Download presentation
Presentation is loading. Please wait.
1
Introduction of Lentigen’s HIV-1 Based Lentiviral Vector System
Hatem Zayed, PhD Jessica Boehmer
2
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
3
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
4
Structure of HIV Virus (Simple but Fatal)
Thank you Tim for your kind introduction and for this opportunity for me to share my experiences at VIRxSYS Corporation for the last 6 and half years in developing biological products; namely the process of taking a initial scientific concept all the way into clinical trials and developing pipeline products for HIV vaccines and cancer therapy. I would like to make this discussion as informal as possible. So if you have any questions please feel free to stop me at any time. Nucleocapsid
5
Life cycle of HIV 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
6
Comparing Onco-retrovirus to Lentivirus
LTR LTR gag pol U5 R U3 R env U3 y Only infects dividing cells Lentivirus (HIV-1) U3 U5 R y LTR gag pol env Vif Vpr Tat Rev Vpu Nef Infects both dividing and non-dividing cells
7
Retroviral Recombination: lessons learned from oncoretroviruses
gag pol U3 U5 R y LTR env U5 R y U3 Gene of Interest gag pol U3 U5 R y LTR env Recombination can generate replication Competent viruses
8
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
9
1st Generation Vectors Limited homology between vector and helper sequences Separation of helper plasmids Still retains HIV accessory genes in the packaging plasmid
10
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
11
3rd Generation Vectors Self-inactivating (SIN) vectors
12
Constructing The Self-Inactivated (SIN) Lentiviral Vector
y LTR gag pol env Vif Vpr Tat Rev Vpu Nef HIV-1 Provirus R Gene of Interest BGH PA Transducing Vector y LentiMax™
13
Helper Constructs y U3 U5 R gag pol env HIV-1 Provirus Gag-Pol
LTR gag pol env Vif Vpr Tat Rev Vpu Nef HIV-1 Provirus Gag-Pol Human Globin pA CMV P SV40 pA VSV-G
14
Genome Replication y An 3’ gag pol env U5 R U3 + Strand RNA 5’ R
Reverse transcription Integration Provirus (DNA) R U3 U5 y LTR +1 Transcription
15
SIN (Self Inactivation)
LTR LTR U5 R U3 R env Provirus (DNA) U3 gag pol U5 R Deletion y + Strand RNA An U5 R transcription Provirus (DNA) R gag pol env Reverse transcription Integration U3 y X
16
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.
17
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
18
VSV-G Pseudotyped Lentiviral Vectors
Efficiently Transduce Many Cell Types R GFP BGH PA HeLa MCF10A HEK 293 GTM3
19
VSV-G Pseudotyped Lentiviral Vectors Can Transduce Primary Rat Hepatocytes
GFP BGH PA MOI: 100 50 25 10
20
Bioluminescence imaging done after 1 month (7 mice).
Luciferase EF-1α-PyMT = 3.7 x 106 particles/site Vectors: 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
21
Lenti-KitTM LTR SIN P LacZ pA For cDNA Pri-miRNA P LacZ copGFP P LacZ
AscI NotI LTR SIN P LacZ WPRE pA For cDNA Pri-miRNA P LacZ IRES copGFP WPRE P LacZ IRES Puro WPRE SCMV EF1a/HTLV PacI BamHI ClaI For shRNA copGFP P WPRE LacZ LacZ SCMV H1: BamHI/PacI U6: ClaI/pacI
22
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 qPCR Titer
23
Experimental Design
24
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 #
25
Factors to Consider Transduction Method
MOI (Multiplicity of Infection) Sensitivity to cytotoxicity
26
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
27
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
28
Cytotoxicity Quality of Viral vectors Inherent nature of target cells
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
29
LentiMax™ Production System
29
30
Order Rec’d—Triage/Analysis
Order Flow Order Rec’d—Triage/Analysis Clone Picks Sent for Sequence Analysis Customer forwards cDNA for gene or shRNA sequence Sequence Discrepancy Reports Received & Analyzed Lentigen Receives cDNA or sequence Plasmid Preparation Cloning/Structural Analysis
31
Order Flow Certificate of Analysis Generated Gel Verification
Product Shipped Production of Viral Particles Customer Shipment Alert QC Testing (Sterility & Titer) Customer Receives Product
Similar presentations
