| The year 1953 saw the isolation of the Adenovirus which was
soon recognized as an invaluable tool for investigating mammalian molecular biology. Several of the distinguished features
of Adenovirus have made it the preferred vehicle for gene
transfer and transgene expression in mammalian cells. The following
presents a small overview of the biology of Adenovirus.
Adenoviruses are associated with a number of disorders (eg.
common cold), most of which are mild. The pathology is primarily
from inflammation and loss of infected epithelial cells.
Viruses of subgroup C (serotype 2, 5) cause various respiration
infections in confined groups (elderly, military recruits and
Adenovirus is a non-enveloped 80-110 nm diameter virus presenting
an icosahedral symmetry. Human Adenoviruses contain
a linear, double stranded DNA genome, with a terminal
protein (TP) attached covalently to the 5’ termini. The DNA,
which has a length of approximately 36,000 bp, is wrapped in
a histone-like protein and has inverted terminal repeats (ITRs)
of 50-200 bp, which act as origins of replication.
The hexon, penton base, and knobbed fiber are the most
important capsid proteins with regards to gene delivery. Hexon
is the major protein forming the 20 triangular faces of the viral
capsid. The 240 hexon capsomers in the capsid are trimers,
each interacting with six other trimers. The 12 vertices are
formed by the penton capsomere, a complex of five copies of
the penton base, and three copies of fiber. Each penton capsomere
interacts with five hexon capsomeres, one from each of
the five faces that converge at the vertex. The knobbed fiber
protrudes from the fiber base.
Adenovirus has been adapted so that it can be used as both gene
delivery and gene therapy tools. Recombinant Adenovirus
(rAd) generated with the technologies offered by Qbiogene
have their E1 and E3 regions deleted. The E1 deletion prevents
the recombinant Adenovirus from replicating and therefore no
cell lysis occurs. Once packaged into a complementing cell line,
i.e. a cell line that provides the E1 products in trans (e.g. QBIHEK
293A cells), viral replication is made possible. The E3
region, not essential for viral growth, is also deleted. These two
deletions allow the introduction of the transgene of interest into
Gene delivery, gene therapy and protein over-expression
with recombinant Adenovirus
Gene delivery consists in introducing DNA and RNA into cells,
tissues, or organisms, in order to study regulation and function
of genes and proteins. The biggest hurdle that gene delivery technologies have to overcome is the cell membrane, which is
impermeable to negatively charged macromolecules such as
DNA and RNA. Numerous different gene delivery methods
using either chemical, physical or biological pathways have
been used in recent years and are constantly being improved
upon. The advantage of recombinant Adenovirus lies in its
potential to bind and efficiently enter mammalian cells
through its naturally occurring receptor (CAR) (see figure 2).
Recombinant Adenovirus is largely used in gene therapy which
aims at treating both genetic (e.g. cancer, haemophilia) and
infectious diseases (e.g. AIDS) by introducing new genetic
material into selected cells.
Recombinant Adenovirus can also be used in vaccination by
expressing a gene product that triggers an immune response.
Recombinant Adenovirus technology is also used to overexpress
proteins of interest and to subsequently study their
functions. In contrast to prokaryotic or insect-based systems,
the use of human cells permits the complex post-translational
protein modifications required to ensure the proper folding
and post-translational modifications of the protein. In addition,
the CMV5 promoter, being substantially stronger than the
standard CMV promoter, allows high expression of the protein
Qbiogene Adenoviral Vector Systems
Four different Adenoviral expression systems are offered for
the production of recombinant adenovirus. Two of these systems
are based on homologous recombination in E. coli, one
uses homologous recombination in human QBI-HEK 293A
cells, and our most recent system takes advantage of site-specific
transposition in E. coli.
The major differences between the four systems lie in the mode
of recombination and the transfer vectors. With each of the kits,
several transfer vectors are available. Some transfer vectors
come with a promoter and a poly A site, others benefit from a
user-supplied expression cassette. Several of our transfer vectors
are available with an MCS (multiple cloning site), whereas
others have a single cloning site. In addition, the strength of the
promoter, and therefore the level of expression of the protein, is
an important consideration for the researcher in the choice of
the kit to use.
Adsorption and entry into the cell
The adsorption of the virus to target cell receptors involves
high-affinity binding via the knob portion of the fibre. The
prime receptor for human Adenovirus serotype 5 is identical to
that for coxsackie B virus and has been named the Coxsackie/
Adenovirus receptor (CAR). After the attachment step, interaction
between the penton base and αv integrins on the cell
surface leads to internalisation of the virus through endocytosis.
Once inside the cell, the virus escapes the endosome with
help of the penton base, and translocates to the nuclear pore
complex, where the viral DNA is released into the nucleus and
transcription begins. Transcription, replication and viral packaging
take place in the nucleus of the infected cell.
A complex series of splicing accompanies transcription, and
genes are transcribed from both strands. Adenovirus transcription
is a two-phase event, early and late, occurring before and
after viral DNA replication, respectively. The early transcribed
regions are E1, E2, E3 and E4. The E1 gene products can be
further subdivided into E1A and E1B. E1 gene products are
involved in the replication of the virus. The E2 region is subdivided
into E2A and E2B. These proteins provide the machinery
for viral DNA replication and the ensuing transcription of
late genes. Most of the E3 proteins are involved in modulating
the immune response of infected cells, a function not essential
for viral growth in vitro. The gene products encoded by the E4
region (called ORFs 1-6/7) are involved in the metabolism of
virus messenger RNA and provide functions that promote
virus DNA replication and shut-off of host protein synthesis.
Furthermore, E4 products prevent viral DNA concatenation.
Advantages of using Recombinant Adenovirus
There are many advantages in using an Adenovirus to introduce
genetic material into host cells.
- Represents a homologous system for human genes: adenoviral
vectors use a human virus as vector and human cells as host. Therefore, human proteins have identical post-translational modifications as native proteins
- Has the ability to infect most mammalian cell types (both replicative
- Allows propagation in suspension thus permitting production of
- May be grown at high titer (1010 VP/mL, which can be concentrated
up to 1013 VP/mL)
- Accommodates reasonably large transgenes (up to 7.5 kb)
- Supports simultaneous expression of multiple genes
- Allows a high expression of the recombinant protein (up to 35% of
total cellular protein)
- Is well tolerated, with post-infection viability of the host cells
- Remains epichromosomal, i.e. does not integrate into the host
chromosome, inactivating genes or activating oncogenes
- Can be generated without any special equipment
- Can be used in combination with an inducible expression
All these advantages, and the extensive knowledge of viral
genetics, have made recombinant Adenovirus the vector of
choice for functional genomics research, protein-over-expression,
pre-clinical studies and clinical trials.
The plasmids, that carry part of the Ad5 genome and are used
in the recombination step, are all of the first generation, i.e.
∆E1/E3. The gene of interest, or the expression cassette, is inserted into the deleted E1 region.
Each kit is available with the principal components and controls
for the generation of 5 recombinant viruses and includes a comprehensive applications manual.