Lentiviral Vectors: design, production, and titration · Lentiviral Vectors: design, production, and titration ONPRC Lentiviral Vector Core Molecular and Cellular Biology Core Greg

Lentiviral Vectors:design, production,

and titration

ONPRC Lentiviral Vector CoreMolecular and Cellular Biology Core

Greg Dissen

Gag LTRLTR Pro

U

PolEnvVif

Nef

R

RRE

RevTat

HIV provirus

SDψ

∆∆∆

2nd and 3rd generation viral vectors

1. Viral backbone was stripped to allow room for transgenes

2. Development of the Self-Inactivating (SIN) vector

Lentiviral Vector System

2. Modification of 3’LTR “Self Inactivating”

U3 R U5-456 +1 +60 +181

AP-1

NF-AT?

USFEts

TCF-1α

NF-κBNF-ATc

SP1

TATA

-418EcoRV

-18PvuII

∆U3 R U5pHR’ SIN-18

400 bp Deletion

Wild-Type HIV 3’ LTR

5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE

Lentiviral Vector System (3rd generation)

∆U3 R U5DU3 R U5

SDΨ

∆U3 R U5∆U3 R U5

Integration into theHost Genome

Self InactivationNo LTR promoter interference

Gag LTRLTR Pro

U

PolEnvVif

Nef

R

RRE

RevTat

HIV provirus

SDψ

Lentiviral Vector Systems

∆U3 R U5DU3 R U5

∆

∆U3 R U5DU3 R U5

2nd Generation vector

3rd Generation vector

Requires Tat for production

Tat is not required

SIN

SIN

Constitutive promoterRSV or CMV

RSV

Gag LTRLTR Pro

U

PolEnvVif

Nef

R

RRE

TatRev

HIV provirus

SD ψ

Lentiviral Vector Generations

1st Generation:

2nd Generation:

3rd Generation:

HIV provirus:

pMDLgpRRE or pLP1

pRSV-Rev or pLP2

HIV-1 core proteinsEnzymes and Accessory factorsFrom separate plasmidAnd env plasmid

pLV

pMD.G+

+

+pLV

pMD.G

pLV

pMD.G

Packaging reducedgag, pol, tat, revAnd env plasmid

Requirement for tatEliminated, revMoved to separate plasmid

Packaging plasmids

4th generation?

Clontech

5’LTR cPPT wPRE SIN3’LTR

centralpolypurinetract

woodchuckhepatitis virusposttranscriptionalregulatoryelement

RRE

Revresponsiveelement


pHR’ SIN-18

∆U3 R U5DU3 R U5

3rd GenConstitutivePromoter:RSVCMV

SDΨ

PackagingRegion

SpliceDonorSite

Both cPPT and wPRE increaseTransduction efficiency and transgene expression

Rev is essential for viral replicationBinds mRNAs removing them from splicesome = full-length and partially spliced

8 KB



∆U3 R U5DU3 R U5

SDΨ

RNA Polymerase II

Constitutive:CMVSV40hEFpPGK

Tissue Specific

Reporter

Reporter Vector



∆U3 R U5DU3 R U5

SDΨ

New components:

∆U3 R U5DU3 R U5

IRES

Allows the production of two proteins from one mRNA. A Bicistronic RNA.

Internal Ribosome Entry Site:

∆U3 R U5DU3 R U5

2nd

promoter

Allows the production of two mRNAs from one vector.



∆U3 R U5DU3 R U5

SDΨ

New components:

∆U3 R U5DU3 R U5

IRESMCS

Multiple Cloning Site for transgenes to be expressed:

∆U3 R U5DU3 R U5

IRES

Transgene

2nd

promoter

2nd

promoter


∆U3 R U5DU3 R U5

IRES

Transgene

Insertion of a heterologous Intron

∆U3 R U5DU3 R U5Transgene

Intron

A heterologous intron had been found to increase expression of transgenesin transgenic mice.

2nd

promoter

Rat insulin II intron A


∆U3 R U5DU3 R U5

IRES

NGF

∆U3 R U5DU3 R U5

IRES

NGF

Intron

200

150

100

50

0IntronNo IntronLV

ng/m

l med

ium

CMV

Small peptideLigand is Produced andExpression isEnhancedFrom vector ContainingHeterologousIntron

Lentiviral Vector System (3rd generation)IRES

∆U3 R U5DU3 R U5

Jag

hEFp

eGFP∆U3 R U5DU3 R U5

IntronCMVp

∆U3 R U5DU3 R U5

∆U3 R U5DU3 R U5

∆U3 R U5DU3 R U5

Jag

GAPDH

GFP

1

2

3

4

5

1 2 3 4 5

140 kD

42 kD

27 kD

Lentiviral Vector System (3rd generation)IRES

∆U3 R U5DU3 R U5

Jag

hEFp

eGFP∆U3 R U5DU3 R U5

IntronCMVp

∆U3 R U5DU3 R U5

∆U3 R U5DU3 R U5

∆U3 R U5DU3 R U5

Jag

GAPDH

GFP

1

2

3

4

5

1 2 3 4 5

GAPDH (36kDa)

eGFP (27kDa)

FXYD (14kDa)

polybrene LV FIG LV GIF

1:10 1:20 1:30 1:10 1:20 1:30

Infection of Hib5 (6 Well plate, 200,000 cells/well)

LV FIG (1:30) LV GIF (1:30)

FXYD1 EGFP

IRES

FXYD1EGFP

IRESCMV CMV

miR30=Retinoblastoma (Rb)Targeting sequence:AGCAGTTCGATATCTACTGAAA

Stegmeier, 2005

pPRIMEPotent RNA Interferenceusing MicroRNA Expression

pPRIME-CMV-GFPmiR30-shRNA

Pol II driven shRNAwas more active than the Pol IIIconstruct

pPRIME-TET-GFPCMV-dsREDCMV-NeoT-REX-GFP

cPPT Xho I EcoRI

CMV ∆U3 R U5DU3 R U5 3’miR305’miR30GFPCMV CM R WRERRE

Lentiviral Vector System: Gene Suppression



∆U3 R U5DU3 R U5

SDΨ

New components:

∆U3 R U5DU3 R U5

U6 promoterT3

U6-siRNA MCS Cassette

* *

486 bp



∆U3 R U5DU3 R U5

SDΨ

New components:

∆U3 R U5DU3 R U5

U6 promoterT3

U6-siRNA MCS Cassette

* *

486 bp

∆U3

U6-siRNA

Virus Production

1. Cells: human Embryonic Kidney 293T/17Cells have been transformed with temperature sensitive large T antigen Strain was selected specifically for its high transfectability

2. Cells are grown in antibiotic free conditions DMEM (1.5 g/l Na Bicarbonate), 4.5 g/l Glucose, Defined fetal bovine serum, 10% CO2 Advantage to antibiotic free medium = immediately know when there is a problem/contamination

3. Cells are plated to achieve 70 confluency in 10 cm dishes that havebeen coated with poly-L-lysine (6 to 11 x 106 cells/dish)

Day 1

pLV7,438 bp

SIN3’ LTRWPRE

eGFP

hCMV-PcPPT5’LTR RRE

pMD.G6,010 bp

Poly A

VSV G

hβ GlobinIVS2

hCMV-P

pMDLgpRRE8,895 bp

Pol

GAG

hCMV-P

RREPoly A

pRSV-Rev4,174 bp

Poly A

RSV

REV

PackagingTransgene Envelope


TransfectionpLV

pMDLpg.RRE pRSV-RevpMD.G

CaPO4 CaPO4

pLV7,438 bp

SIN3’ LTRWPRE

eGFP

hCMV-PcPPT5’LTR RRE

pMD.G6,010 bp

Poly A

VSV G

hβ GlobinIVS2

hCMV-P

pMDLgpRRE8,895 bp

Pol

GAG

hCMV-P

RREPoly A

pRSV-Rev4,174 bp

Poly A

RSV

REV

PackagingTransgene Envelope


TransfectionpLV

pMDLpg.RRE pRSV-RevpMD.G

Transfected 293T cellsCaPO4 CaPO4

M1

M1

99.18%

99.8%

HEPES - Transfection

BES - Transfection

M1

0.29%Control

Transfection

Infection

pLVpMDLpg.RRE pRSV-Rev

pMD.G

Conditioned Medium

Transfected 293T cells

Infected 293T cellsFACs detects Infected cells, Expressed asPercentage of total

FACs Titer:

Virus Production

Titer Analysis Possibilities:FACS for gene expression product:

Real-Time PCR for integrated viral DNA in host genome

Reverse transcription Real-Time PCR for viral RNA

Dependent on Promoter activityConstitutive promoter = useful Titers that predict infection rateTissue specific promoters might not give useful titers

Dependent on infection and integration into the host genomeReal-Time PCR Titers predict infection rate

Dependent only on the presence of the viral RNADoes not predict infection rate of the viral particles

Acknowledgements

Molecular and Cellular Biology CoreOregon National Primate Research Center

Eliot Spindel, MD., [email protected]

Yibing Jia, M.S.DNA Sequencing, Realtime PCR & [email protected]

CoreyAyne Singleton, M.S.Cell culture, Genomic DNA preparation, Lentivirus [email protected]

Greg Dissen, Ph.D.Director, Lentiviral [email protected]

0

10

20

30

40

50

60

70

0 0.5 1 1.5 20

10

20

30

40

50

60

70

0 0.5 1 1.5 20

10

20

30

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50

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70

0 0.5 1 1.5 2

CMVshort GnRHlong GnRH

Fluorescence in 293-T embryonic kidney cells

viral prep (µl)

% fl

uore

scen

ce

Noriega

0

10

20

30

40

50

60

0 50 100 150 200 250

CMVshort GnRHlong GnRH

viral prep (µl)

Fluorescence in GT1-7 neuronal cells%

fluo

resc

ence

Noriega

Replication Competent Lentivirus (RCL)

Gag LTRLTR Pro

U

PolEnvVif

Nef

R

RRE

TatRev

HIV provirus

SD ψ

Wild-Type Virus

3rd Generation Lentiviral Vector

+Replication competent LTRGag,pol, rev, env, tat

Source:Carry over from packaging orEnvelope plasmidsOrEndogenous viruses

Replication Competent Lentivirus (RCL)Protocol:

1. Infect 1 million SupT-1 cells with 5 million viral TUs

2. Pass the cells 3 times over 2-3 weeks

3. Test the medium for p24 protein with ELISA kit (commercial)

+ StdTest

Preps

Lentiviral Vectors: design, production, and titration · Lentiviral Vectors: design, production, and titration ONPRC Lentiviral Vector Core Molecular and Cellular Biology Core Greg

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