Biomedical Engineering Reference
In-Depth Information
helper-dependent
, so that missing functions must be
supplied
in trans
. This can be accomplished by co-
introducing a helper virus or transfecting the cells
with a helper plasmid, each of which must carry the
missing genes. Usually steps are taken to prevent the
helper virus completing its own infection cycle, so
that only the recombinant vector is packaged. It is
also desirable to try and prevent recombination oc-
curring between the helper and the vector, as this can
generate wild-type replication-competent viruses as
contaminants. An alternative to the co-introduction
of helpers is to use a
complementary cell line
, which
is transformed with the appropriate genes. These
are sometimes termed 'packaging lines'. For many
applications, it is favourable to use vectors from
which all viral coding sequences have been deleted.
These
amplicons
(also described as '
gutless vectors
')
contain just the
cis
-acting elements required for
packaging and genome replication. The advantage
of such vectors is their high capacity for foreign DNA
and the fact that, since no viral gene products are
made, the vector has no intrinsic cytotoxic effects.
The choice of vector depends on the particular
properties of the virus and the intended host,
whether transient or stable expression is required
and how much DNA needs to be packaged. For
example, icosahedral viruses such as adenoviruses
and retroviruses package their genomes into pre-
formed capsids, whose volume defines the maximum
amount of foreign DNA that can be accommodated.
Conversely, rod-shaped viruses such as the baculo-
viruses form the capsid around the genome, so there
are no such size constraints. There is no ideal virus
for gene transfer - each has its own advantages and
disadvantages. However, in recent years, a number
of hybrid viral vectors have been developed incorpo-
rating the beneficial features of two or more viruses.
The interested reader can consult recent reviews on
this subject (Robbins
et al
. 1998, Reynolds
et al
.
1999). Also note that most of the principles discussed
above also apply to the use of plant viruses as vectors
(Chapter 12).
Adenovirus
Adenoviruses are DNA viruses with a linear, double-
stranded genome of approximately 36 kb. The genome
of serotype Ad5, from which many adenovirus
vectors are derived, is shown in Fig. 10.5. There are
six early-transcription units, most of which are
essential for viral replication, and a major late
transcript that encodes components of the capsid.
Adenoviruses have been widely used as gene trans-
fer and expression vectors, because they have many
advantageous features, including stability, a high
capacity for foreign DNA, a wide host range that
includes non-dividing cells, and the ability to pro-
duce high-titre stocks (up to 10
11
plaque-forming
units (pfu)/ml) (reviewed by Berkner 1992). They
are suitable for transient expression in dividing cells
because they do not integrate efficiently into the
genome, but prolonged expression can be achieved
in post-mitotic cells, such as neurons (e.g. see
Davidson
et al
. 1993, LaSalle
et al
. 1993). Adeno-
viruses are particularly attractive as gene therapy
vectors, because the virions are taken up efficiently
by cells
in vivo
and adenovirus-derived vaccines have
been used in humans with no reported side-effects.
However, the recent death of a patient following
an extreme inflammatory response to adenoviral
TL
L1
L2
L3
L4
L5
MLT
VA
E1a
E1b
plX
E3
3'
5'
5'
3'
0
Ψ
10
20
30
40
50
60
70
80
90
100
IVa2
E2a
E2b
E4
Fig. 10.5
Map of the adenovirus genome, showing the positions of the early transcription units (E), the major late transcript
(MLT), the tripartite leader (TL) and other genes (VA, pIX, IVa2). Terminal repeats are shown in pink,
ψ
is the packaging site.
