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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. AdenoExpress™. Available with Biological Response Genes Cytokines GM-CSF

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adenoexpress
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
adenoexpress2
AdenoExpress™

Available with Biological Response Genes

  • Cytokines
      • GM-CSF
      • IFN-a
      • IFN-g
      • IL-2
      • IL-12
adenoexpress3
AdenoExpress™
  • Angiostatic genes
      • Angiostatin
      • Endostatin
  • Tumor suppressors
      • P53
  • Co-Stimulatory
      • B7.1
adenoexpress4
AdenoExpress™

Two Formats Available

adenoexpress5
AdenoExpress™

Coronal Plane Section of Rat Striatums

Injected with Ad5.CMV-GFP

Courtesy of Dr. Michael Y. Chen, NIH

adenoexpress6
AdenoExpress™

Rat Spinal Cord

Injected with Ad5.CMV-LacZ

Courtesy of Dr. Michael J. Iadarola, NIH

adenoexpress7
AdenoExpress™

Mature Dentritic Cells

Infected with Ad5.CMV-GFP

Courtesy of Dr. Peter Hevezi, EOS Biotech

slide8
Visualizing gene expression by whole-body fluorescence imagingYang et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12278-12282AntiCancer Inc, San Diego, CA
ad5 cmv5 gfp
Ad5.CMV5-GFP

External and internal images of Ad5.CMV-GFP

ad5 cmv5 gfp11
Ad5.CMV5-GFP

External whole-body image of Ad5.CMV5-GFP

in the brain 24h after gene delivery.

ad5 cmv5 gfp12
Ad5.CMV5-GFP

External whole-body image of Ad5.CMV5-GFP

gene expression in the liver

adenovirus custom services23
Adenovirus Custom Services

From cloning of your cDNA into an Adenovirus transfer vector

to

cGMP Manufacturing

adenovirus custom services24
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.
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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).
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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.
adenovirus custom services27
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.
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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.
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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.

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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.
adenovirus custom services31
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.
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Adenovirus Custom Services

Recombinant Adenovirus Titration

  • Plaque Assay (Plaque Forming Units, PFU)
  • Tissue Culture Infectious Dose 50 (TCID50)
  • OD260 (Viral Particle, VP)
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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.
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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.
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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.
slide38
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
slide39
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

slide40
Qu’est-ce que le contrôle de la qualité
  • Vérification de la conformité du produit aux exigences spécifiées
slide41
Pourquoi le contrôle de la qualité?

Assurer une reproductibilité du produit

slide42
Outils du contrôle de la qualité
  • Mode opératoire normalisé
  • Fiches analytiques
  • Assistant QC
slide44
Qualités de l’assistant QC
  • Autonome
  • Structuré
  • Polyvalent
  • Minutieux
  • Appliqué
  • Honnête
slide45
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
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