7Transcription Units E1, E2, E4 Encode essential regulatory proteins involved in:Transactivation of viral and cellular promotersDNA replicationCell cycleApoptosisEssential for replication in cell culture
8Transcription Units E3 Escaping host immune defenses Preventing T cell cytotoxicityTumor necrosis factor (TNF) actionDispensable for replication in cell culture
9Transcription Units Late genes Encodes proteins required for packaging the viral genomeEssential for replication in cell culture
12Adenovirus Particles Docking at the Cell Surface Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
13Internalization of Adenovirus Into the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
14Adenovirus Trapped Within an Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
15Adenovirus Starting to Break Out of the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
16Adenovirus Escaping From the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
17Two Adenoviruses at a Nucleopore After Escaping the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
18Adenoviruses Accumulate in the Nuclei Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
19Advantages of using a recombinant Adenovirus 1. Broad host rangeCan infect a broad range of mammalian cellsAllows for the expression of recombinant proteins in most mammalian cell lines and tissuesHave been used extensively to express human as well as non-human proteins
20Advantages of using a recombinant Adenovirus 2. Infection and expression of genes in replicative and non-replicative cellsRetroviruses can only infect replicative cellsTransfection cannot be done in non-replicative cellsBest system to study gene expression in primary non-replicative cellsAllows for a direct comparison of results obtained with transformed cell lines and primary cells
21Advantages of using a recombinant Adenovirus 3. Replicates efficiently to high titersProduction of 1010 to 1011 VP/mLCan be concentrated up to 1013 VP/mL
22Advantages of using a recombinant Adenovirus 4. Helper independent Ad can accommodate up to 7.5 kb of foreign DNAAd 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
23Advantages of using a recombinant Adenovirus 5. Homologous system for human genesHuman virus as a vector and human cells as a hostProper folding and exact post-translational modifications of human proteinsMost human proteins are expressed at high levels and are fully functional.
24Advantages of using a recombinant Adenovirus 6. No insertional mutagenesis; remains epichromosomalRetroviruses integrate randomly into the host chromosome and can inactivate genes or activate oncogenesAd remains epichromosomal and therefore does not interfere with other host genes.
25Advantages of using a recombinant Adenovirus 7. Propagation in suspension cultures293 cells can be adapted to grow in suspensionAllow production scale-up: > 20L
26Advantages of using a recombinant Adenovirus 8. Simultaneous expression of multiple genesInsertion of two genes in a double expression cassetteCo-infection using different recombinant viruses each expressing a different proteinModulation of the expression by changing the MOI
27Construction of a Recombinant Adenovirus Deletion of E1 and E3 regionsRenders the virus replication defective: E1 being essential, it is complemented in a specially designed packaging cell line (293)Makes room for gene of interestGene of interest is cloned into a transfer vectorGene of interest is transferred into the viral genome by homologous recombination
28The Most Powerful Adenoviral Expression System Available AdenoVator™ SystemThe Most Powerful Adenoviral Expression System Available
29AdenoVator™ SystemFast and reliable: Only three easy steps to generate recombinant AdenovirusFully compatible with our AdEasy™ Vector SystemHighest 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 BFPComprehensive kit components, instruction manual and specialized after-sales technical support
30AdenoVator™ Principle Exploit the robust and efficient E. coli homologous recombination systemNo in vitro ligation or tedious manipulation of a large DNA3 step processClone cDNA in a transfer vectorIn vivo homologous recombination in bacteriaVirus production in 293 cells
32Increased Protein Expression AdenoVator™Increased Protein Expression
33AdenoVator™: 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.
34AdenoVator™: 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.
37Protein Over-Expression in Mammalian Cells The pAdenoVator-CMV5 transfer vectorAllows 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.
38Protein 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.
39Co-Expression with GFP & BFP AdenoVator™Co-Expression with GFP & BFP
40AdenoVator™: 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).
43AdenoVator™: 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.
44Adenoviral vector preparations AdenoExpress™AdenoExpressAdenoviral vector preparationsReady-to-use in vitro and in vivoFully CharacterizedQuality controlled
45AdenoExpress™ Titration by Infectious Unit (IU) Plaque Assay, TCID50 Viral ParticlesOD260Ratio VP:IU 30SterileMycoplasma FreeEndotoxin Free