Download Document

Gene Delivery Systems
Nicolas Hoffmann
Pierre Jolicoeur
Applications
Transfection Reagent (90%)
Functional Genomics
-2-
Protein Over-Expression (5%)
Gene Therapy Research (5%)
Adenovirus Gene Delivery
Systems
Adenovirus Biology: an overview
-3-
Adenoviral Vectors: a valuable research
tool
Applications and Uses
Products and Services
Adenovirus Structure
-4-
Electron Micrograph of Purified
Adenoviruses Particles
-5-
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Transcription Units
-6-
Transcription Units
E1, E2, E4
-7-
Encode essential regulatory proteins
involved in:
Transactivation of viral and cellular
promoters
DNA replication
Cell cycle
Apoptosis
Essential for replication in cell culture
Transcription Units
E3
Escaping host immune defenses
Preventing T cell cytotoxicity
-8-
Tumor necrosis factor (TNF) action
Dispensable for replication in cell culture
Transcription Units
Late genes
Encodes proteins required for packaging
the viral genome
-9-
Essential for replication in cell culture
Adenovirus Attachment
- 10 -
Adenovirus Internalization
- 11 -
Adenovirus Particles Docking at the
Cell Surface
- 12 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Internalization of Adenovirus Into the
Endosome
- 13 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Trapped Within an
Endosome
- 14 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Starting to Break Out of the
Endosome
- 15 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenovirus Escaping From the
Endosome
- 16 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Two Adenoviruses at a Nucleopore
After Escaping the Endosome
- 17 -
Courtesy of Dr. Pierre Boulanger, Montpellier School of Medicine, France
Adenoviruses Accumulate in the
Nuclei
- 18 -
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
- 19 -
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
- 20 -
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 1010 to 1011 VP/mL
Can be concentrated up to 1013 VP/mL
- 21 -
Advantages of using a recombinant
Adenovirus
4. Helper independent Ad can
accommodate up to 7.5 kb of foreign
DNA
- 22 -
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
- 23 -
Human virus as a vector and human cells
as a host
Proper
folding
and
exact
posttranslational 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
- 24 -
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
- 25 -
Allow production scale-up: > 20L
Advantages of using a recombinant
Adenovirus
8. Simultaneous expression of multiple
genes
- 26 -
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
- 27 -
Gene of interest is cloned into a transfer
vector
Gene of interest is transferred into the viral
genome by homologous recombination
AdenoVator™ System
- 28 -
The Most Powerful Adenoviral
Expression System Available
AdenoVator™ System
- 29 -
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 coexpressed 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
- 30 -
3 step process
Clone cDNA in a transfer vector
In vivo homologous recombination in
bacteria
Virus production in 293 cells
AdenoVator™ Principle
- 31 -
AdenoVator™
- 32 -
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.
- 33 -
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.
- 34 -
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
- 35 -
Protein Over-Expression in
Mammalian Cells
- 36 -
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,
- 37 -
• 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
- 38 -
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™
- 39 -
Co-Expression with GFP & BFP
AdenoVator™: Co-Expression with
GFP and BFP
- 40 -
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
- 41 -
AdenoVator™: Co-Expression with
BFP
- 42 -
AdenoVator™: Co-Expression with
GFP and BFP
Allows for the easy identification of
infected cells with an inverted
fluorescent microscope,
- 43 -
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
- 44 -
Ready-to-use in vitro and in vivo
Fully Characterized
Quality controlled
AdenoExpress™
- 45 -
Titration by
Infectious Unit (IU)
Plaque Assay, TCID50
Viral Particles
OD260
Ratio VP:IU  30
Sterile
Mycoplasma Free
Endotoxin Free