Biomedical Engineering Reference
In-Depth Information
Use of AAV vectors as gene therapy vectors is attractive owing to their large packaging
capacity of up to 4.9 kb [33] and the apparent lack of induction of cellular immune responses
[34]. However, humoral responses to AAV have been detected in animal models, and the
presence of neutralizing antibodies greatly reduces the success of vector readministration
[35, 36]. Since up to 90% of the population is seropositive for AAV and a significant
percentage could have preformed neutralizing antibodies to AAV-2 [37], this could limit the
usefulness of AAV for gene therapy applications [32].
AAV requires the presence of a helper virus for propagation, usually adenovirus (Ad)
or herpesvirus. In the absence of help, AAV integrates into a particular locus on q13.3qter
human chromosome 19 and becomes latent. Although this process requires AAV rep proteins
which are deleted in AAV vectors, transgene expression has been reported for months and
up to several years in some
in vivo
models, possibly due to long-lived double-stranded
episomal rAAV genomes or by random integration into the host cell genome For a recent
review of AAV vector developments, see Buning
et al
. [38].
11.2.3.6 Adenoviruses
Ads are non-enveloped viruses with a linear double-stranded DNA genome. There are 50
distinct serotypes in humans. They are associated with the common cold and they can cause
respiratory, intestinal and eye infections in humans [32, 39]. Ads have been widely used
for gene transfer since they have a broad host range and can infect both proliferating and
non-dividing cells. However, the Ad genome does not integrate into the host cell genome,
so infection with Ad vectors will only lead to transient gene expression.
Based on the time of transcription of each gene after infection, the Ad genome is divided
into the early (E) and late (L) regions, with an ITR at either end [40]. Ad enters the host cell
via specific cell surface receptors;
in vitro
, these include the coxsackievirus and Ad receptor
(CAR [41]).
In vivo
, the receptor usage remains controversial [42, 43]. Ad is internalized
rapidly via receptor-mediated endocytosis, facilitated via receptors including integrins
α
v
β
3
and
α
v
β
5 [44].
A number of Ad vectors have been developed, including replication-competent and
replication-defective vectors, mostly based on serotype 2 (Ad2) or serotype 5 (Ad5). The
first generation of replication-defective Ad vectors was E1- and E3-deleted. The second
generation includes E1-, E3- and E4- or E2-deleted vectors based on Ad5. E1-deleted Ad5
vectors can be grown in specific cell lines such as human embryonic kidney 293 cells [45]
transformed with Ad E1, to supply E1 in
trans
. The left-hand ITR and packaging signals
from the left-hand 300 bp of the genome are required for replication in 293 cells [46].
First-generation Ad vectors were strongly immunogenic, with immunosuppressive drugs
needed to extend transgene expression [47, 48]. However, second-generation Ad vectors
have overcome Ad immunogenicity to some extent by introduction of a mutation in the Ad
E2a gene [49], or deletion in E4 [50] with only modest success [51].
To reduce immunogenicity, a gutted (or gutless) Ad vector was developed containing
only the ITR required for replication and 5
-
cis
-acting Ad encapsulation signals necessary
for packaging [52]. However, this vector is difficult to produce, requiring the use of helper
virus to provide all the viral proteins in
trans
[32].
Outlined below is a protocol for co-transfection of FG293 cells with Ad5 shuttle vector
pDC516 and pBHGfrt genomic plasmid (see Protocol 11.1), followed by a protocol to purify
these Ad5 plaques by end-point dilution (see Protocol 11.2). Protocols for bulk preparation
of crude lysates of high titre Ad5 stock (see Protocol 11.3) and preparation of CsCl
2
purified
recombinant Ad5 (see Protocol 11.4) follow.
