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
Children’s 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)
repcap
ITR
Wild-type AAV
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
> 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, Children’s 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, Children’s 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 >10% 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 < 1%)

9. Vector Lot Serotype Transgene size (bp Backbone size (bp)
HEK DNA pg /109vg
Plasmid DNA
pg /109vg
06002
AAV2
4297
6980
27.0
11.0
003A
20.9
13.0
NHP
38.5
13.3
0802
AAV6
7.5
15.8
0803
6.3
17.7
avg (± sd)
14.2 (± 2.6)
0801
4406
2620
16.9
88.3
N0701
4679
2638
16.4
122
0701
10.1
77.6
0702
4811
12.8
103
0703
8.7
147
107.6 (± 27.6)
Use of oversized vector plasmid backbone reduced residual plasmid DNA 7.5-fold (p<0.001)

10. Orthogonal purification steps required to remove empty capsids
Col L1
Col L2
Col L3
Col L4
MWM
CsCl
Orthogonal purification steps required to remove empty capsids
(5e10 vg)
kDa 94 76 67 53 43 30
VP1 (87)
VP2 (73)
VP3 (61)

11. Formulation ionic strength and vector aggregation
Osmolarity = ∑ci
Ionic strength (µ) = ½ ∑cizi2

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
AAV vector characterization:
Certification for clinical use
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
Safety:
Adventitious viruses
Mycoplasma
WT AAV
USP sterility
Endotoxin pH
Osmolality
Aggregation
Appearance
METHOD
SDS-PAGE SS / WB
restriction digest / SB
DNA sequencing / 4-fold redundancy
SDS-PAGE SS / CB
optical Density 260 / 280nm
Q-PCR
ICP-MS
ELISA
transduction / 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
dynamic light scattering
visual inspection
2x1010 vg
VP1
VP2
VP3
MWM

13. Template for Clinical AAV Vector Quality Control Testing

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