Presentation on theme: "Gene Delivery Systems Nicolas Hoffmann Pierre Jolicoeur."— Presentation transcript:
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
Adenovirus Particles Docking at the Cell Surface Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Internalization of Adenovirus Into the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Trapped Within an Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Starting to Break Out of the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Escaping From the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Two Adenoviruses at a Nucleopore After Escaping the Endosome Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenoviruses Accumulate in the Nuclei Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
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
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
Advantages of using a recombinant Adenovirus 3. Replicates efficiently to high titers Production of to VP/mL Can be concentrated up to VP/mL
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
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.
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.
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
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
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
AdenoVator™ System The Most Powerful Adenoviral Expression System Available
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
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
AdenoVator™ Increased Protein Expression
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.
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.
AdenoVator™: Increased Protein Expression
Protein Over-Expression in Mammalian Cells
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.
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.
AdenoVator™ Co-Expression with GFP & BFP
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).
AdenoVator™: Co-Expression with GFP
AdenoVator™: Co-Expression with BFP
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.
AdenoExpress™ AdenoExpress Adenoviral vector preparations Ready-to-use in vitro and in vivo Fully Characterized Quality controlled
AdenoExpress™ Titration by Infectious Unit (IU) Plaque Assay, TCID 50 Viral Particles OD 260 Ratio VP:IU 30 Sterile Mycoplasma Free Endotoxin Free