Lentiviral Vectors: design, production, and titration
Transcript of Lentiviral Vectors: design, production, and titration
![Page 1: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/1.jpg)
Lentiviral Vectors:design, production,
and titration
ONPRC Lentiviral Vector CoreMolecular and Cellular Biology Core
Greg Dissen
![Page 2: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/2.jpg)
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
![Page 3: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/3.jpg)
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
![Page 4: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/4.jpg)
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
![Page 5: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/5.jpg)
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
![Page 6: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/6.jpg)
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
![Page 7: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/7.jpg)
Packaging plasmids
4th generation?
Clontech
![Page 8: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/8.jpg)
5’LTR cPPT wPRE SIN3’LTR
centralpolypurinetract
woodchuckhepatitis virusposttranscriptionalregulatoryelement
RRE
Revresponsiveelement
Lentiviral Vector Systems
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
![Page 9: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/9.jpg)
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector Systems
∆U3 R U5DU3 R U5
SDΨ
RNA Polymerase II
Constitutive:CMVSV40hEFpPGK
Tissue Specific
Reporter
Reporter Vector
![Page 10: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/10.jpg)
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System (3rd generation)
∆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.
![Page 11: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/11.jpg)
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
Lentiviral Vector System (3rd generation)
∆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
![Page 12: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/12.jpg)
Lentiviral Vector System (3rd generation)
∆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
![Page 13: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/13.jpg)
Lentiviral Vector System (3rd generation)
∆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
![Page 14: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/14.jpg)
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
![Page 15: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/15.jpg)
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
![Page 16: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/16.jpg)
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
![Page 17: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/17.jpg)
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
![Page 18: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/18.jpg)
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
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
![Page 19: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/19.jpg)
5’LTR cPPT hCMV-P eGFP wPRE SIN3’LTRRRE
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
U6-siRNA
![Page 20: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/20.jpg)
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
![Page 21: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/21.jpg)
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
Lentiviral Vector System
TransfectionpLV
pMDLpg.RRE pRSV-RevpMD.G
CaPO4 CaPO4
![Page 22: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/22.jpg)
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
Lentiviral Vector System
TransfectionpLV
pMDLpg.RRE pRSV-RevpMD.G
Transfected 293T cellsCaPO4 CaPO4
![Page 23: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/23.jpg)
M1
M1
99.18%
99.8%
HEPES - Transfection
BES - Transfection
M1
0.29%Control
![Page 24: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/24.jpg)
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:
![Page 25: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/25.jpg)
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
![Page 26: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/26.jpg)
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]
![Page 27: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/27.jpg)
![Page 28: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/28.jpg)
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
40
50
60
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
![Page 29: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/29.jpg)
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
![Page 30: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/30.jpg)
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
![Page 31: Lentiviral Vectors: design, production, and titration](https://reader030.fdocuments.us/reader030/viewer/2022020108/58a2f8131a28abd1778ba542/html5/thumbnails/31.jpg)
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