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Introduction of Lentigen’s
HIV-1 Based Lentiviral
Vector System
Hatem Zayed, PhD
Jessica Boehmer
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
Gene Delivery Vehicles
• Non-viral
– DNA (plasmid)
– DNA-Liposomes
– Molecular conjugates
• Viral
– Retroviral vector
• Onco-retroviral vector
– MuLV
• Lentiviral vector
–
–
–
–
–
– Gene gun
– Electroporation
–
–
–
–
HIV
SIV
FIV
EAIV
BAIV
Adenoviral vector
AAV
Herpes viral vector
Others
Structure of HIV Virus (Simple but Fatal)
Nucleocapsid
Life cycle of HIV
1. 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
Comparing Onco-retrovirus to Lentivirus
Onco-retrovirus
LTR
gag
R
LTR
pol
U3 R
env
U3
U5
y
Only infects dividing cells
Lentivirus (HIV-1)
Vif
LTR
Rev
Vpu
LTR
R
U3 R
U5
y
gag
U3
pol
Vpr Tat
env
Nef
Infects both dividing and non-dividing cells
Retroviral Recombination: lessons
learned from oncoretroviruses
LTR
gag
R
pol
LTR
env
U3 R
U3
U5
y
R
Gene of Interest
U5
y
LTR
Recombination can generate replication
Competent viruses
gag
R
U3
pol
env
U3 R
U3
U5
y
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 cisacting elements of HIV
1st Generation Vectors
• Limited homology between vector and helper
sequences
• Separation of helper plasmids
• Still retains HIV accessory genes in the
packaging plasmid
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
3rd Generation Vectors
Self-inactivating (SIN) vectors
Constructing The Self-Inactivated (SIN)
Lentiviral Vector
Vif
LTR
Rev
Vpu
R
LTR
U3 R
HIV-1 Provirus
gag
U5
U3
pol
Vpr Tat
env
Nef
y
R
Gene of Interest
Transducing Vector
y
BGH PA
LentiMax™
Helper Constructs
Vif
LTR
Rev
Vpu
R
U3 R
HIV-1 Provirus
gag
U5
Gag-Pol
VSV-G
LTR
U3
pol
Vpr Tat
env
y
Nef
Human Globin pA
CMV P
SV40 pA
Genome Replication
R U5
+ Strand RNA
U3 R
5’
y
gag
pol
An 3’
env
R
Reverse transcription
Integration
Provirus
(DNA)
LTR
R
LTR
U3 R
U5
+1
y
Transcription
R U5
SIN (Self Inactivation)
Provirus
(DNA)
LTR
R
env
U5
y
gag
pol
LTR
U3 R
U3
Deletion
R
U5
transcription
R U5
An
+ Strand RNA
Reverse transcription
Integration
U3
env
Provirus
(DNA)
X
y
gag
pol
R
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.
LentiMax™ Vector Application
• 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
VSV-G Pseudotyped Lentiviral Vectors
Efficiently Transduce Many Cell Types
R
GFP
HeLa
MCF10A
HEK 293
GTM3
BGH PA
VSV-G Pseudotyped Lentiviral Vectors Can Transduce
Primary Rat Hepatocytes
R
BGH PA
GFP
MOI:
100
50
25
10
Luciferase
Vectors:
EF-1α-PyMT = 3.7 x 106 particles/site
EF-1α-Luciferase = 5 x 106 particles/site
Bioluminescence imaging done after 1 month (7 mice).
MIPS Project #3811, UMB-Lentigen, Ricardo A. Feldman, March 1, 2007
Lenti-KitTM
AscI
NotI
LTR
SIN
LacZ
P
WPRE
pA
For
cDNA
Pri-miRNA
P
LacZ
IRES
copGFP
WPRE
P
LacZ
IRES
Puro
WPRE
SCMV
EF1a/HTLV
For shRNA
P
SCMV
copGFP
BamHI
ClaI
WPRE
PacI
LacZ
LacZ
H1: BamHI/PacI
U6: ClaI/pacI
Comparing Lentigen’s Lentiviral Vector Kit Product
to Other Commercial Kit Products
1.60E+09
1.40E+09
qPCR Titer
1.20E+09
1.00E+09
8.00E+08
6.00E+08
4.00E+08
2.00E+08
0.00E+00
Lentigen
A
Company
B
C
Experimental Design
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 #
Factors to Consider
• Transduction Method
• MOI (Multiplicity of Infection)
• Sensitivity to cytotoxicity
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
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
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
LentiMax™ Production System
Order Flow
Order Rec’d—Triage/Analysis
Customer forwards cDNA
for gene or shRNA
sequence
Lentigen Receives
cDNA or sequence
Cloning/Structural Analysis
Clone Picks Sent for
Sequence Analysis
Sequence Discrepancy
Reports Received & Analyzed
Plasmid Preparation
Order FlowCertificate of Analysis
Generated
Gel Verification
Product Shipped
Production of Viral
Particles
Email Customer
Shipment Alert
QC Testing
(Sterility & Titer)
Customer Receives
Product