Biology Reference
In-Depth Information
Recombinant Ad and AAV vectors are widely used for gene therapy
approaches (see Chapter 8) and to date are the most common VNPs that
are under investigation in clinical trials. Large-scale production is thus an
important factor. For the most part, Ad and AAV are expressed using tissue
culture methods. For safer use in gene-delivery applications, replication-
deficient vectors are preferable. First-generation Ad vectors (genes E1 and
E3 deleted) and second-generation Ad vectors (E1, E3, and/or E4 deleted)
are based on type 5 Ad and rendered replication deficient through deletion of
viral proteins involved in replication and early gene regulation. Propagation
of replication-deficient Ads is facilitated by co-transfection of cells with so-
called helper vectors or plasmids that supply the necessary replications
proteins
.
AAV is a defective virus and relies by nature on the presence of
helper viruses, such as Ad (serotype 5) or HSV (serotype 1) (reviewed in
Lai
in trans
(in
trans
means “acting from another molecule”)
, 2002).
Insertion of the gene of interest can be achieved either by ligating
the sequence into backbone vector fragments or through homologous
recombination events. Human embryonic kidney (HEK) 293 cells are
typically used for propagation of recombinant Ads or AAVs. A range of
optimized propagation techniques have been developed (see the following
reviews and references therein: Choi
et al.
, 2007a,b; Clark, 2002; Grieger &
Samulski, 2005; Jozkowicz & Dulak, 2005; Lai
et al.
et al.
, 2002; Lu, 2004; Segura
et al.
, 2008). Also, there is substantial commercial support and a wide
variety of kits and reagents are available.
Ads and AAVs grow to high titers in tissue culture and yields obtained are
typically a few milligrams per 1 l cell culture. Compared with other systems,
yields are only moderate. Heterologous expression of plant or bacteriophage
VLPs gives rise to yields that are 10-100 times higher than those obtained
for Ads and AAV. Bioreactor techniques have been developed for large-scale
production (Iyer
, 1999). Yields in standard tissue culture are usually
higher compared with large-scale bioreactor production; on the downside
tissue culture techniques are labor-intensive and have limited potential for
large-scale production.
et al.
.
IN VITRO
ASSEMBLY, DISASSEMBLY, AND RE-ASSEMBLY
MethodS
As mentioned above, coat proteins expressed in
typically do not self-
assemble into intact capsids; this is, for example, the case for CCMV (Zhao
et al.
E. coli
, 1995). However, the proteins can be purified from
E. coli
lysates and
then assembled into intact VLPs using
In vitro
assembly methods have been established for a broad range of viruses. Intact
in vitro
assembly conditions.
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