Adeno -associated Virus Vectors to Support Clinical Studies J . Fraser Wright, Ph.D.

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. repcap ITR 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. De-gowning -0.10” WG Clinical Vector Core Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia General layout 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. Use of oversized vector plasmid backbone reduced residual plasmid DNA 7.5-fold (p<0.001)

10. MWM Col L1 Col L2 Col L3 Col L4 CsCl MWM 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. 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 MWM VP1 VP2 VP3 2x1010 vg - 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 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 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

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