes» NSE, synapsin (neuron specific enolase) - neurons
• Altering envelope proteins to increase binding to specific receptors found only on the desired cell types is possible also, but could be technically challenging
HIV-1 Provirus and Lentiviral Vector Packaging System
pHIV7-GFP
gagpol
vif
vpr
vpuenv nef
revtat
U3 R U5 U3 R U5
5’ LTR 3’ LTR
CMV RRE flap CMV EGFPR RU3U5 U5ψ
pCgp
CMV VSV-G pApCMV-G
CMV pArevpCMV-rev
CMV gag/pol RRE pA
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
Lentiviral vectors available
CMV-GFPCMV eGFP MND-GFP
MND eGFP
PGK-GFPPGK eGFP
pHIV7-GFPCMV RRE flap CMV EGFPR RU3U5 U5ψ
MBP-GFPMBP eGFP
FCIVUbiquitin IRES Venus
MBP eGFP
GFAP-GFPGFAP eGFP
PGK-GFP-HAPGK eGFP HA
U6-PGK-GFP PGK eGFPU6
5’LTR flap attB1 attB2 Ubi-c rtTA3 IRES Venus WPRE3’SIN LTRTRE 5’miR30DsRed shRNA 3’miR30
+DoxpSLIK-TR-miRc2
Lentiviral Vectors Available in the Hope Center Core
Vector name Promoter ApplicationsFCIV Ubiquitin High level expression, mostly neuronsCMV-GFP CMV High level expression, some non-neuronalCMV-lckGFP CMV Membrane targeted (McIver et al., 2005)PGK-GFP PGK (phosphoglycerate kinase) (Zufferey et
al., 1998; Follenzi et al., 2000)High level expression, mostly neurons
PGK-lckGFP PGK Membrane targeted (McIver et al., 2005)g ( , )MBP-GFP MBP (myelin basic protein) (Gow et al., 1992) Oligo lineage expression(McIver et al.,
2005)
MBP-lckGFP MBP Membrane targeted (McIver et al., 2005)SYN-GFP Synapsin Neuronal-specific expressionGFAP-GFP glial fibrillary acidic protein (GFAP) Astrocyte-specific expressionMND-GFP myeloproliferative sarcoma virus enhancer
(Challita et al., 1995),Ubiquitous expression
shRNA vectors pLKO1 U6 (Stewart et al., 2003) Pol III promoter U6 expresses in all cell
types; no reporter
U6-PGK-GFP U6 + PGK for GFP Modified pLK01 with GFP reporter driven by PGK
AarI digestion
U6-PGK-GFP PGK eGFPU6
----GAAACACCGATAAGCAGGTGGCATGCCACCTGCTACCTTTTTTCT--------CTTTGTGGCTATTCGTCCACCGTACGGTGGACGATGGAAAAAAGA----
AarI AarI
shRNA vector for simple construction of shRNA vectors
5’-pACCGNNNNNNNNNNNNNNNNNNNNNCTTCCTGTCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAAGGACAGTNNNNNNNNNNNNNNNNNNNNNAAAAp-5’
----GAAACACCGNNNNNNNNNNNNNNNNNNNNNCTTCCTGTCANNNNNNNNNNNNNNNNNNNNNTTTTTTCT--------CTTTGTGGCNNNNNNNNNNNNNNNNNNNNNGAAGGACAGTNNNNNNNNNNNNNNNNNNNNNAAAAAAGA----
----GAAAC TTTTTTCT--------CTTTGTGGC AAGA----
Annealed double strand oligonucleotides
Start of transcription
sense loop antisense termination signal
Ligation
Primary neuronal-astrocyte cultures
Pregnant mouse@ E15
Dissect fetal neocortices
PND 1-2 mouse pups
Astrocyte cultures Neuronal + astrocyte cultures
Dissect out neocortices
Plate onto astrocyte
monolayer
Allow to mature 12-14 days
Plate onto culture vessel
Allow to mature 2-4 weeks
Effects of Promoters on Transduction of Mouse Cortical Neurons
50 μM
CMV-GFP FCIV MND-GFP PGK-GFP
Fluo
resc
ence
Mer
geN
euN
LV-GFAP-GFP in Astrocytes
Fluorescence Fluorescence + GFAP Fluorescence + Hoech
GFAP Hoechst Phase Contrast
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
lentiviral Vector Transduction of Neuroblastoma cellsCMV-GFP MND-GFP FCIV PGK-GFP
Phas
e co
ntra
stA
50 μMFluo
resc
ence
Fluo
resc
ence
Phas
e co
ntra
st
CMV-GFP CMV-GFP FCIV PGK-GFPB
200 μM 50 μM 200 μM 200 μM
Paul Kotzbauer Lab
Lentiviral Vector Transduction in Rat Cerebellar Granule Cells
CMV-GFP PGK-GFP
Fluo
resc
ence
200 μM
Phas
e co
ntra
st
200 μM
David Harris Lab
pMBP-GFP in Oligodendrocytes
Fluorescence Phase contrast
Mark Goldberg Lab
Adeno-associated Virus (AAV)
• Advantages– Nonpathogenic – Can be made at very high titer and diffuses more easily than LV in vivo– 90% episomal (extrachromosomal) circle, may reduce the chance of
integration mutagenesis• AAV2 vector
– First recombinant AAV vector and still most widely usedy– Over 20 clinical trials
• Disadvantages– Small packaging capacity– AAV2 may be neuron-specific and has limited diffusion and tissue
specificity– AAV2 purification is somewhat labor-intensive, other pseudotypes may
be moreso– Other pseudotypes may be better for specific applications
Wild Type AAV and Recombinant AAV Vectors
Ad
wtAAV
Ad
Ad
Rep/Cap Rep/Cap
rAAV
Rep/Cap
293 cell Target cell
Helper
Ad
AAV plasmidE1AE1B
E2A, E4, VA
rep, capmRNA
Different Serotypes of AAV Vectors Used in CNS
Tissue Transduction Reference
Cerebellum AAV5 Alisky et al, 2000, Neuroreport
Substantia nigra, AAV1, AAV5, AAV2 Burger et al, 2004, Mol. Therstriatum
Ventricle, Striatum AAV4, AAV5, AAV2 Davidson, 2000, PNAS
Cerebrum AAV1, AAV2 Wang et al, 2003, Gene Ther.
Striatum AAV7>AAV8>AAV5>AAV2=AAV6 Harding et al, 2006, Hum. Gene. Ther.
Cortex, Striatum, AAV7, AAV8, AAV9 Cearley et al, 2005, Hippocampus Mol. Ther.
.
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
AAV in cultured neurons
AAV 2/1GFP NeuN Both
GFP GFAP Both
AAV in cultured neurons
AAV 2/2GFP NeuN Both
GFP GFAP Both
AAV in cultured neurons
AAV 2/5GFP NeuN Both
GFP GFAP Both
• Show spread of – PGK Lck– PGK
EF1a
ctx
LV PGK-eGFP LV EF1a-eGFP
ctx
AAV2-eGFP
– EF1a– AAV2
cc
ventstr
cc
Mark Goldberg Lab
Neuron specificity of viral vectors
AAV2-GFPLV EF1a-GFPLV PGK-GFP
GFPNeuN Mark Goldberg Lab
Viral Vector applications• Over expression of genes of interest
– Cell-type specific expression– Lineage labeling– Reporters: e.g., proteasome activity, Ca2+ levels in
different cellular compartments– Time specific expression
Regulatable expression (e g tet on/off)• Regulatable expression (e.g., tet on/off)• Cre expression to silence in specific transgenic animals, or
use cre/lox vectors to direct expression only in cells that express cre
• Gene silencing– shRNA, dominant negatives
• Cell silencing (e.g.,allostatin receptor)• Use your imagination!?
SOCIETY FOR NEUROCHEMISTRY 2011VIRAL VECTORS WORKSHOP
3/21/2011
How to use viral vectors1. Think of a brilliant experiment that requires
transgene expression or gene silencing.2. Decide what viral vector works best for you
• Where do you want to express or knockdown the gene of interest?
• How specific does expression/knockdown need to be?• How long do you need expression to last?• Does toxicity matter?• More practical concerns:More practical concerns:
– Will you make the vector in your lab or collaborate or puchase the vector?
– Do you have resources? (Time, $$$, expertise)3. Decide on promoter, vector pseudotype if
appropriate. You may want to test various options in your system of choice.
4. Design cloning strategy and subclone inserts into appropriate backbone, then package vector.
5. Perform brilliant experiment.6. Write paper, get grants, rest on laurels.
How to use viral vectors: The Easy Way (Shameless Promotion of our Viral Core)
1. Think of a brilliant experiment that requires transgene expression or gene silencing.
2. Not sure which vector to use. Thinking about LV and/or AAV?
3. Contact Hope Center Viral Vectors Core4. Get great advice.4. Get great advice.5. Get help cloning.6. Have us make your vector.7. Perform brilliant experiment.8. Write paper, get grants, rest on laurels.
SOCIETY FOR NEUROCHEMISTRY 2011 3/21/2011 VIRAL VECTORS WORKSHOP
11
VIRAL VECTORS OVERVIEW‐ SUPPLEMENTAL INFORMATION
HSV‐1
• dsDNA, enveloped virus • Does not integrate • Cloning capacity <150 kB for amplicon vectors • Complex to produce; preps may have helper vector contamination • Expression in vivo takes 1‐2 days by some reports • Long‐lasting expression • Available in different pseudotypes with different promoters
Adenovirus
• dsDNA, nonenveloped • Does not integrate • Cloning capacity up to 36kB, most systems less • Relatively easy to produce in a lab with cloning and cell culture expertise • Expression onset varies; may take 1 week for maximal • Long‐lasting expression • Available in different pseudotypes with different promoters • Have some immunogenicity and toxicity in cell lines and in vivo
Alpha viruses (Semliki Forest Virus, Sindbis Virus, Venezuelan Equine Encephalitis
• ssRNA, enveloped • no integration • Cloning capacity @ 6.5 kB max • Expression onset very rapid as +RNA virus • Expression for a few days, not long‐lasting • No alternative pseudotypes or promoters
VECTOR TYPE Cloning Capacity Customization Production OTHER HSV‐1 Large up to 150 kB Yes Complex Adenovirus Medium ~ 36 kB max Yes OK Toxicity Alphaviruses Max 6.5 kb No OK Short‐lived expression Lentivirus (LV) ~ 7 kB Yes OK Adeno Associated Virus (AAV)
~ 5 kB Yes Complex Nontoxic; very useful in vivo
SOCIETY FOR NEUROCHEMISTRY 2011 3/21/2011 VIRAL VECTORS WORKSHOP
12
• May have some toxicity
HIV‐1 (lentiviral vectors; FIV and SIV also used)
• ssRNA, enveloped • Some integration into host genome • Cloning capacity <10 kb (@ 7 kb most vectors, with promoter) • Relatively easy to produce in a lab with cloning and cell culture expertise • Expression onset varies; may take 1‐2 weeks for maximal • Long‐lasting expression • Available in different pseudotypes with different promoters
Adeno‐associated virus
• ssDNA, nonenveloped • yes some integration, usually into a specific site • Cloning capacity @ 5 kB max • Expression onset varies; may take 1‐2 weeks for maximal • Long‐lasting expression • Available in different pseudotypes with different promoters • No significant toxicity
SOCIETY FOR NEUROCHEMISTRY 2011 3/21/2011 VIRAL VECTORS WORKSHOP
13
WHERE YOU CAN GET VIRAL VECTORS IF YOU DON’T WANT TO MAKE THEM IN YOUR OWN LAB
[Disclaimer: these lists are very likely NOT complete ….]
CORES THAT PROVIDE SERVICES TO INVESTIGATORS FROM OTHER INSTITUTIONS:
Baylor College of Medicine http://vector.bcm.tmc.edu/index.htm
University of North Carolina http://genetherapy.unc.edu/services.htm
University of Florida http://www.gtc.ufl.edu/core/vector-core-lab.htm
University of Iowa http://www.uiowa.edu/~gene/
University of Michigan http://www.med.umich.edu/vcore/
University of Pennsylvania http://www.med.upenn.edu/gtp/vectorcore/
Washington University/Hope Center http://hopecenter.wustl.edu/cores/viralVectors
ACADEMIC CORES THAT PROVIDE SERVICES ONLY TO INVESTIGATORS WITHIN THEIR INSTITUTION:
Harvard University http://www.dfhcc.harvard.edu/core-facilities/vector/
Tufts University http://mcri.tuftsmedicalcenter.org/CoreFacilities/AdenoviralVectorCore/
University of Pittsburgh http://www.vectorcore.pitt.edu/vectorcore.php
MANY COMMERCIAL VENDORS SUPPLY VECTORS AND WILL DO CUSTOM CLONING
SOCIETY FOR NEUROCHEMISTRY 2011 3/21/2011 VIRAL VECTORS WORKSHOP
14
Hope Center Viral Vectors Core
http://hopecenter.wustl.edu/cores/viralVectors
Services
The Washington University Hope Center Viral Vectors Core provides lentiviral and AAV vectors and other services to academic investigators on a fee‐for‐service basis. The Core has special expertise in the use of viral vectors for neuroscience applications. Services for Hope Center members and Washington University neuroscience investigators are subsidized by the Hope Center for Neurological Disorders and the P30 NIH Neuroscience Blueprint Interdisciplinary Center Core Grant awarded to Washington University. The Core also supervises shared molecular and cell culture equipment in the Hope Center. The goals of the Core are to provide the highest quality viral vectors available, to serve as a repository for the viral resources and expertise available at Washington University and to develop new vectors and reporters for neuroscience research.
The Core will assist investigators with the design and construction of lentiviral vectors and adeno‐associated virus (AAV) vectors and with viral packaging. The Core has special expertise in vectors for neuroscience applications. We can provide viral vectors expressing marker proteins for use in testing viral expression in different cell types and organs. Basic reporter vectors are available for research purposes to Washington University and most academic investigators; some transgenes and vectors may require that the investigator obtain permission from the source lab. Investigators should obtain Recombinant DNA approval from their institution’s Biosafety office prior to using these vectors in their laboratories.
How to Obtain Core Services
1. Please complete a "Request for New Virus" form from Hope Center Viral Vectors Core website: http://hopecenter.wustl.edu//cores/viralVectors
2. Contact Dr. Mingjie Li, Core Coordinator (e‐mail: [email protected], phone: 747‐1123) to discuss your needs.
Support
The Viral Vectors Core facility is supported by user fees and by a Neuroscience Blueprint Core grant (P30 NS057105) from NIH to Washington University and by NIH Program Project Grant, NS032636, "Cell‐Cell Interaction and Hypoxic Brain Injury."
SOCIETY FVIRAL VEC
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SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
Viral vector tools to investigate the roleof neuroinflammation in models of
neurodegeneration
Malú G. Tansey, PhDAssociate Professor
Department of PhysiologyEmory University School of Medicine
ASN 2011St. Louis, MO
Malú G. Tansey, Ph.DDisclosure of Interest
Research Support
1. Michael J. Fox Foundation
2. Parkinson’s and Movement Disorders Foundation
Speakers Bureau
None
Clinical TrialsNone
3. American Health Assistance Foundation (AHAF)
4. NIH/NINDS
ConsultantKaryopharm Inc.
I own no stocks or equity in any pharmaceutical company
Previous EmploymentXencor Inc.
Overview• Neuroinflammation + Neurodegeneration
• Parkinson’s disease (PD)
D ti i fl t t t i ff d • Do anti-inflammatory strategies afford neuroprotection?– Viral vectors to express DN-TNF and inhibit
soluble Tumor Necrosis Factor (solTNF)– Viral vectors to express RGS10 in microglia and
limit cytokine production and neurotoxicity
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
MicrogliaMicroglia
• Monocyte-derived macrophages of the CNS• Innate immunity and immune surveillance• Activation results in:• Activation results in:
• Change in cell morphology• Upregulation of cell surface receptors (CD68, CD45,
II, F4/80, etc.)• Increased production of reactive oxygen and nitroge
species (ROS/RNS)• Release of cytokines – TNF, IL-1β, IL-6• Recruitment of other cells in peripheral blood
• Long lasting microgliosis in neurodegenerative diseases
Microglia in Human BrainPost-mortem Tissue
Anti-Iba1 Anti-Iba1
Age-matched Control Parkinson’s disease
Parkinson’s Disease• Most common neurodegenerative movement
disorder• Clinical symptoms: tremor, rigidity, bradykinesia• Current therapies treat symptoms; no therapies
yet proven to slow down disease progression• Pathology: loss of substantia nigra DA neurons, Pathology: loss of substantia nigra DA neurons,
Lewy Body inclusions
Courtesy of Dennis Burns, UT Southwestern
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
NeurodegenerationPD, AD, HD ? PD, AD, HD, ALS
NeuroinflammationWhat is the new evidence ?
Healthy Control
Parkinson’s Incidence of idiopathic Parkinson’s Disease among chronic
NSAID users is 46% lower than in non-users (Chen et al., 2003; Chen et al., 2005; Samii 2009)
Chronic systemicDisease
Environmental Exposures
Question:Do molecular divergent triggers converge on neuroinflammation
to promote neurodegeneration ?
R&D InScope
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
What factors and mechanisms contribute to dopaminergic neuron loss in Parkinson’s Disease?
ETIOLOGYGenetic Factors
Environmental FactorsGene-Environment
Interactions
PATHOGENESIS
Mitochondrial dysfunction
Oxidative Stress
Inflammation
Excitotoxicityα-synLRRK2UCHL1
Protein handling dysfunction with LB formation
Neuronal dysfunction
Cell death (apoptosis)of nigrostriatal DA
neuronsAdapted from Schapira
and Olanow 2004
InflammationParkinPINK1DJ-1MAPT (tau)
Tumor Necrosis Factor (TNF)
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
Model: TNF-induced neurodegeneration
Hypothesis: TNF is a critical mediator of dopaminergic neurondeath regardless of the trigger that elicits its production.
TNF monomer
DN-TNF monomer with disrupted receptor interfaces
TNFR monomer
Soluble DN-TNF
Soluble TNF
+Inactive soluble heterotrimers
Dominant-Negative in genetics:"A mutation producing a rogue protein that interferes with the function of the native protein."
Conflict of Interest Declaration: M. Tansey is an ex-employee of Xencor buthas no financial stake in the company stock options.
Melissa McCoy
MJFF Role of Inflammation in PD2002
Keith Tansey
y
Teri Martinez Kelly Ruhn
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
In Vivo Rat Models
Adapted from McCoy et al 2006
TH / Map 2 • Single injection of 6-OHDA (20ug) or chronic infusion of LPS (2ug, 0.01mg/kg) into striatum
• Continuous infusion of XENP 345 (~0.1mg/kg/day) in SNpc for 2-3 weeks
•Estimation of nigral DA neurons by unbiased stereology.
In Vitro Rat Models
Inhibition of soluble TNF alone is sufficient to rescueDA neurons from LPS or 6-OHDA-induced death
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Saline 6-OHDA 6-OHDA + DN-TNF LPS LPS + DN-TNF
Saline 6-OHDA 6-OHDA DN-TNF
LPS DN-TNF
LPS
****
* *0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Saline 6-OHDA 6-OHDA + DN-TNF LPS LPS + DN-TNF
Saline 6-OHDA 6-OHDA DN-TNF
LPS DN-TNF
LPS
****
* *
DA
neu
rons
in S
Npc
Adapted from McCoy et al. 2006
McCoy et al. J Nsci 2006
McCoy et. al 2008
GFAPGFPGFP
Lentiviral delivery of DN-TNF attenuates DA neuron loss in hemiparkinsonian rats
• Single injection, pre-terminal lesion of 20 ug 6-OHDA into the right striatum causing a hemi-lesion.• Intra-nigral delivery of 2 ulof lenti-DN-TNF or lenti-GFP.
6-OHDA
Lenti-DN-TNF or GFP
Striatum
McCoy et al Mol Ther 2008
Inhibition of soluble TNF with lenti-DN-TNF is sufficient to rescue locomotor deficits in hemiparkinsonian rats
McCoy et al Mol Ther 2008
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
3/21/2011
Schematic of Experimental Design for co-inhibition andDelayed TNF inhibition studies in progressive rat model of PD
Harms et al., Mol Ther, 2010
Harms et al Mol Ther 2010
60
80
100
120
tral
ater
al)
Co-Inhibition Delayed
n th
e SN
pc
Progression of 6-OHDA cell loss from 2 5 weeksa
bc
b bc
Delayed TNF inhibition halts progressive phase of nigral DA neuron death
0
20
40
60
5 weeksSaline 6-OHDA 6-OHDA/GFP 6-OHDA/DN-TNF
(% o
f Con
TH+
Neu
rons
in Progressive phase
2 weeks 5 weeks 5 weeks
c c
Harms et al., Mol Ther, 2010
ICV injection of Lenti-DN-TNF in 3xTgAD mouse model attenuated intracellular Aβ
McAlpine et al Neurob of Disease 2009
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Summary• Expression Levels
– Virus spread (lenti vs. AAV)
– Regional differences (SNpc versus st i t m)
• Translational potential of viral vectors– AAV-hAADC, GCH1,
TH (DA synthesis)striatum)
– Cell-type specificity (glia vs. neurons)
– Transgene silencing– Inducible vectors
(Tet-ON/Tet-OFF)
– AAV-Neurturin/CERE-120 (trophic factor)
Progression of a typical neurodegenerative disease: AD
Lab membersDN-TNF:Melissa McCoy, Ph.DAshley Harms, Ph.D3xTgAD:Fiona McAlpine, Ph.DRGS10:Jae Lee, PhDMelissa McCoy, Ph.DAshley Harms, Ph.D
Acknowledgements Funding SupportCollaborators -MJ Fox Foundation DN-TNF: -NIH NINDS D. Szymkowski (Xencor) - High Q/CHDI (HD)
Lenti-DN-TNF: -AHAF (AD)A. Blesch (UCSD/Heidelberg)
AAV-DN-TNF:M. Li (Wash U)
SOCIETY FOR NEUROCHEMISTRY 2011 VIRAL VECTORS WORKSHOP
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Malú G. Tansey, Ph.DAssociate ProfessorDepartment of PhysiologyEmory University School of Medicine605L Whitehead Biomedical Res Bldg615 Michael StreetAtlanta, GA 30324404-727-6126 Office
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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CNS-Directed Gene Therapy for Inherited Metabolic Diseases
Mark S. Sands, Ph.D.Washington University School of Medicine
Viral Vectors
Adeno-Associated Virus Vectors*
Lentiviral Vectors
Adenoviral Vectors
Herpes Virus Vectors
Rabies Virus Vectors
Direct CNS Gene Transfer
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AAV 2(1st generation AAV vector)
- Shown to transduce a wide variety of cells in vivo
- Direct I.C. injection in the murine model of mucopolysaccharidosisVII
Frisella et al, Mol. Ther. 3:351
AAV 2(1st generation AAV vector)
NeuN GFAP
β-Gluc
Merge Merge
β-Gluc
Frisella et al, Mol. Ther. 3:351
AAV 2(1st generation AAV vector)
Frisella et al, Mol. Ther. 3:351
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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AAV 2(1st generation AAV vector)
Frisella et al, Mol. Ther. 3:351
AAV Pseudotyping(serotypes 2, 4, 5)
Davidson et al, PNAS (2000) 97:3428
AAV Pseudotyping(serotypes 2 vs. 5)
Lin et al., Mol. Ther. (2005) 12:422
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AAV Pseudotyping(serotypes 2 vs. 5)
Lin et al., Mol. Ther. (2005) 12:422
AAV in Different Species(AAV1, Feline CNS)
Vite et al., Ann Neurol (2005) 57:355
AAV in Different Species(AAV1, Feline CNS)
Vite et al., Ann Neurol (2005) 57:355
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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Overcoming the Blood Brain Barrierg
Intravenous AAV2/9
StriatumCingulate
gyrusAnterior
commissureInternalcapsule
Foust et al., Nat Biotech (2009) 27:59
Corpuscollosum
Hippocampus Midbrain Cerebellum
Cell-Specific AAV Capsids
Chen et al., Nat Med (2009) 15:1215
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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Cell-Specific AAV Capsids
Chen et al., Nat Med (2009) 15:1215
Axonal Transport
AAV and Axonal Transport
V
ONON
A
MPS VII MPS VII + AAV
Hennig et al, Mol. Ther. (2004) 10:106
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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AAV and Axonal Transport
Hennig et al, J Neurosci, (2003) 23:3302
AAV and Axonal Transport
Griffey et al., Mol. Ther. (2005) 12:413
AAV and Axonal Transport
Griffey et al., Mol. Ther. (2005) 12:413
SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP
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AAV and Axonal Transport
Sands and Haskins, Acta Paediatrica (2008) 97:22
AAV and Axonal Transport
Cearley and Wolfe, J. Neurosci (2007) 27:9928
AAV and Axonal Transport(Murine VTA)
Cearley and Wolfe, J. Neurosci (2007) 27:9928
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Conclusions
1) AAV vectors transduce multiple cell types in the CNS
2) AAV vectors mediate persistent expression2) AAV vectors mediate persistent expression
3) AAV vectors can be pseudotyped to alter the tropism
4) AAV can be transported axonally