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

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

– Gene gun– Electroporation

• Viral– Retroviral vector

• Onco-retroviral vector– MuLV

• Lentiviral vector– HIV– SIV– FIV– EAIV– BAIV

– Adenoviral vector– AAV– Herpes viral vector– Others

Structure of HIV Virus (Simple but Fatal)

Nucleocapsid

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

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

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

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

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

Transducing VectorR

Gene of InterestBGH PA

U3

U5

R

RLTR LTR

gag polenv

U3

Vif

Vpr Tat

Rev Vpu

Nef

HIV-1 Provirus

LentiMax™

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

+ 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

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

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.

• 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

MCF10AHeLa

GTM3HEK 293

VSV-G Pseudotyped Lentiviral Vectors Efficiently Transduce Many Cell Types

R GFP

BGH PA

100 50 25MOI: 10

VSV-G Pseudotyped Lentiviral Vectors Can Transduce Primary Rat Hepatocytes

R GFP

BGH PA

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

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

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

Experimental DesignExperimental 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 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

Order FlowGel Verification

Production of Viral Particles

QC Testing (Sterility & Titer)

Certificate of Analysis

Generated

Product Shipped

Email Customer Shipment Alert

Customer Receives Product