In otherwise successful gene therapy trials insertional
mutagenesis has resulted in leukemia. The identification of
new short synthetic genetic insulator elements (GIE) which
would both prevent such activation effects and shield the
transgene from silencing, is a main challenge. Previous attempts
with e.g. b-globin HS4, have met with poor efficacy
and genetic instability. We have investigated potential
improvement with two new candidate synthetic GIEs in
SIN-gamma and lentiviral vectors. With each constructs two
internal promoters have been tested: either the strong Fr-
MuLV-U3 or the housekeeping hPGK.We could identify a
specific combination of insulator 2 repeats which translates
into best functional activity, high titers and boundary effect in
both gammaretro and lentivectors. In target cells a dramatic
shift of expression is observed with an homogenous profile
the level of which strictly depends on the promoter strength.
These data remain stable in both HeLa cells over three
months and cord blood HSCs for two months, irrespective of
the multiplicity of infection (MOI). In comparison, control
native and SIN vectors expression levels show heterogeneous,
depend on the MOI and prove unstable. We have
undertaken genotoxicity assessment in comparing integration
patterns ingenuity in human target cells sampled over
three months using high-throughput pyro-sequencing. Data
will be presented. Further genotoxicity assessment will include
in vivo studies. We have established insulated vectors
which harbour both boundary and enhancer-blocking effect
and show stable in prolonged in vitro culture conditions.
Work performed with support of EC-DG research FP6-NoE,
CLINIGENE: LSHB-CT-2006-018933