Biology Reference
In-Depth Information
transgene expression. As their fi rst-generation counterparts,
HC-Ad vectors remain episomally in the nucleus and thus have a
very low risk to cause insertional mutagenesis [
9
].
Beyond classical gene replacement/addition therapy
approaches recent results suggest that HC-Ad vectors may also be
favorable tools for genetic vaccination, potentially because of the
absence of immunocompetition between vector-derived and trans-
genic antigen epitopes as it occurs with fi rst-generation vectors
[
10
,
11
]. Comprehensive overviews over the various features of
HC-Ad vectors can be found in ref. [
12
-
14
].
1.2 116 Cell-Based
HC-Ad Vector
Production
Due to the fact that HC-Ad vectors are devoid of all viral coding
sequences, their production depends on a helper virus, which
delivers the adenoviral gene products required for replication and
packaging in trans. This helper virus is a replication-defi cient fi rst-
generation vector (
E3), which can only replicate and
deliver the gene products required for HC-Ad vector production
in cells that transcomplement
E1
. Various systems have been
developed during the last years to allow for preferential packaging
of HC-Ad vector genomes in the presence of helper virus genomes
[
15
-
19
]. The most widely used system is based on a helper virus
whose packaging signal
Δ
E1/
Δ
is fl anked by cre-recombinase recognition
sites (loxP, “fl oxed packaging signal”) [
15
]. This allows for
replication and packaging of HC-Ad vector genomes in the
presence of helper virus in cells which transcomplement
E1
and at
the same time express cre-recombinase to excise the packaging
signal from the helper virus. Obviously, the effi ciency with which
the packaging signal is excised from the helper virus genomes is a
major determinant for the degree of contamination of a HC-Ad
vector preparation with helper virus. To our best knowledge the
most reliable producer cell line for HC-Ad vectors up to date is the
116 cell line generated by Palmer and Ng [
20
]. While being based
on 293 cells to allow for transcomplementing E1, this cell line is
characterized by a high constitutive expression of the Cre-
recombinase. In addition, 116 cells can be maintained as adherent
monolayers or in suspension culture. The latter signifi cantly
facilitates upscaling of vector production.
Ψ
1.3 HC-Ad Vector
Plasmids
The only adenovirus-derived sequences of a HC-Ad vector are the
cis
-acting sequences comprising the inverted terminal repeats
(ITRs) and the packaging signal (
), which are required to replicate
(ITR) and encapsidate the vector genomes during production.
Therefore, the transgene capacity of HC-Ad vectors is about 36 kB
which corresponds to the genome size of wild-type adenovirus.
Importantly, in order to achieve effi cient packaging into stable
vector capsids the HC-Ad vector genome should be 28−30 kB in
size (including the transgene expression cassette) [
21
,
22
]. If the
vector genome is smaller than 27 kB or larger than 37 kB there is
a high risk of vector genome rearrangements or low yields. Typical
Ψ
