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)

• 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

Infrastructure

Organization Chart

Production Team

Supervisor

Certification LaboratorySupervisor

Molecular DiagnosticsSupervisor

IU VPF Manager Administrator

Management

Technicians Technicians Technicians

Erol CetinokQA Specialist

Rafat Abonour, M.D.Director, IUSCC Clinical Research Office

Stephen Willaims, M.D.IU Simon Cancer Center Director

Quality Assurance

Legend1: Biological safety cabinets2: Incubators3: Refrigerators4: -20°C Freezers5: -70°C Freezers6: Storage racks Lab benches or shelves Sinks Pass through autoclave Clean room pass over line

R4-029 Floor Plan

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Funded in part by a NCRR Construction grant

Institutions Receiving VectorInstitutions Receiving Vector

• Case Western ReserveCase Western Reserve• Cincinnati Children’s HospitalCincinnati Children’s Hospital• Columbia UniversityColumbia University• Dana-FarberDana-Farber• Fred Hutchinson Fred Hutchinson • Indiana UniversityIndiana University• LA Children's HospitalLA Children's Hospital• MD AndersonMD Anderson• New England DeaconessNew England Deaconess• NIHNIH• Stanford UniversityStanford University• University of MichiganUniversity of Michigan• University of WashingtonUniversity of Washington• Washington UniversityWashington University

Retroviral Vectors Lentiviral Vectors• University of WisconsinUniversity of Wisconsin• University of WashingtonUniversity of Washington

Producing Lentiviral Vectors

LENTIVIRAL VECTORSLENTIVIRAL VECTORS• Vector integrates Vector integrates

• Gene transfer rate can exceed 90% Gene transfer rate can exceed 90%

• Less dependent on cell cycle Less dependent on cell cycle

• Vectors (VSVG psedotyped) can be Vectors (VSVG psedotyped) can be concentrated to high titerconcentrated to high titer

• Possible risk of insertional mutagenesisPossible risk of insertional mutagenesis

• Possible risk of replication competent virusPossible risk of replication competent virus

ProPro

ConCon

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

CMV gag RRE polyApro pol

RSV REVpRev

5’LTR gag SIN-3’LTRpsi

RRE CMV GFPTransfer vector

Packaging Construct

CMV VSV-Gß-globin intron

pMD.G

5’LTR pro pol

vif

vpr

vpuEnv

tatrev

3’LTR

nef

psiHIV

Lentiviral Vector Plasmids

gag

Production of LentivirusProduction of Lentivirus

CollectCollectSupernatantSupernatant

Hollow Fiber Tangential Flow FiltrationHollow Fiber Tangential Flow Filtration

Continue concentration100-200 fold

Perform Diafiltration 6 volume exchange

Add Benzonase

Concentration by Ultrafiltration to 2 Liters

Conduct Transient Transfection10 liters, serum free

Store overnight at 4 CHarvest second 10 liters day 2

CaPO4

*Serum free Media

Change

20 Liter Production

*16 to 24 hours post transfection

Final Product (Vial + Certify)

20 Liter Production Runs20 Liter Production Runs

200-400 fold concentration with recovery of IU 80%

Vector CertificationVector Certification

Master Cell Bank

Production Run

Filter and Vial

Certification

Certification

Sterility, Mycoplasma, In vitro, In vivo, bovine, porcine, Cell Identify, Human viruses

Sterility, Mycoplasma, RCL, Titer, Endotoxin, SV40/E1A transfer Vector integrity

RELEASE TESTING

MCB

SterilityMycoplasmaIn Vitro viral assayIn Vivo viral assayBovine virusesPorcine virusesHuman virusesCell identity

Vector Supernatant

SterilityMycoplasmaEndotoxinIn Vitro viral assayVector Insert StabilityTransfer of E1A, SV40RCL (supernatant)RCL (co-culture)P24 TiterInfectious TiterTransgene expression

Core Services for GTRP InvestigatorsProduce 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 predict1 Infectious particles per 1000 Virions

Kahl et al. J. Virol 78: 1421, 2004

Vectors contain transduction inhibitors or defective particles

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

VSV-G RRV

Infectious TiterPhysical Titer (p24)RNA Molecules/mL

•HIV-1 Vectors not Uniform-Vector particles of Expected Size (80-140 nm)-Smaller particles (30-50 nM)-Aggregates

HIV-1 Vector HIV-1(CSCGW/VSVG) (R7-GFP)

50 nm

D

500 nm100 nm

Transmission Electron Microscopy (TEM) of HIV-1 Vectors

Concentration of Vectors Removes Smaller Particles

Vector/beads ---- Stain ---- Particle size distribution (Count ~ 200 particles/5 grid spaces)

aggregates not quantitated

Pseudotyping Envelope Influences HIV-1 Vector Composition

POSTERFactors 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

IU CollaboratorsIU CollaboratorsKaren Pollok, Ph.D.Karen Pollok, Ph.D.Laura Haneline, Ph.D.Laura Haneline, Ph.D.Christie Orschell, Ph.DChristie Orschell, Ph.DEddy Srour, Ph.D. Eddy Srour, Ph.D. Vince Gatone, Ph.D.Vince Gatone, Ph.D.Mike Vasko, Ph.D.Mike Vasko, Ph.D.

CollaboratorsCollaboratorsDavid Sanders, PurdueDavid Sanders, PurduePhil Zoltick, U. of PennPhil Zoltick, U. of PennRick Morgan, NIH, NCIRick Morgan, NIH, NCIJeremy Luban, ColumbiaJeremy Luban, Columbia

IU- VPFIU- VPFKen CornettaKen CornettaScott CrossScott CrossLisa DuffyLisa DuffyLaksmi Sastry, PhDLaksmi Sastry, PhDDaniela Bischof, PhDDaniela Bischof, PhD

Clara HazelgroveClara HazelgroveSue KoopSue KoopLina SegoLina SegoJing YaoJing YaoSamantha GriffinSamantha GriffinAparna JastiAparna JastiLorraine MathesonLorraine MathesonErol CetinokErol Cetinok

Lab AlumniLab AlumniGuiandre JosephGuiandre JosephChristoph KahlChristoph KahlShangming Zhang, M.D.Shangming Zhang, M.D.

Support byNHLBI: HHSN26820074820 and P01 HL53586 (Dinauer)NCI: N02-RC-67002NCRR: U42 RR11148Lilly Endowment: Indiana Genomics Initiative (INGEN)

Department ofMedical and

Molecular Genetics

Understanding defective particles versus Understanding defective particles versus defective infection.defective infection.

gag/polgag/pol

envenv

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