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Safe selection of genetically manipulated human primary keratinocytes with very high growth potential using CD24.
[junctional epidermolysis bullosa]
Stable
and
safe
corrective
gene
transfer
in
stem
keratinocytes
is
necessary
for
ensuring
success
in
cutaneous
gene
therapy
.
There
have
been
numerous
encouraging
preclinical
approaches
to
cutaneous
gene
therapy
in
the
past
decade
,
but
it
is
only
recently
that
a
human
volunteer
suffering
from
junctional
epidermolysis
bullosa
could
be
successfully
grafted
using
his
own
non-selected
,
genetically
corrected
epidermal
keratinocytes
.
However
,
ex
vivo
correction
of
cancer
-prone
genetic
disorders
necessitates
a
totally
pure
population
of
stably
transduced
stem
keratinocytes
for
grafting
.
Antibiotic
selection
is
not
compatible
with
the
need
for
full
respect
for
natural
cell
fate
potential
and
avoidance
of
immunogenic
response
in
vivo
.
In
order
to
surmount
these
problems
,
we
developed
a
strategy
for
selecting
genetically
modified
stem
cell
keratinocytes
.
Driving
ectopic
expression
of
CD
24
(
a
marker
of
post-mitotic
keratinocytes
)
at
the
surface
of
clonogenic
keratinocytes
permitted
their
full
selection
.
Engineered
keratinocytes
expressing
CD
24
and
the
green
fluorescent
protein
(
GFP
)
tracer
gene
were
shown
to
retain
their
original
growth
and
differentiation
potentials
both
in
vitro
and
in
vivo
over
300
generations
.
Also
,
they
did
not
exhibit
signs
of
genetic
instability
.
Using
ectopic
expression
of
CD
24
as
a
selective
marker
of
genetically
modified
human
epidermal
stem
cells
appears
to
be
the
first
realistic
approach
to
safe
cutaneous
gene
therapy
in
cancer
-prone
disease
conditions
.
Diseases
Validation
Diseases presenting
"ectopic expression"
symptom
esophageal adenocarcinoma
esophageal carcinoma
hirschsprung disease
holt-oram syndrome
junctional epidermolysis bullosa
kindler syndrome
pendred syndrome
primary effusion lymphoma
severe combined immunodeficiency
typhoid
werner syndrome
x-linked adrenoleukodystrophy
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