Artificial cells as a novel approach to gene therapy - Artificial Cells, Cell Engineering and Therapy - page 246

active factor IX from non-hepatic cells opens up the possibility of treating other
diseases that normally also require liver-specific proteins, e.g. -antitrysin,
apolipoproteins and other coagulation factors. However, each product must be
verified biologically for efficacy as not all products may function as well as
factor IX expressed from a non-hepatic source.

Promoter and enhancer

Sustained expression of therapeutic genes in vivo is an important consideration
in any form of gene therapy. Some viral genomes such as retrovirus and
lentivirus can integrate into the host's genome in vivo and achieve lifetime
expression. However, non-viral form of plasmid transfection generally used
for the genetic modification of encapsulated cells does not alter the genome of
the recipient. Inactivation or loss of plasmid DNA from transfected cells could
result in declining levels of expression even to background level. The human
cytomegalovirus (hCMV) promoter is one of the most powerful and popular
promoters that can drive high-level expression both in vitro and in vivo.
However, the high expression provided by hCMV is often unstable owing to
the high ratio of CpG motifs that bind cytokines resulting in inactivation of
hCMV promoter and contain repressor-binding sites (Yew et al., 2001).
Alternative hybrid promoters such as the mouse CMV/hEF1 promoter and
hCMV/Ubb promoter, which contain CpG-free motifs, have been constructed
and demonstrated to be as capable of driving expression as hCMV (Yew et al.,
2001). Thus, the choice of appropriate promoters and enhancers is a crucial
consideration for the establishment of a `universal' cell stock for

encapsulation.

10.3.3 Design of microenvironment

When the cells are encapsulated, they enter into a new microenvironment,
changing from being in a monolayer to a suspension in a polymer scaffold bound
by limiting membranes. Theoretically, fibroblasts would continue proliferating,
eventually breaking down the capsules, whereas myoblasts could differentiate
into myotubes, thus avoiding the overcrowding problem. In reality, after
encapsulation, the viability of encapsulated fibroblasts decreased to 50% after 4-
week culture in vitro but without rupturing the capsules, while the encapsulated
myoblasts did not proliferate and differentiate as well as in the unencapsulated
state (Chang et al., 1994). Hence, the microenvironment of the microcapsules is
less than optimal for these cell types. As many groups have reported (Peirone et
al., 1998a; Orive et al., 2003; Ponce et al., 2005), after sometime within the
microcapsules, the cells formed clusters that inhibited the exchange of oxygen,
nutrients and metabolic waste, leading to apoptosis or necrosis within (Orive et
al., 2003). Such deleterious effects not only limit the therapeutic efficacy of the