Biomedical Engineering Reference
In-Depth Information
Samulski 2000). The fact that AAV uses concate-
meric replication intermediates has been used to
circumvent perhaps the most serious disadvantage
of AAV vectors, which is the limited capacity for
foreign DNA. This strategy involves cloning a large
cDNA as two segments in two separate vectors,
which are co-introduced into the same cell. The 5
p5
p19
p40
polyA
rep
cap
ori, res, pac, int
Fig. 10.6
Map of the adeno-associated virus genome,
showing the three promoters, the
rep
and
cap
genes, and the
cis
-acting sites required for replication (
ori
), excision (rescue),
(
res
), packaging (
pac
) and integration (
int
). Terminal repeats
are shown as pink blocks.
′
portion of the cDNA is cloned in one vector, down-
stream of a promoter and upstream of a splice donor
site. The 3
portion is cloned in another vector,
downstream of a splice acceptor. Concatemerization
results in the formation of heterodimers and tran-
scription across the junction yields an mRNA that
can be processed to splice out the terminal repeats of
the vector. In this way, cDNAs of up to 10 kb can be
expressed (Nakai
et al
. 2000, Sun
et al
. 2000).
′
developed vectors in which both genes were deleted
and the transgene was expressed from either an endo-
genous or a heterologous promoter (McLaughlin
et al
. 1988, Samulski
et al
. 1989). From such experi-
ments, it was demonstrated that the repeats are the
only elements required for replication, transcription,
proviral integration and rescue. All current AAV
vectors are based on this principle.
In vitro
mani-
pulation of AAV is facilitated by cloning the inverted
terminal repeats in a plasmid vector and inserting
the transgene between them. Traditionally, recom-
binant viral stocks are produced by transfecting this
construct into cells along with a helper plasmid to
supply AAV products, and then infecting the cells
with adenovirus to stimulate lytic replication and
packaging. This has generally yielded recombinant
AAV titres too low to use for human gene therapy
and contaminated with helper AAV and adenovirus.
The recent development of transfection-based ade-
noviral helper plasmids, packaging lines and the use
of affinity chromatography to isolate AAV virions
has helped to alleviate such problems (reviewed by
Monahan & Samulski 2000).
AAV vectors have been used to introduce genes
efficiently into many cell types, including liver (Snyder
et al
. 1997), muscle (Pruchnic
et al
. 2000) and
neurons (Davidson
et al
. 2000). However, deletion
of the
rep
region abolishes the site specificity of provi-
ral integration, so the vector integrates at essentially
random positions, which may increase the risk of
insertional gene inactivation (Weitzman
et al
. 1994,
Yang
et al
. 1997, Young
et al
. 2000). It is also
unclear whether the persistence of the vector and
prolonged transgene expression are due primarily
to vector integration or to episomal maintenance
of concatemeric double-stranded DNA (dsDNA)
copies of the genome (for discussion see Monahan &
Baculovirus
Baculoviruses have rod-shaped capsids and large,
dsDNA genomes. They productively infect arthro-
pods, particularly insects. One group of baculoviruses,
known as the nuclear polyhedrosis viruses, have an
unusual infection cycle that involves the production
of
nuclear occlusion bodies
. These are proteinaceous
particles in which the virions are embedded, allow-
ing the virus to survive harsh environmental condi-
tions, such as desiccation (reviewed by Fraser 1992).
Baculovirus vectors are used mainly for high-level
transient protein expression in insects and insect
cells (King & Possee 1992, O'Reilley
et al
. 1992). The
occlusion bodies are relevant to vector development
because they consist predominantly of a single
protein, called polyhedrin, which is expressed at
very high levels. The nuclear-occlusion stage of the
infection cycle is non-essential for the productive
infection of cell lines; thus the polyhedrin gene
can be replaced with foreign DNA, which can be
expressed at high levels under the control of the
endogenous polyhedrin promoter. Two baculo-
viruses have been extensively developed as vectors,
namely the
Autographa californica
multiple nuclear
polyhedrosis virus (AcMNPV) and the
Bombyx mori
nuclear polyhedrosis virus (BmNPV). The former is
used for protein expression in insect cell lines, particu-
larly those derived from
Spodoptera frugiperda
(e.g.
Sf9, Sf21). The latter infects the silkworm and has
been used for the production of recombinant protein
