Biomedical Engineering Reference
In-Depth Information
repeat (LTR) promoters in retroviral vectors such as the pBABE series) may be exploited
[6]. Many early vector plasmids used the SV40 promoter; however, in recent years the Rous
sarcoma virus (RSV) promoter and the cytomegalovirus (CMV) immediate early promoter
have been more often utilized since they have been shown to drive a higher level constitu-
tive expression, although with different temporal characteristics [7]. Where tissue-specific
expression is desirable, a number of tissue-specific promoters have been employed with
some success. For example, therapeutic levels of factor VIII were achieved after gene ther-
apy with a liver-specific high-capacity adenoviral vector [8] in murine and canine models of
hemophilia; and a number of cell-type specific promoters have been described for most other
tissues, including endothelium, cardiac, skeletal, smooth muscle, epithelia, and skin with
varying results (reviewed in Sadeghi and Hitt [9]). One drawback of using tissue-specific
promoters can be that they are not as strong expressers as the viral promoters; therefore,
the use of viral enhancer elements has also been employed [10].
It is often desirable to be able to regulate gene expression, and the tetracycline (Tet)-
regulated expression system has been extensively used
in vitro
and
in vivo
[11, 12]. These
elements have also been incorporated into a number of gene therapy vectors (e.g. [13, 14]).
11.2.2.2 Other features
Viral vector plasmids usually lack some or all of the genes required for propagation, but
a packaging signal is maintained, together with other essential virally encoded regulatory
sequences; this is discussed in more detail below.
Expression of a selectable marker and/or a second therapeutic gene may be desirable
depending on the gene therapy strategy. These genes may be under the control of a separate
promoter [15] or an internal ribosome entry site (IRES) may be included for multi-cistronic
transcription [16]. As with plasmids designed for other applications, it is also desirable that
the plasmids developed for use in gene therapy applications contain sequences required for
efficient propagation of the vector in bacteria and a multi-cloning site (MCS) for cloning of
insert DNA. For viral vector plasmids these genes may be encoded on more than one separate
plasmid to further minimize the potential for replication-competent virus production.
11.2.3 Viral vectors
The life cycle of a generic virus comprises attachment of the virus via specific host cell
surface receptors, followed by entry into the cell. The virus then becomes uncoated to allow
release of viral genetic material into host cells (infection) followed by expression of viral
proteins and assembly of new viral particles (replication). Gene therapy vectors have been
developed to exploit these life cycles, using modified genomes that carry the therapeutic
gene cassette in place of large amounts of the viral genome. This is sometimes called 'trans-
duction,' which is defined as non-replicative or abortive infection to introduce functional
genetic information expressed from recombinant vectors into target cells [17, Figure 11.1].
Within the virus genome are genes required for replication and infection as well
as
cis
-acting regulatory sequences. Removal of most of the viral genes and regulatory
sequences is advantageous since it improves safety by minimizing the risk of reconstitution
into productive viral particles by recombination events, and increases the size of insert
DNA that can be incorporated. However, at least some of the excised viral genes are
still essential for propagation of vectors. Such genes are expressed on separate plasmids
