Welcome back to Applied Biological Materials Cell Culture series! In this video, we will
introduce the recombinant adenovirus expression system.
Viral vectors are tools commonly used to express exogenous genetic materials in vivo or in vitro
Among them, the most commonly used viral vectors are retrovirus, lentivirus,
adenovirus, and adeno-associated virus (AAV). Adenovirus or "Ad" has two common serotypes,
which are type 2 and type 5. Ad is a non-enveloped virus with a linear, double-stranded DNA genome.
Its nucleocapsid consists of many proteins with 3 major types: fiber, penton, and hexon.
The hexon is the structural basis of adenovirus and composes most of the viral capsid. Both
the fiber and penton based proteins are for receptor binding and internalization of the
adenovirus into host cells. All serotypes of adenoviruses use coxsakie- and adenovirus- receptor
(CAR) as the primary receptor on the host cell. After initial attachment via
CAR, the penton binds to the integrin receptors located on the surface of the cell. Next,
the binding induces the virus uptake by the host cell via endocytosis. Once inside the
host cell, the capsid of the virus is dismantled and the core protein-coated viral genome is
transported into the nucleus. In contrast to other viruses, adenovirus DNA does not
integrate into the host genome and instead remains in an episomal state.
Once the adenovirus enters the host cell, the first gene to be expressed is the E1A
gene. The presence of the E1A gene triggers the expression of the delayed early genes
in E1B, E2, E3, and E4 transcription units. The expression of adenoviral E1B, E2, E3 and
E4 genes will alter the expression of a group of cellular genes in the host cell to facilitate adenovirus
replication. ITR refers to the inverted terminal repeat sequences at the end of each genome
which contains all the elements necessary for replication and packaging.
Recombinant Ad expression vectors exploit the high nuclear transfer efficiency and the
low pathogenicity of the virus to deliver genes to the host cell. Adenovirus vectors
can be used in vaccines where the vector expresses a foreign antigenic protein. Also, adenovirus vectors
can be used in gene therapy where the vector expresses a functional
protein to correct a genetic defect. For the first generation vectors, E1 gene
region is replaced by the transgene or therapeutic gene to be delivered to the target cell with
a high activity promoter such as cytomegalovirus immediate early (CMV) promoter. In order to
propagate the recombinant virus that lacks E1 transgene expression, a complementing producer
cell line such as HEK293 or PER.C6 is required. Both cell lines are stable enough to express
the viral E1A and E1B proteins. Despite the E1 and E3 deletion, there is evidence
that shows viral genes are still expressed at low levels in cells transduced with first-generation vectors
This can cause direct toxicity and immune response in vivo. The second-generation
vectors are constructed with additional deletions of E2 or E4 function, which improved transgene
persistence and decrease inflammatory response. However, these vectors still exhibit leaky
expression of viral genes. The advantage is that the second generation recombinant
vectors can accommodate bigger transgene sizes. The construction of the vector genome usually
involves the direct cloning recombination or transposition techniques in E. coli cells.
Next, the vector is processed and released from the plasmid or cosmid backbone and then
transfected into the complimenting producer cell lines for virus packaging.
Helper-dependent adenovirus also known as "HD-Ad" vectors, or ‘gutless’ adenovirus vectors have had
all viral coding genes deleted. Therefore, HD-Ad vectors have greatly
reduced toxicity and immunogenicity in vivo, enabling long-term transgene expression.
Because they lack viral coding genes, they require a
helper virus in order to be produced. During the production stage, E1- and recombinase
expressing producer cells are transfected with recombinant HD-Ad vector DNA. The HD-Ad
vector DNA carries the transgene and co-infects with the helper virus. Inside the producing
cells, the packaging signal in the helper-virus is excised by recombinase, making it unable
to be packaged into viral particles. However, the helper virus still expresses
regulatory and structural proteins required for replication and packaging of the HD-Ad
vector DNA. The disadvantage of helper dependent adenovirus vectors is that an extra step is
required for viral purification. While most replication-incompetent adenovirus
vectors are used in research, it is not uncommon to have replication-competent adenovirus for
clinical trials. In fact, conditionally replication-competent adenoviruses are often exploited for their
cytotoxicity and are used exclusively on targeting tumor cells. Cancer cells are more permissive
to adenovirus replication because the gene expression pattern of cancer cells greatly
facilitates adenovirus replication. A general procedure of adenovirus amplification
begins with the transfection of producer cells with recombinant replication-defective adenovirus
vector carrying the transgene. The vector viruses are replicated and packaged in the
producer cells. After several rounds of infection using the producer cells with the vector lysate
for amplification, transgene vector particles are purified and then used to infect the targeted
cells. Many different viruses are being developed
as gene transfer vectors, but the most advanced ones are adenovirus, retrovirus (including
lentivirus) and adeno-associated virus. One of the major advantages of adenoviral
vectors is that they provide the most efficient gene transfer among other viral vector systems
for many cell types. Since, they do not integrate into host cell genome, there
are low disturbances in genes. As well, they are
not suitable for long term correction of chronic disorders but suitable for therapeutics that
needs high transient expression. Adenovirus vectors can be for clinical use,
such as for gene therapy and vaccination. RD Ad-based vectors have also been used to
treat cancer and tuberculosis. At abm, we offer a comprehensive library of
adenoviruses that contain full-length ORFs for human, mouse, and rat genes. For more
information on adenovirus techniques or cell culture techniques please check out our knowledge
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