Gene transfer that relies on integrating vectors often suffers
from epigenetic or regulatory effects that influence the expression
of the therapeutic gene and=or of cellular genes located
near the vector integration site in the chromosome.
Insulator elements act to block gene activation by enhancers,
while chromatin domain boundary or barrier sequences
prevent gene-silencing effects. At present, the modes of action
of insulator and barriers are poorly understood, and their use
in the context of gene therapies remains to be documented.
Using combinations of reporter genes coding for indicator
fluorescent proteins, we constructed assay systems that allow
the quantification of the insulator or barrier activities of genetic
elements in individual cells. This presentation will illustrate
how these assay systems were used to identify short
DNA elements that insulate nearby genes from activation by
viral vector elements, and=or that block the propagation of a
silent chromatin structure that leads to gene silencing. We
will show that some barrier elements do not merely block
repressive effects, but that they can act to stabilize and sustain
transgene expression. We will illustrate that this may be
beneficial when transgenes are introduced into stem or precursor
cells using non-viral vectors, where later differentiation
may lead to the silencing of the therapeutic gene. We will
show that these elements can be used to maintain efficient
transgene expression upon the differentiation of murine
precursor cells towards myofibers, in a model of cell therapy
for muscle dystrophies.