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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.
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Lentiviral Vector Production CoreIndiana University Vector Production FacilityPrincipal 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 Administrator IU VPF Manager Production Team Certification Laboratory Molecular Diagnostics Supervisor Supervisor Supervisor Technicians Technicians Technicians
Production (Room C) 1 2 1 2 3 Production (Room B) 6 Main Lab (Room A) 2 5 Ante Room R4-029 Floor Plan 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
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 Institutions Receiving Vector Retroviral Vectors Lentiviral Vectors • University of Wisconsin • University of Washington
LENTIVIRAL VECTORS • Vector integrates • Gene transfer rate can exceed 90% • Less dependent on cell cycle • Vectors (VSVG psedotyped) can be concentrated to high titer Pro • Possible risk of insertional mutagenesis • Possible risk of replication competent virus Con
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 Env 3’LTR pro pol vpr tat rev nef Lentiviral Vector Plasmids Transfer vector psi GFP CMV 5’LTR gag RRE SIN-3’LTR Packaging Construct gag CMV RRE polyA pro pol pRev RSV REV ß-globin intron pMD.G CMV VSV-G
Production of Lentivirus Collect Supernatant Hollow Fiber Tangential Flow Filtration CaPO4 *Serum free Media Change 20 Liter Production Final Product (Vial + Certify) *16 to 24 hours post transfection
20 Liter Production Runs 200-400 fold concentration with recovery of IU 80%
Vector Certification Sterility, Mycoplasma, In vitro, In vivo, bovine, porcine, Cell Identify, Human viruses Certification Master Cell Bank Production Run Sterility, Mycoplasma, RCL, Titer, Endotoxin, SV40/E1A transfer Vector integrity Filter and Vial Certification
RELEASE TESTING Sterility Mycoplasma In Vitro viral assay In Vivo viral assay Bovine viruses Porcine viruses Human viruses Cell identity 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 large- scale 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 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 500 nm HIV-1 Vector HIV-1 (CSCGW/VSVG) (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 Vector/beads ---- Stain ---- Particle size distribution (Count ~ 200 particles/5 grid spaces) aggregates not quantitated
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
Department of Medical and Molecular Genetics Collaborators David Sanders, Purdue Phil Zoltick, U. of Penn Rick Morgan, NIH, NCI Jeremy Luban, Columbia 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 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. Lab Alumni Guiandre Joseph Christoph Kahl Shangming Zhang, M.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