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Lentiviral Vector Production Core Indiana University Vector Production Facility Principal Investigator: Ken Cornetta, M. D. Co-Principal Investigator : Lakshmi Sastry, Ph.D. Co-Principal Investigator : Daniela Bischof, Ph.D. Philosophy • Focus on bringing forth novel improvements in integrating vectors • Work aimed at Phase I/II products • Major aim is to serve academic community • In addition to production, focus on developing new release testing (viral vector specific) Infrastructure • Prior experience with retroviral vector production through the now defunct National Gene Vector Laboratory program • Currently maintain > 50 SOP related to vector production, certification and facility organization • DMF for lentiviral vectors filed with FDA • Audit by BCG for retro production in 2000 • Audit by BCG in 2005 for lenti production • Audit by FDA in 2003 without major deficiencies Organization Chart Management IU VPF Manager Production Team Supervisor Technicians Certification Laboratory Supervisor Technicians Administrator Molecular Diagnostics Supervisor Technicians Quality Assurance Stephen W illaims, M.D. IU Simon Cancer Center Director Rafat Abonour, M.D. Director, IUSCC Clinical Research Office Erol Cetinok QA Specialist R4-029 Floor Plan 5 1 2 2 Production (Room C) 1 Production (Room B) 2 Main Lab (Room A) Ante Room Legend 1: Biological safety cabinets 2: Incubators 3: Refrigerators 4: -20°C Freezers 5: -70°C Freezers 6: Storage racks Lab benches or shelves Sinks Pass through autoclave Clean room pass over line 3 6 Funded in part by a NCRR Construction grant Institutions Receiving Vector Retroviral Vectors • • • • • • • • • • • • • • Case Western Reserve Cincinnati Children’s Hospital Columbia University Dana-Farber Fred Hutchinson Indiana University LA Children's Hospital MD Anderson New England Deaconess NIH Stanford University University of Michigan University of Washington Washington University Lentiviral Vectors • University of Wisconsin • University of Washington Producing Lentiviral Vectors LENTIVIRAL VECTORS Pro • • • • Vector integrates Gene transfer rate can exceed 90% Less dependent on cell cycle Vectors (VSVG psedotyped) can be concentrated to high titer • Possible risk of insertional mutagenesis Con • Possible risk of replication competent virus Types of Lentiviral Vectors 1. HIV-1/HIV-2, FIV, SIV, EIAV 2. HIV-1 vectors most commonly used 3. Process developed for HIV-1 vector production HIV psi 5’LTR vpu vif gag pro pol vpr Env tat rev 3’LTR nef Lentiviral Vector Plasmids Transfer vector Packaging Construct pRev pMD.G 5’LTR psi gag RRE CMV gag CMV RRE pro RSV CMV REV ß-globin intron VSV-G GFP pol SIN-3’LTR polyA Production of Lentivirus Collect Supernatant Hollow Fiber Tangential Flow Filtration CaPO4 *Serum free Media Change 20 Liter Production Conduct Transient Transfection 10 liters, serum free Store overnight at 4 C Harvest second 10 liters day 2 Concentration by Ultrafiltration to 2 Liters Add Benzonase Perform Diafiltration 6 volume exchange *16 to 24 hours post transfection Continue concentration 100-200 fold Final Product (Vial + Certify) 20 Liter Production Runs 200-400 fold concentration with recovery of IU 80% Vector Certification Master Cell Bank Certification Sterility, Mycoplasma, In vitro, In vivo, bovine, porcine, Cell Identify, Human viruses Production Run Filter and Vial Certification Sterility, Mycoplasma, RCL, Titer, Endotoxin, SV40/E1A transfer Vector integrity RELEASE TESTING MCB Sterility Mycoplasma In Vitro viral assay In Vivo viral assay Bovine viruses Porcine viruses Human viruses Cell identity Vector Supernatant Sterility Mycoplasma Endotoxin In Vitro viral assay Vector Insert Stability Transfer of E1A, SV40 RCL (supernatant) RCL (co-culture) P24 Titer Infectious Titer Transgene expression Core Services for GTRP Investigators Produce clinical-grade lentiviral vectors for use in heart, lung, and blood clinical studies • Provide pilot runs for pre-clinical evaluation prior to production of largescale vector runs • Generate vectors under cGMP using envelopes and media tailored to the investigators needs • Assist in release testing to certify vectors for clinical use • Assist investigator with FDA required documentation Factors Influencing Quality of Lentiviral Vectors • Assessed by Potency, Safety and Stability • Influenced by production and processing methods Physical titers predict 1 Infectious particles per 1000 Virions 1.00E+11 1.00E+10 1.00E+09 Infectious Titer Physical Titer (p24) RNA Molecules/mL 1.00E+08 1.00E+07 1.00E+06 1.00E+05 VSV-G RRV Vectors contain transduction inhibitors or defective particles Kahl et al. J. Virol 78: 1421, 2004 Transmission Electron Microscopy (TEM) of HIV-1 Vectors D 50 nm 100 nm HIV-1 Vector (CSCGW/VSVG) 500 nm HIV-1 (R7-GFP) •HIV-1 Vectors not Uniform -Vector particles of Expected Size (80-140 nm) -Smaller particles (30-50 nM) -Aggregates Concentration of Vectors Removes Smaller Particles % Total Particles 100 75 CGW-V-SF(UC) 50 CGW-V-SF-C(C) CGW-V_SF-C(F) 25 0 30-50 50-80 80-120 Size (nm) Vector/beads ---- Stain ---- Particle size distribution (Count ~ 200 particles/5 grid spaces) aggregates not quantitated Pseudotyping Envelope Influences HIV-1 Vector Composition POSTER Factors Influencing Lentiviral Vector Quality-Dynamic Light Scattering (DLS) Analysis of HIV-1 Vectors (Formulation/Storage) Challenges/Future Goals • Increase scale of production • Packaging cell lines • Simplification of RCL testing • Continued development of release testing Lab Alumni Guiandre Joseph Christoph Kahl Shangming Zhang, M.D. Department of Medical and Molecular Genetics IU- VPF Ken Cornetta Scott Cross Lisa Duffy Laksmi Sastry, PhD Daniela Bischof, PhD Clara Hazelgrove Sue Koop Lina Sego Jing Yao Samantha Griffin Aparna Jasti Lorraine Matheson Erol Cetinok Collaborators David Sanders, Purdue Phil Zoltick, U. of Penn Rick Morgan, NIH, NCI Jeremy Luban, Columbia IU Collaborators Karen Pollok, Ph.D. Laura Haneline, Ph.D. Christie Orschell, Ph.D Eddy Srour, Ph.D. Vince Gatone, Ph.D. Mike Vasko, Ph.D. Support by NHLBI: HHSN26820074820 and P01 HL53586 (Dinauer) NCI: N02-RC-67002 NCRR: U42 RR11148 Lilly Endowment: Indiana Genomics Initiative (INGEN) Understanding defective particles versus defective infection. gag/pol env + -