AdenoExpress™

1. AdenoExpress™ Available with • Reporter genes: • Ad5.CMV-GFP • Ad5.CMV5-GFP • Ad5.CMV-LacZ • Null Vectors: • Ad5.Null • Ad5.CMV-Null • Wild Type Viruses: • Adenovirus Wild Type Serotype 5 • Adenovirus Wild Type Serotype 2

2. AdenoExpress™ Available with Biological Response Genes • Cytokines • GM-CSF • IFN-a • IFN-g • IL-2 • IL-12

3. AdenoExpress™ • Angiostatic genes • Angiostatin • Endostatin • Tumor suppressors • P53 • Co-Stimulatory • B7.1

4. AdenoExpress™ Two Formats Available

5. AdenoExpress™ Coronal Plane Section of Rat Striatums Injected with Ad5.CMV-GFP Courtesy of Dr. Michael Y. Chen, NIH

6. AdenoExpress™ Rat Spinal Cord Injected with Ad5.CMV-LacZ Courtesy of Dr. Michael J. Iadarola, NIH

7. AdenoExpress™ Mature Dentritic Cells Infected with Ad5.CMV-GFP Courtesy of Dr. Peter Hevezi, EOS Biotech

8. Visualizing gene expression by whole-body fluorescence imagingYang et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12278-12282AntiCancer Inc, San Diego, CA

9. Ad5.CMV5-GFP External and internal images of Ad5.CMV-GFP

10. Ad5.CMV5-GFP

11. Ad5.CMV5-GFP External whole-body image of Ad5.CMV5-GFP in the brain 24h after gene delivery.

12. Ad5.CMV5-GFP External whole-body image of Ad5.CMV5-GFP gene expression in the liver

13. Cumate Inducible Expression System

14. Cumate Inducible Promoter Regulatory Elements

15. Cumate Inducible Promoter Strategy 2

16. Applications of the Cumate Inducible Promoter

17. Generation of Recombinant Adenoviruses Expressing Toxic Proteins

18. Generation of Recombinant Adenoviruses Expressing Toxic Proteins

19. Inducible Expression in Stable Cell Lines

20. Adenovirus-Based Expression Libraries

21. Adenovirus-Based Expression Libraries

22. Adenovirus Custom Services

23. Adenovirus Custom Services From cloning of your cDNA into an Adenovirus transfer vector to cGMP Manufacturing

24. Adenovirus Custom Services Cloning of Gene into an Adenovirus Transfer Vector • Subcloning of your gene of interest into an adenovirus transfer vector. • Verification of the orientation of the subcloned insert by restriction mapping.

25. Adenovirus Custom Services Construction of a Recombinant Adenovirus • Production of a recombinant adenovirus by in vivo homologous recombination. • Plaque purification • Screening (Southern, Western, or by PCR).

26. Adenovirus Custom Services Plaque Purification of a Recombinant Adenovirus Prior to Amplification • Plaque purification assay and isolation of a pure recombinant adenovirus (when Ad provided by customer). • Identification of a pure recombinant adenovirus by Southern, Western or by PCR screening.

27. Adenovirus Custom Services Amplification of a Recombinant Adenovirus • Amplification of a recombinant adenovirus in 293 cells at a desired volume. • Infected cells are harvested and three freezing and thawing cycles are performed, followed by centrifugation to remove cell debris from recombinant adenovirus.

28. Adenovirus Custom Services Double Cesium Chloride Gradient Purification of a Recombinant Adenovirus Amplified viral stocks should be purified in order to: • Remove all defective particles from the preparation, • Remove the condition media containing undesired contaminants as well as serum, • Resuspend the virus in a buffer more suitable for later use and long term storage, • Concentrate the virus to high levels.

29. Adenovirus Custom Services Double Cesium Chloride Gradient Purification of a Recombinant Adenovirus • Purification on a CsCl step gradient followed by a continuous CsCl gradient. • Dialysis of viral stock against desired buffer and aliquoting. P.S. Purification of a virus is essential before undertaking any in vivo project.

30. Adenovirus Custom Services Concentration of a Purified Recombinant Adenovirus • Concentration of purified virus via a filter centrifugation up to 1013 VP/mL. • Viral particle titration (VP) by OD260 measurement.

31. Adenovirus Custom Services Identity Test of Viral Stock • Infection of non-permissive cells (ex. HeLa) with amplified viral stock. • Extraction of cellular proteins from infected cells. • Western blotting on extracted proteins.

32. Adenovirus Custom Services Recombinant Adenovirus Titration • Plaque Assay (Plaque Forming Units, PFU) • Tissue Culture Infectious Dose 50 (TCID50) • OD260 (Viral Particle, VP)

33. Adenovirus Custom Services Time Course and MOI Assay for Protein Production • Infection of HeLa cells using the recombinant adenovirus with three different MOIs. • Harvesting infected cells at two time points, typically at 48 and 72 hours.

34. Adenovirus Custom Services Large-Scale Protein Production in HeLa Cells • Infection of HeLa cells in serum-free media with the recombinant adenovirus. • Protein expression in HeLa cells, according to characteristics of the recombinant adenovirus or custom specifications. • Harvesting infected cells and concentration by centrifugation. • Delivery of a cell pellet and/or culture supernatant.

35. Adenovirus Custom Services Clinical Grade (cGMP) Production • Pilot project production of adenoviral vectors under GLP, • Adenoviral vector production, clinical (cGMP) grade, • Complete safety testing, • cGMP compliance and FDA registration.

37. CONTRÔLE DE LA QUALITÉ

38. Plan • Qu’est-ce que la qualité • Qu’est-ce que le contrôle de la qualité • Pourquoi le contrôle de la qualité? • Outils du contrôle de la qualité • Tâches de l’assistant QC • Qualités de l’assistant QC • Documentation et Assurance de la qualité

39. Qu’est-ce que la qualité Ensemble des propriétés et des caractéristiques d’un service ou d’un produit qui lui confèrent l’aptitude à satisfaire des besoins exprimés ou implicites

40. Qu’est-ce que le contrôle de la qualité • Vérification de la conformité du produit aux exigences spécifiées

41. Pourquoi le contrôle de la qualité? Assurer une reproductibilité du produit

42. Outils du contrôle de la qualité • Mode opératoire normalisé • Fiches analytiques • Assistant QC

43. Tâches de l’assistant QC

44. Qualités de l’assistant QC • Autonome • Structuré • Polyvalent • Minutieux • Appliqué • Honnête

45. Documentation et Assurance de la qualité • Vérification de la documentation pour s’assurer du respect des méthodes et des spécifications • Assurance de la qualité: ensemble des activités nécessaires pour donner une confiance adéquate que les produits satisferont aux exigences spécifiées