In otherwise successful gene therapy trials for the treatment
of SCID patients and others, insertional mutagenesis
has resulted in leukemia development. Besides the integration
of vectors that including strong enhancers, more recently,
SIN-vectors have been shown to partially retain oncogenic
potential. The identification of genetic elements which would
both prevent such activation effects and shield the transgene
from silencing, is a main challenge. Previous attempts met
with difficulties in producing the vectors and poor efficacy of
the insulators (GIE). The improvement of integrating vectors
safety has been investigated using new candidate synthetic
GIEs. The latter have been introduced in retroviral and lentiviral
vectors. Native LTRs, SIN-LTRs, and SIN-insulated
constructs have been designed and compared, using two sets
of internal promoter, i.e. strong and housekeeping. We could
establish that a specific insulator translates at best into functional
activity and boundary effect in both vector types. We
could also determine that other genetic elements are key determinants
in order to achieve accurate expression and viral
titre, from these insulated vectors. A dramatic shift in the expression
profile is observed in target cells, with a homogenous
pattern including data on both cell-lines and primary HSCs
from cord blood. The assessment of potential genotoxicity will
be presented, based on the comparison of the integration
patterns ingenuity in human target cells sampled over a three
months period with both reference LTRs and SIN versus test
insulated vectors, using high-throughput pyro-sequencing.