Transcript Slide 1

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
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2
2
Production (Room C)
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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
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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
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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
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