- 1 - adenoexpress available with reporter genes: ad5.cmv-gfp ad5.cmv5-gfp ad5.cmv-lacz null...

- 1 -

AdenoExpress™

Available withReporter 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

CytokinesGM-CSF

IFN-

IFN-

IL-2

IL-12

- 3 -

AdenoExpress™

Angiostatic genesAngiostatin

Endostatin

Tumor suppressorsP53

Co-StimulatoryB7.1

- 4 -

AdenoExpress™

Format Concentration

(VP/mL)

Volume

(μL)

Total Qty

(VP)

Small 1X1011 100 1X1010

Large 1X1013 50 5X1011

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 CellsInfected with Ad5.CMV-GFP

Courtesy of Dr. Peter Hevezi, EOS Biotech

Visualizing gene expression by whole-body fluorescence imaging

Yang et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12278-12282

AntiCancer 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-GFPin the brain 24h after gene delivery.

- 12 -

Ad5.CMV5-GFP

External whole-body image of Ad5.CMV5-GFPgene expression in the liver

- 13 - Cumate Inducible Expression System

- 14 -

Cumate Inducible Promoter Regulatory Elements

- 15 -

Cumate Inducible Promoter Strategy 2

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:

1. Remove all defective particles from the preparation,

2. Remove the condition media containing undesired contaminants as well as serum,

3. Resuspend the virus in a buffer more suitable for later use and long term storage,

4. 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

1. Plaque Assay (Plaque Forming Units, PFU)

2. Tissue Culture Infectious Dose 50 (TCID50)

3. 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.

- 36 -

- 37 - CONTRÔLE DE LA QUALITÉ

- 38 -

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

PlanPlan

- 39 -

Qu’est-ce que la qualitéQu’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 qualitéQu’est-ce que le contrôle de la qualité

Vérification de la conformité du produit aux exigences spécifiées

- 41 -

Pourquoi le contrôle de la qualité?Pourquoi le contrôle de la qualité?

Assurer une reproductibilité du produit

- 42 -

Outils du contrôle de la qualitéOutils du contrôle de la qualité

Mode opératoire normalisé

Fiches analytiques

Assistant QC

- 43 -

Tâches de l’assistant QCTâches de l’assistant QC

C on fo rm e

R ecom m en ce le tes t

H ors sp é c ifica tion s

V é rifica tion d es sp é c ifica tion s

L ec tu re d es ré su lta ts

E ffec tu e le tes t

H ora ire d es tes ts

R eço it u n é ch an tillon

- 44 -

Qualités de l’assistant QCQualités de l’assistant QC

Autonome

Structuré

Polyvalent

Minutieux

Appliqué

Honnête

- 45 -

Documentation et Assurance de la qualitéDocumentation 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