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 of the barrier activities of
genetic elements in individual cells. This presentation will
illustrate how these assay systems were used to identify short
DNA elements that can insulate nearby genes from activation
by viral vector enhancer elements, and=or that can block the
propagation of a silent chromatin structure that leads to gene
silencing. We will show that small elements of the order of
100-400 nucleotides can be designed to achieve both insulator
and boundary function, as needed for safer integrating viral
vectors.