Introduction of Lentigen’s HIV-1 Based Lentiviral Vector System
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Introduction of Lentigen’s Introduction of Lentigen’s HIV-1 Based Lentiviral HIV-1 Based Lentiviral
Vector SystemVector SystemHatem Zayed, PhD Hatem Zayed, PhD Jessica Boehmer Jessica Boehmer
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Goals of today presentation Introduction to lentiviral vectors Development of safer lentiviral
vectors for gene therapy. Superior lentiviral kits using
LentiMax™ vectors Experimental design How to order
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Gene Delivery Vehicles• Non-viral
– DNA (plasmid)– DNA-Liposomes– Molecular conjugates
– Gene gun– Electroporation
• Viral– Retroviral vector
• Onco-retroviral vector– MuLV
• Lentiviral vector– HIV– SIV– FIV– EAIV– BAIV
– Adenoviral vector– AAV– Herpes viral vector– Others
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Structure of HIV Virus (Simple but Fatal)
Nucleocapsid
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Life cycle of HIV1. Attachment/Entry
2. Reverse Transcription
and DNA Synthesis
3. Transport to Nucleus
4. Integration
5. Viral Transcription
6. Viral Protein Synthesis
7. Assembly of Virus
8. Release of Virus
9. Maturation
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Comparing Onco-retrovirus to Lentivirus
gag pol
U3
U5
R
RLTR LTR
gag polenv
U3
Vif
Vpr Tat
Rev Vpu
Nef
U3
U5
R
RLTR LTR
envU3
Onco-retrovirus
Lentivirus (HIV-1)
Only infects dividing cells
Infects both dividing and non-dividing cells
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Retroviral Recombination: lessons learned from oncoretroviruses
gag pol U3
U5
R
RLTR LTR
envU3
U5
R
U3 Gene of Interest
gag pol U3
U5
R
RLTR
envU3
Recombination can generate replicationCompetent viruses
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1st Generation Lentivirus Vectors
Transient transfection of three plasmids in 293T :
Packaging plasmid: all HIV viral genes, except env
Envelope plasmid: G envelope glycoprotein of vesicular stomatitis virus (VSV G)
Transducing vector: gene or cDNA of interest and the minimal cis-acting elements of HIV
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1st Generation Vectors
• Limited homology between vector and helper sequences
• Separation of helper plasmids• Still retains HIV accessory genes in the
packaging plasmid
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2nd Generation Vectors
Elimination of accessory genes from packaging plasmid
• No effect on vector titer• Retains property of transduction of many
dividing and non-dividing cells• Increased safety margin
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3rd Generation Vectors
Self-inactivating (SIN) vectors
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Constructing The Self-Inactivated (SIN) Lentiviral Vector
Transducing VectorR
Gene of InterestBGH PA
U3
U5
R
RLTR LTR
gag polenv
U3
Vif
Vpr Tat
Rev Vpu
Nef
HIV-1 Provirus
LentiMax™
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CMV P Human Globin pA
SV40 pA
Gag-Pol
VSV-G
U3
U5
R
RLTR LTR
gag polenv
U3
Vif
Vpr Tat
Rev Vpu
Nef
HIV-1 Provirus
Helper Constructs
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+ Strand RNA 5’R
An 3’gag pol
env
U5R U3 R
Reverse transcriptionIntegration
Provirus(DNA)
R
U3
U5
R
y U5
RLTR LTR
Genome Replication
+1
Transcription
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SIN (Self Inactivation)
U5
RDeletion
Provirus(DNA)
U3
U5
R RLTR LTR
gag polenv
U3
+ Strand RNA An
U5Rtranscription
X
Provirus(DNA)
R
gag polenv
Reverse transcriptionIntegrationU3
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Safety of Lentigen’s LVs
– No HIV proteins are expressed from the vector, only gene or sequence of interest is expressed from gutted backbone
– The 3’ U3 region of the 3’LTR is modified to inactivate the original promoter/enhancer activity of the LTR, resulting in a self-inactivating (SIN) viral vector.
– There are no significant regions of homology between the vector and helper constructs that would result in their recombination.
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• Creation of stable cell lines • Expression of genes in primary cells • Gene of RNAi delivery into neurons or hard to transfect
cell types • Gene Therapy Applications • RNAi expressing cell lines—stable knockdown of gene
expression • Efficient generation of transgenic animals • Animal experiments that require localized gene delivery • Detection and localization of proteins in live cells • Drug discovery—creation of cell lines that express
reporter genes in response to chemical stimulants • Rapid production of proteins from cell lines
LentiMax™ Vector Application
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MCF10AHeLa
GTM3HEK 293
VSV-G Pseudotyped Lentiviral Vectors Efficiently Transduce Many Cell Types
R GFP
BGH PA
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100 50 25MOI: 10
VSV-G Pseudotyped Lentiviral Vectors Can Transduce Primary Rat Hepatocytes
R GFP
BGH PA
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MIPS Project #3811, UMB-Lentigen, Ricardo A. Feldman, March 1, 2007
Vectors: EF-1α-PyMT = 3.7 x 106 particles/siteEF-1α-Luciferase = 5 x 106 particles/site
Bioluminescence imaging done after 1 month (7 mice).
Luciferase
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Lenti-KitTM
P LacZ WPRE
P LacZ IRES copGFP WPRE
P LacZ IRES WPREPuro
WPREcopGFPP
AscI NotI
LacZ
PacIBamHI
ClaI
SCMVH1: BamHI/PacIU6: ClaI/pacI
LTR SIN
pA
SCMVEF1a/HTLV
ForcDNA
Pri-miRNA
For shRNA LacZ
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Comparing Lentigen’s Lentiviral Vector Kit Product to Other Commercial Kit Products
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
1.40E+09
1.60E+09
Lentigen A B C
Company
qPC
R T
iter
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Experimental DesignExperimental Design
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Common Terminology• Infection: process of virus entrance and
replication– used for wild type viruses– Virus replicates and produces many progeny viruses– You say “HIV-1 infects CD4+ T cells”
• Transduction: process of vector entrance– used for viral vectors– Vector does not replicate and produces progeny
vectors– You say “Lenti-GFP vectors transduce T cells”
• Titer: amount of infectious particles• MOI: Ratio of infectious particle # to cell #
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Factors to Consider
• Transduction Method• MOI (Multiplicity of Infection)• Sensitivity to cytotoxicity
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Methods for Vector Transduction
• Conventional method:– Small volume – Rocking– With or without polybrene (4~8 ug/ml)– 2~4 hrs or O/N
• Spin transduction: 2,000 x g, 1~4 hrs• Retronectin
– Coat retronectin on a plate– For hematopoietic stem cell transduction
• Magnetic nanoparticle
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MOI (Multiplicity of Infection)
• Depending on the permissivity of cells– B cells are very difficult to transduce
• MOI of 5 one hit per cell• Use a reporter vector to find proper MOI
– MOI 5, 10, 50, 100• Use polybrene to enhance transduction
– Some cells are very sensitive to the toxicity of polybrene
– Extensive wash after transduction
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Cytotoxicity
• Quality of Viral vectors– Ratio of defective particles to infectious
particles: p24/TU– Purity of vector particles:
• Contaminants: proteins, DNA, cell debris
• Inherent nature of target cells– Permissivity– Sensitivity to transduction enhancement
reagents
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LentiMax™ Production System
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Order FlowOrder Rec’d—Triage/Analysis
Lentigen Receives cDNA or sequence
Customer forwards cDNA for gene or shRNA sequence
Cloning/Structural Analysis
Clone Picks Sent for Sequence Analysis
Sequence DiscrepancyReports Received & Analyzed
Plasmid Preparation
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Order FlowGel Verification
Production of Viral Particles
QC Testing (Sterility & Titer)
Certificate of Analysis
Generated
Product Shipped
Email Customer Shipment Alert
Customer Receives Product