Adeno-associated Virus Vectors to Support Adeno-associated Virus Vectors to Support Clinical Studies 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
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Adeno-associated Virus Vectors to Support Clinical Studies J. Fraser Wright, Ph.D. Principal Investigator, AAV Clinical Vector Laboratory Center for Cellular.
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Adeno-associated Virus Vectors to Support Clinical StudiesAdeno-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 MeetingNHLBI Gene Therapy Resource Program SessionBoston, May 28, 2008
• 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
repcapITRITR ITRITR
Wild-type Wild-type AAVAAV
• 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.
The NHBLI GTRP Clinical AAV Vector Laboratory, part of The NHBLI GTRP Clinical AAV Vector Laboratory, part of The Center for Cellular and Molecular Therapeutics, The Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, provides:Children’s Hospital of Philadelphia, provides:
Clinical Vector CoreClinical Vector Core
Center for Cellular and Center for Cellular and Molecular Therapeutics, Molecular Therapeutics, Children’s Hospital of Children’s Hospital of Philadelphia Philadelphia
General layoutGeneral layout
Purification-0.05” WG
Cell culture-0.10” WG
De-gowning-0.10” WG
Gowning-0.125” WG
Staging-0.15” WG
QC Lab0.00” WG
PT1
PT2
PT3
PT1
Overview of AAV2 Vector Overview of AAV2 Vector Biosynthesis MethodBiosynthesis Method
1. . Initiation and propagation of HEK293 Initiation and propagation of HEK293 cells from a Master Cell Bank vial cells from a Master Cell Bank vial
↓↓2.2. Seeding of HEK293 cells in roller bottles Seeding of HEK293 cells in roller bottles
↓↓3. 3. Transfection of HEK293 cellsTransfection of HEK293 cells
↓↓4. 4. Post transfection medium exchange Post transfection medium exchange
in serum free mediumin serum free medium
Overview of AAV VectorOverview of AAV VectorPurification ProcessPurification Process
1. 1. Vector harvest concentration by TFFVector harvest concentration by TFF↓↓
2. 2. Harvest lysis by microfluidization Harvest lysis by microfluidization and clarification by filtrationand clarification by filtration
↓↓3. 3. Vector purification by Vector purification by
ion exchange chromatographyion exchange chromatography↓↓
in vitro assay for viral contaminantagar cultivable and non-cultivable ICA with Adfour media, direct inoculationLALpotentiometryosmometrydynamic light scatteringvisual inspection
AAV vector characterization: AAV vector characterization: Certification for clinical useCertification for clinical use
- Characterize AAV vectors - Characterize AAV vectors manufactured for clinical studiesmanufactured for clinical studies
- Assess clinical lot consistency- Assess clinical lot consistency
- Ensure successful process - Ensure successful process transfer from research to clinical transfer from research to clinical manufacturing / comparabilitymanufacturing / comparability
2x1010 vg
VP1VP2 VP3
MWM
Template for Template for Clinical AAV Vector Clinical AAV Vector Quality Control Quality Control TestingTesting
AcknowledgementsAcknowledgements
GTRP Co-Investigators: Katherine HighGuang Qu
Clinical Vector Core: Bernd HauckOlga ZelenaiaJitin BajajXingge Liu
Process Development Guang QuJinmin Zhou
Regulatory Affairs Jennifer McDonnellGreg Podsakoff