Biomedical Engineering Reference
In-Depth Information
-peptide, allowing identification of appropriate recombinant through classic “blue/
white” selection in bacteria. Again, plasmids containing the appropriate recombi-
nants can be verified through restriction analysis, isolated, linearized, and transfected
into E1-complementing mammalian cells to produce recombinant adenovirus.
The major drawback of this vector system is its space limitation for carrying
foreign DNA and its inability to deliver long-term and stable transgene expression
in vivo
due to immune responses to transgene products and/or viral proteins
[24]
.
Initially, it was thought that, in the absence of replication, these vectors would be
safe and weakly immunogenic, providing high-efficiency, long-term gene expression.
However, it has been discovered through preclinical and clinical studies that replica-
tion is only one of the several viral features that must be inactivated to produce a
vector for gene transfer
in vivo
[24]
.
5.2.5.3 Second-Generation Adenoviral Vector
The purpose of second-generation vectors development is to overcome the limita-
tions of first-generation Ad vectors (
Fig. 5.6
). Second-generation Ad vectors gener-
ally describe vectors with some additional deletions in other important viral genes.
This additional deletion of important genes is based on the backbone of the first-
generation E1-deleted vectors. Deletion for
E1
/
E2
or
E1
/
E4
genes in ad vectors could
also be called second-generation vectors.
E2
and
E4
are both required for normal
viral replication, generating second-generation Ad vectors. These second-generation
vectors can only be propagated in cell lines that complement both
E1
and
E2
or
E1
and
E4
[25-29]
.
The replication of adenovirus DNA is dependent on the three main factors:
1.
Products of the
E2
gene
2.
E2a that encodes the viral DNA-binding protein
3.
E2b that encodes the preterminal protein and DNA polymerase
[30]
The E2a product is not only essential for viral DNA replication, but also upregu-
lates transcription from the major late promoter. This major late promoter controls
the synthesis of all viral structural proteins. Thus, E2a-deleted vectors are safer than
first-generation vectors.
Zhou and Beaudet applied the tTA-inducible system to enhance E2a promoter
activity in complementing cells
[29]
. In this tTA expression system, the tTA (trans-
activator protein) binds to the
tet
operators and induces transcription from the pro-
moter in the absence of tetracycline, but not in its presence
[31,32]
. This change in
the system leads to increasing the titer of E2a-deleted vectors. The
E1
and
E4
genes
play important roles in the regulation of viral and cellular gene expression. Second-
generation vectors having deletion of
E1
/
E4
are found to have reduced hepatotoxic-
ity and improved stability
in vivo
[33,34]
. Intravascular delivery of an E1/E4-deleted
adenovirus in a patient with ornithine transcarbamylase deficiency caused a severe
immune system reaction that led to multiple organ system failure
[35]
. This incident
has led to serious concerns regarding the safety of adenovirus delivered intravascu-
larly. The second-generation vector systems have provided some degree of improved
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