Workshop W02 in Neuroscience Research Society March Saint ... · Viral Vectors in Neuroscience...

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


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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


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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


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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.

.


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AAV in cultured neurons

AAV 2/1GFP NeuN Both

GFP GFAP Both



GFP GFAP 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!?


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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


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


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


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

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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

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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

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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)

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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

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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

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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

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ater

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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)

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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

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SOCIETY FOR NEUROCHEMISTRY2011 VIRAL VECTORS WORKSHOP

3/21/2011

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


NeuN GFAP

β-Gluc

Merge Merge

β-Gluc




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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

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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

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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

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Hennig et al, J Neurosci, (2003) 23:3302


Griffey et al., Mol. Ther. (2005) 12:413


Griffey et al., Mol. Ther. (2005) 12:413

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Sands and Haskins, Acta Paediatrica (2008) 97:22


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

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Workshop W02 in Neuroscience Research Society March Saint ... · Viral Vectors in Neuroscience...

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