Biology Reference
In-Depth Information
expression cassettes are signifi cantly smaller than 10 kB. Thus,
HC-Ad vector genomes are harboring so-called “stuffer” DNA in
order to have genome sizes in the preferable range. These stuffer
sequences should be derived from noncoding eucaryotic DNA,
should be free of repetitive and promoter elements, and should not
exhibit homology with the helper virus. A wide variety of different
plasmids harboring HC-Ad vector backbones to accommodate
differently sized transgene expression cassettes is available.
1.4
Helper Virus
In addition to HC-Ad vector plasmids also a variety of helper virus
plasmids is available [
11
,
20
,
23
]. In general, the helper virus
should—in addition to the E1 deletion and the fl oxed packaging
signal—not harbor any sequences which show homology to the
HC-Ad vector.
For HC-Ad vector production only purifi ed and fully charac-
terized helper virus stocks should be used. Helper viruses are pro-
duced as fi rst-generation vectors (
see
elsewhere in this topic,
see
Chapter
12
). We found it mandatory to characterize the genome
of the helper virus stocks to the greatest extent possible (including
restriction digestion, southern blotting and sequencing) in order
to ensure that only helper virus stocks with the correct confi gura-
tion and sequence of the fl oxed packaging signal are used.
We have made excellent experience with a helper virus based
on pESHV (unpublished), which is an E1-deleted helper virus
with a fl oxed packaging signal derived from Adlc8cluc [
15
].
In order to generate capsid-modifi ed, retargeted HC-Ad vectors it
is mandatory to use an accordingly capsid-modifi ed helper virus
[
23
,
24
]. It is suffi cient to use this helper virus only during the
large-scale production step.
The production process of HC-Ad vectors is divided into several
stages. First, small amounts of infectious HC-Ad vector are rescued
from its corresponding plasmid. Second, the small HC-Ad vector
amounts are then serially amplifi ed until a certain titer is reached
that allows for a medium-scale preparation. Third, this medium-
scale preparation is purifi ed, characterized, and used to generate a
large-scale preparation. To obtain a high-quality HC-Ad vector
preparation within a reasonable time it is mandatory to have a well-
organized cell-culture schedule.
1.4.1 Brief Outline of
HC-Ad Vector Production
2
Materials
2.1 Cultivation
116 Cells
1. 116 producer cells.
2. Minimal essential medium (MEM) for cultivating adherent
116 cells, supplemented with 10 % FCS, 100 U/mL penicillin-
streptomycin, 2 mM L-glutamine, and 0.1 mg/mL
hygromycin B.
