Introduction of Lentigen’s HIV-1 Based Lentiviral Vector System

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. 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 Gene Delivery Vehicles

4. Structure of HIV Virus (Simple but Fatal) 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. R R gag pol U5 U5 Vif Rev Vpu LTR LTR U3 R U3 gag pol y env Vpr Tat Nef Comparing Onco-retrovirus to Lentivirus Onco-retrovirus LTR LTR U3 R env U3 y Only infects dividing cells Lentivirus (HIV-1) Infects both dividing and non-dividing cells

7. Retroviral Recombination: lessons learned from oncoretroviruses R R R gag gag pol pol LTR LTR U3 R U5 U5 U5 env U3 y Gene of Interest Recombination can generate replication Competent viruses U3 y LTR U3 R env U3 y

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. Vif Rev Vpu LTR LTR R U3 R HIV-1 Provirus U3 gag pol U5 env Vpr Tat Nef y R BGH PA Gene of Interest Constructing The Self-Inactivated (SIN) Lentiviral Vector Transducing Vector y LentiMax™

13. Vif Rev Vpu LTR LTR R U3 R HIV-1 Provirus U3 gag pol U5 env Vpr Tat Nef y SV40pA Helper Constructs Gag-Pol Human Globin pA CMVP VSV-G

14. R R U3 gag pol R U5 U3 R An 3’ y U5 U5 R env Reverse transcription Integration LTR LTR Provirus (DNA) Transcription +1 Genome Replication 5’ + Strand RNA y R

15. R R gag gag pol pol U5 U5 Deletion transcription R U5 An + Strand RNA Reverse transcription Integration R env Provirus (DNA) X U3 SIN (Self Inactivation) LTR LTR U3 R env Provirus (DNA) U3 y y

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. MCF10A HEK 293 R BGH PA HeLa GFP GTM3 VSV-G Pseudotyped Lentiviral Vectors Efficiently Transduce Many Cell Types

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

20. 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. LacZ P copGFP IRES WPRE copGFP P Lenti-KitTM AscI NotI LTR SIN LacZ P WPRE pA For cDNA Pri-miRNA LacZ P Puro IRES WPRE BamHI ClaI SCMV EF1a/HTLV PacI WPRE For shRNA LacZ LacZ SCMV H1: BamHI/PacI U6: ClaI/pacI

22. 1.60E+09 1.40E+09 1.20E+09 1.00E+09 qPCR Titer 8.00E+08 6.00E+08 4.00E+08 2.00E+08 0.00E+00 Lentigen A B C Company Comparing Lentigen’s Lentiviral Vector Kit Product to Other Commercial Kit Products

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 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

30. Order Flow Customer forwards cDNA for gene or shRNA sequence Lentigen Receives cDNA or sequence Cloning/Structural Analysis Order Rec’d—Triage/Analysis Clone Picks Sent for Sequence Analysis Sequence Discrepancy Reports Received & Analyzed Plasmid Preparation

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