Biomedical Engineering Reference
In-Depth Information
(d) Selectable markers incorporated into vectors for
ex vivo
propagation could be anti-
genic if they enter the hemopoietic cell lineage. For example, if the protein were to be
expressed, it could lead to humoral immune responses, which would be detrimental
to patients [3].
3
Specificity
: Ideally a vector needs to be capable of targeting and entering a specific
pre-empted cell type or tissue, depending on the intended clinical application. This can
be done by adding targeting ligands or by using tissue-specific promoters, as discussed
later. Unpredictable side effects due to the ectopic expression of the transgene in normal
tissues should be avoided at all costs.
4
Regulation
: This is highly desirable, since it allows for
(a) activation of transgene when needed
(b) maintenance of transgene expression within a therapeutic window
(c) possibility to silence the gene if necessary; there has been some success
in vitro
and
in animal models using antibiotic-responsive promoters (e.g. TetON [4]), or use of
a hypoxia switch [5].
5
Delivery of genes of any size or function
: There is often a limit to the amount of foreign
DNA that can be inserted into viral vectors depending on the size of the wild-type viral
genome and the number of viral genes that can be deleted whilst retaining infectivity.
Inserts much larger than the endogenous DNA that has been removed will not be effi-
ciently packaged by viral structural proteins. In contrast, there is in theory no limit to
the size of DNA that can be delivered by non-viral methods of gene transfer.
6
Long term and elevated expression
: Viral transfer of DNA is more efficient than
non-viral transfer, and viruses have evolved strategies to avoid degradation within the host
cell. However, duration of transgene expression could be limited by the immunogenicity
of viral genes. DNA delivered by non-viral methods is sometimes rapidly removed from
cells by lysosomal degradation, although there are now a number of different strategies
to overcome this (see below).
7
Cost effective to produc
: Vectors should be cheap to produce in large quantities. Vectors
for non-viral transfer of DNA can be prepared rapidly with less stringent quality control
assays required before use, and usually have less batch-to-batch variation in potency.
On the other hand, complex purification procedures and quality control measures are
often required during production of sufficient quantities of viruses for gene therapy
applications. Viral vectors and non-viral vectors vary greatly in their design and ability
to deliver DNA to a cell. There are advantages and disadvantages to both systems, which
are summarized in Table 11.1.
11.2.2 Plasmid design
11.2.2.1 Promoters
It is essential when optimizing a vector for gene therapy that the therapeutic gene is
expressed with maximum efficiency at the right time and in the right cells; thus, a great
deal of research has been carried out into the best promoters to use. Many strategies have
employed viral promoters. Endogenous viral promoters (for example, the long terminal
