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 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) 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

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

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

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

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 y LTR gag pol env Vif Vpr Tat Rev Vpu Nef HIV-1 Provirus R Gene of Interest BGH PA Transducing Vector y LentiMax™

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

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

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

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 BGH PA HeLa MCF10A HEK 293 GTM3

VSV-G Pseudotyped Lentiviral Vectors Can Transduce Primary Rat Hepatocytes GFP BGH PA MOI: 100 50 25 10

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

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

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

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 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

LentiMax™ Production System 29

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

Order Flow Certificate of Analysis Generated Gel Verification Product Shipped Production of Viral Particles Email Customer Shipment Alert QC Testing (Sterility & Titer) Customer Receives Product