1. Nicolas Hoffmann Pierre Jolicoeur
Gene Delivery Systems
Nicolas Hoffmann
Pierre Jolicoeur

2. Applications Transfection Reagent (90%) Functional Genomics
Protein Over-Expression (5%)
Gene Therapy Research (5%)

3. Adenovirus Gene Delivery Systems
Adenovirus Biology: an overview
Adenoviral Vectors: a valuable research tool
Applications and Uses
Products and Services

4. Adenovirus Structure

5. Electron Micrograph of Purified Adenoviruses Particles
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

6. Transcription Units

7. Transcription Units E1, E2, E4
Encode essential regulatory proteins involved in:
Transactivation of viral and cellular promoters
DNA replication
Cell cycle
Apoptosis
Essential for replication in cell culture

8. Transcription Units E3 Escaping host immune defenses
Preventing T cell cytotoxicity
Tumor necrosis factor (TNF) action
Dispensable for replication in cell culture

9. Transcription Units Late genes
Encodes proteins required for packaging the viral genome
Essential for replication in cell culture

10. Adenovirus Attachment

11. Adenovirus Internalization

12. Adenovirus Particles Docking at the Cell Surface
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

13. Internalization of Adenovirus Into the Endosome
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

14. Adenovirus Trapped Within an Endosome
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

15. Adenovirus Starting to Break Out of the Endosome
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

16. Adenovirus Escaping From the Endosome
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

17. Two Adenoviruses at a Nucleopore After Escaping the Endosome
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

18. Adenoviruses Accumulate in the Nuclei
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France

19. Advantages of using a recombinant Adenovirus
1. Broad host range
Can infect a broad range of mammalian cells
Allows for the expression of recombinant proteins in most mammalian cell lines and tissues
Have been used extensively to express human as well as non-human proteins

20. Advantages of using a recombinant Adenovirus
2. Infection and expression of genes in replicative and non-replicative cells
Retroviruses can only infect replicative cells
Transfection cannot be done in non-replicative cells
Best system to study gene expression in primary non-replicative cells
Allows for a direct comparison of results obtained with transformed cell lines and primary cells

21. Advantages of using a recombinant Adenovirus
3. Replicates efficiently to high titers
Production of 1010 to 1011 VP/mL
Can be concentrated up to 1013 VP/mL

22. Advantages of using a recombinant Adenovirus
4. Helper independent Ad can accommodate up to 7.5 kb of foreign DNA
Ad can normally encapsidate a viral DNA molecule slightly bigger than the normal DNA (105%)
To provide additional cloning space, the E1 and E3 early regions of Ad have been deleted

23. Advantages of using a recombinant Adenovirus
5. Homologous system for human genes
Human virus as a vector and human cells as a host
Proper folding and exact post-translational modifications of human proteins
Most human proteins are expressed at high levels and are fully functional.

24. Advantages of using a recombinant Adenovirus
6. No insertional mutagenesis; remains epichromosomal
Retroviruses integrate randomly into the host chromosome and can inactivate genes or activate oncogenes
Ad remains epichromosomal and therefore does not interfere with other host genes.

25. Advantages of using a recombinant Adenovirus
7. Propagation in suspension cultures
293 cells can be adapted to grow in suspension
Allow production scale-up: > 20L

26. Advantages of using a recombinant Adenovirus
8. Simultaneous expression of multiple genes
Insertion of two genes in a double expression cassette
Co-infection using different recombinant viruses each expressing a different protein
Modulation of the expression by changing the MOI

27. Construction of a Recombinant Adenovirus
Deletion of E1 and E3 regions
Renders the virus replication defective: E1 being essential, it is complemented in a specially designed packaging cell line (293)
Makes room for gene of interest
Gene of interest is cloned into a transfer vector
Gene of interest is transferred into the viral genome by homologous recombination

28. The Most Powerful Adenoviral Expression System Available
AdenoVator™ System
The Most Powerful Adenoviral Expression System Available

29. AdenoVator™ System
Fast and reliable: Only three easy steps to generate recombinant Adenovirus
Fully compatible with our AdEasy™ Vector System
Highest level protein expression available using our unique CMV5 promoter (20-30% TCP)
Rapid identification and quantification of co-expressed gene of interest using GFP or BFP
Comprehensive kit components, instruction manual and specialized after-sales technical support

30. AdenoVator™ Principle
Exploit the robust and efficient E. coli homologous recombination system
No in vitro ligation or tedious manipulation of a large DNA
3 step process
Clone cDNA in a transfer vector
In vivo homologous recombination in bacteria
Virus production in 293 cells

31. AdenoVator™ Principle

32. Increased Protein Expression
AdenoVator™
Increased Protein Expression

33. AdenoVator™: Increased Protein Expression
Regular AdV with CMV-based expression cassettes rarely produce protein exceeding 1 to 2% Total Cellular Protein (TCP) after infection of either non-permissive cells or 293 cells.
The main feature of our new vectors is the CMV5 promoter, a combination of enhancer sequences that increases the transcriptional and translational activities of the CMV promoter in recombinant AdV.

34. AdenoVator™: Increased Protein Expression
The CMV5 promoter was constructed by:
Insertion of the adenovirus tripartite leader (Ad-tpl) with the adenovirus major late enhancer bracketed by splice donor and acceptor sites.
The Ad-tpl binds translation-initiating proteins much more efficiently than most messages. This is a strategy developed by the virus for efficient translation of the late proteins and was exploited in these expression vectors.

35. AdenoVator™: Increased Protein Expression

36. Protein Over-Expression in Mammalian Cells

37. Protein Over-Expression in Mammalian Cells
The pAdenoVator-CMV5 transfer vector
Allows for very high levels of recombinant protein production in non-permissive cell lines,
Greatly simplifies the production and purification effort, since the recombinant protein could be produced at levels approaching 15 to 30% of total cellular protein (TCP) in the absence of the synthesis of other viral proteins.

38. Protein Over-Expression in Mammalian Cells
Because of the absence of viral replication and host protein shut-off, the production host can be maintained in good physiological state for prolonged periods of time and continue to synthesize the recombinant protein, if secreted in the medium,
The higher level of expression that can be achieved with these AdV in non-permissive cells will make it possible to express transgenes at significant levels in vivo at lower MOIs, thereby decreasing the toxic side effects associated with the load of adenovirus particles.

39. Co-Expression with GFP & BFP
AdenoVator™
Co-Expression with GFP & BFP

40. AdenoVator™: Co-Expression with GFP and BFP
Markers of gene expression detectable in living cells and whole organisms,
GFP and BFP genes are encoded as the second cistron in a dicistronic expression cassette.
The gene of interest is cloned into the position of the first cistron.
The two cistrons are separated by the encephalomyocarditis virus Internal Ribosomal Entry Site (IRES).

41. AdenoVator™: Co-Expression with GFP

42. AdenoVator™: Co-Expression with BFP

43. AdenoVator™: Co-Expression with GFP and BFP
Allows for the easy identification of infected cells with an inverted fluorescent microscope,
Possible to gauge the level of transgene expression following infection,
Can be used to measure the effectiveness of the gene transfer protocol in vivo.

44. Adenoviral vector preparations
AdenoExpress™
AdenoExpress
Adenoviral vector preparations
Ready-to-use in vitro and in vivo
Fully Characterized
Quality controlled

45. AdenoExpress™ Titration by Infectious Unit (IU) Plaque Assay, TCID50
Viral Particles
OD260
Ratio VP:IU  30
Sterile
Mycoplasma Free
Endotoxin Free