Adeno-associated Virus Vectors to Support Clinical Studies

1

• Adeno-associated Virus Vectors to Support
  Clinical Studies
• J. Fraser Wright, Ph.D.
• Principal Investigator, AAV Clinical Vector
  Laboratory
• Center for Cellular and Molecular Therapeutics
• Childrens Hospital of Philadelphia

American Society for Gene Therapy Annual
Meeting NHLBI Gene Therapy Resource Program
Session Boston, May 28, 2008
2
JF Wright Disclosures

• Consultant for
• Tacere Therapeutic (CA)
• Genzyme Corporation (MA)

3
Adeno associated virus (AAV)

• member of Parvoviridae family
• 25 nm diameter (small), non-enveloped (stable)
• ssDNA genome of 4.7 kb
• dependent upon helper virus for replication
• adenovirus, herpes simplex virus, others
• gt 9 distinct serotypes, many capsid variants
• no known disease association
• attractive vector for gene delivery because
• lack of pathogenicity
• defective self-replication
• long-term transgene expression in animal models
• various serotypes for different tissues

4
The NHBLI GTRP Clinical AAV Vector Laboratory,
part of The Center for Cellular and Molecular
Therapeutics, Childrens Hospital of
Philadelphia, provides

• Availability of a dedicated facility and staff
  with extensive experience in the development,
  manufacturing and certification of AAV vectors
  for clinical studies
• Commitment to support NHLBI Investigators in the
  translation of promising basic / pre-clinical
  research to clinical trials.

5
Clinical Vector Core Center for Cellular and
Molecular Therapeutics, Childrens Hospital of
Philadelphia General layout
De-gowning -0.10 WG
PT3
Purification -0.05 WG
QC Lab 0.00 WG
PT2
Cell culture -0.10 WG
PT1
PT1
Gowning -0.125 WG
Staging -0.15 WG
6
Overview of AAV2 Vector Biosynthesis Method 1.
Initiation and propagation of HEK293 cells from
a Master Cell Bank vial ? 2. Seeding of
HEK293 cells in roller bottles ? 3.
Transfection of HEK293 cells ? 4. Post
transfection medium exchange in serum free
medium
7

• Overview of AAV Vector
• Purification Process
• 1. Vector harvest concentration by TFF
• ?
• 2. Harvest lysis by microfluidization
• and clarification by filtration
• ?
• 3. Vector purification by
• ion exchange chromatography
• ?
• 4. Gradient centrifugation
• ?
• Buffer exchange by TFF,
• formulation, and 0.2µm filtration
• ?
• 6. Final 0.2 µm filtration and vial fill

8
Oversized cis plasmid backbone reduces
encapsidated DNA impurities

• - Using various vector generation platform
• - transfection of HEK293 using plasmid pRC, pDG
• - rAd infection of stably transfected cell lines
  
• - Significant packaging of vector plasmid
  backbone occurs.
• - This vector-related impurity is present at 5
  of vg in
• gradient-purified rAAV
• - Predicted to be gt10 in rAAV co-purified with
  empty capsids
• (Smith et al, 2003 Chadeuf et al, 2005)

Oversized plasmid backbone reduces unintended
encapsidation of plasmid DNA 7.5 fold (to lt 1)

9
Vector Lot Serotype Transgene size (bp Backbone size (bp) HEK DNApg /109vg Plasmid DNA pg /109vg
06002 AAV2 4297 6980 27.0 11.0
003A AAV2 4297 6980 20.9 13.0
NHP AAV2 4297 6980 38.5 13.3
0802 AAV6 4297 6980 7.5 15.8
0803 AAV6 4297 6980 6.3 17.7
avg ( sd) 14.2 ( 2.6)
0801 AAV2 4406 2620 16.9 88.3
N0701 AAV6 4679 2638 16.4 122
0701 AAV6 4679 2638 10.1 77.6
0702 AAV6 4811 2620 12.8 103
0703 AAV6 4811 2620 8.7 147
avg ( sd) 107.6 ( 27.6)
Use of oversized vector plasmid backbone reduced
residual plasmid DNA 7.5-fold (plt0.001)
10
Orthogonal purification steps required to remove
empty capsids
MWM Col L1 Col L2 Col L3 Col L4 CsCl MWM
(5e10 vg)
kDa 94 76 67 53 43 30
VP1 (87) VP2 (73) VP3 (61)
11
Formulation ionic strength and vector aggregation
Ionic strength (µ) ½ ?cizi2
Osmolarity ?ci
12
AAV vector characterization Certification for
clinical use
- Characterize AAV vectors manufactured for
clinical studies - Assess clinical lot
consistency - Ensure successful process transfer
from research to clinical manufacturing /
comparability

• PARAMETER
• Identity
• AAV capsid protein
• vector genome
• genome sequence
• Purity
• Protein impurities
• Residual plasmid DNA
• Residual mammalian DNA
• Residual cesium chloride
• Residual Benzonase
• Potency
• vector genomes
• in vitro transduction
• infectivity titer

METHOD SDS-PAGE SS / WB restriction digest /
SB DNA sequencing / 4-fold redundancy SDS-PAGE
SS / CB optical Density 260 /
280nm Q-PCR Q-PCR ICP-MS ELISA Q-PCR trans
duction / transgene ELISA limiting dilution /
Q-PCR in vitro assay for viral
contaminant agar cultivable and non-cultivable
ICA with Ad four media, direct
inoculation LAL potentiometry osmometry dynami
c light scattering visual inspection
13
Template for Clinical AAV Vector Quality Control
Testing
14
(No Transcript)
15
(No Transcript)
16
Acknowledgements

GTRP Co-Investigators Katherine High Guang
Qu Clinical Vector Core Bernd Hauck Olga
Zelenaia Jitin Bajaj Xingge
Liu Process Development Guang Qu Jinmin
Zhou Regulatory Affairs Jennifer
McDonnell Greg Podsakoff Research
Core Shangzhen Zhou Sonali Joyce Alex Tai