Mammalian artificial chromosomes are natural chromosome-based vectors that may carry
a vast amount of genetic material in terms of both size and number. They are reasonably
stable and segregate well in both mitosis and meiosis. A platform artificial chromosome
expression system (ACEs) was earlier described with multiple loading sites for a modified
lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level
industrial protein production and the treatment of a mouse model for a devastating
human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic
gene lived more than four times longer than untreated counterparts. This novel gene
therapy method is called combined mammalian artificial chromosome-stem cell therapy.
At present, this method suffers from the limitation that a new selection marker gene
should be present for each therapeutic gene loaded onto the ACEs. Complex diseases
require the cooperative action of several genes for treatment, but only a limited
number of selection marker genes are available and there is also a risk of serious
side-effects caused by the unwanted expression of these marker genes in mammalian
cells, organs and organisms. We describe here a novel method to load multiple genes
onto the ACEs by using only two selectable marker genes. These markers may be removed
from the ACEs before therapeutic application. This novel technology could revolutionize
gene therapeutic applications targeting the treatment of complex disorders and cancers.
It could also speed up cell therapy by allowing researchers to engineer a chromosome
with a predetermined set of genetic factors to differentiate adult stem cells, embryonic
stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic
value. It is also a suitable tool for the investigation of complex biochemical pathways
in basic science by producing an ACEs with several genes from a signal transduction
pathway of interest.
