Biomedical Engineering Reference
In-Depth Information
downstream of the polyhedrin promoter. The foreign
gene is cloned in another plasmid between two Tn
7
repeats and introduced into the bacmid-containing
bacteria, which also contains a third plasmid ex-
pressing Tn
7
transposase. Induction of transposase
synthesis results in the site-specific transposition of
the transgene into the bacmid, generating a recom-
binant baculovirus genome that can be isolated for
transfection into insect cells. Transposition of the
transgene into the bacmid interrupts the
lacZ
gene,
allowing recombinant bacterial colonies to be identi-
fied by blue-white screening.
Although baculoviruses productively infect insect
cells, they can also be taken up by mammalian cells,
although without producing progeny virions. The
number of mammalian cell lines reported to be trans-
duced by baculoviruses is growing (reviewed by Kost
& Condreay 1999) and this suggests that recombin-
ant baculoviruses could be developed as vectors for
gene therapy. A number of baculovirus-borne trans-
genes have been expressed using constitutive pro-
moters, such as the cytomegalovirus immediate
early promoter (Hoffman
et al
. 1995) and the Rous
sarcoma virus promoter (Boyce & Bucher 1996).
More recently, cell lines have been generated that
are stably transformed with baculovirus vectors,
although it is not clear whether the viral genome
has integrated in these cells (Condreay
et al
. 1999).
amplicon vectors can be constructed, which carry
only those
cis
-acting elements required for replica-
tion and packaging. These require packaging systems
to provide the missing functions
in trans
(e.g.
Stavropoulos & Strathdee 1998). Therapeutic use of
herpesvirus vectors has been limited, but a number
of genes have been successfully transferred to neu-
rons
in vivo
(e.g. Boviatsis
et al
. 1994, Lawrence
et al
.
1995). Generally, transgene expression is transient,
although prolonged expression has been observed
in some neuronal populations (see reviews by Vos
et al
. 1996, Simonato
et al
. 2000). Note that HSV
is also transmitted across neuronal synapses dur-
ing lytic infections, a phenomenon that can be ex-
ploited to trace axon pathways (Norgren & Lehman
1998).
Retrovirus vectors
Retroviruses are RNA viruses that replicate via a
dsDNA intermediate. The infection cycle involves
the precise integration of this intermediate into the
genome of the host cell, where it is transcribed
to yield daughter genomes that are packaged into
virions.
Retroviruses have been developed as vectors for a
number of reasons (reviewed by Miller 1992). First,
certain retroviruses are acutely oncogenic because
they carry particular genes that promote host cell
division. Investigation of such viruses has shown
that these
viral oncogenes
are in fact gain-of-function
derivatives of host genes,
proto-oncogenes
, which are
normally involved in the regulation of cell growth.
In most cases, the viral oncogenes are found to be
expressed as fusions with essential viral genes, ren-
dering the virus replication-defective. These
acute
transforming retroviruses
therefore demonstrate the
natural ability of retroviruses to act as replication-
defective gene-transfer vectors. Secondly, most re-
troviruses do not kill the host, but produce progeny
virons over an indefinite period. Retroviral vectors
can therefore be used to make stably transformed
cell lines. Thirdly, viral gene expression is driven by
strong promoters, which can be subverted to control
the expression of transgenes. In the case of murine
mammary-tumour virus, transcription from the viral
promoter is inducible by glucocorticoids, allowing
Herpesvirus vectors
The herpesviruses are large dsDNA viruses that
include EBV (discussed above) and the HSVs (e.g.
HSV-I, varicella zoster). Most HSVs are transmitted
without symptoms (varicella zoster virus is excep-
tional) and cause prolonged infections. Unlike EBV,
which is used as a replicon vector, HSV-I has been
developed as a transduction vector. Viral replication
can occur in many cell types in a wide range of
species if the genome is introduced by transfection,
but HSV vectors are particularly suitable for gene
therapy in the nervous system, because the virus is
remarkably neurotropic. As with other large viruses,
recombinants can be generated in transfected cells
by homologous recombination, and such vectors
may be replication-competent or helper-dependent
(Marconi
et al
. 1996). Alternatively, plasmid-based
