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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