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Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I.
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This
study
aimed
to
investigate
the
potency
and
specificity
of
short
-interfering
RNA
(
siRNA
)
treatment
for
TGFBI
-Arg
124
C
ys
lattice
corneal
dystrophy
type
I
(
LCDI
)
using
exogenous
expression
constructs
in
model
systems
and
endogenous
gene
targeting
in
an
ex
vivo
model
using
corneal
epithelial
cell
cultures
.
A
panel
of
19
TGFBI
-Arg
124
Cys-
specific
siRNAs
were
assessed
by
a
dual-luciferase
reporter
assay
.
Further
assessment
using
pyrosequencing
and
qPCR
was
used
to
identify
the
lead
siRNA
;
suppression
of
mutant
TGFBIp
expression
was
confirmed
by
Western
blot
and
Congo
red
aggregation
assays
.
An
ex
vivo
model
of
LCDI
was
established
using
limbal
biopsies
from
corneal
dystrophy
patients
harboring
the
Arg
124
C
ys
mutation
.
Treatment
efficiency
of
the
siRNA
was
assessed
for
the
inhibition
of
the
mutant
allele
in
the
primary
patient
's
corneal
epithelial
cells
using
pyrosequencing
,
quantitative
PCR
(
qPCR
)
,
and
an
ELISA
.
A
lead
siRNA
was
identified
,
and
demonstrated
to
be
potent
and
specific
in
inhibiting
the
TGFBI
-Arg
124
Cys
mutant
allele
at
the
mRNA
and
protein
levels
.
Besides
high
allele
specificity
,
siRNA
treatment
achieved
a
44
%
reduction
of
the
endogenous
Arg
124
Cys
allele
in
an
ex
vivo
model
of
LCDI
.
We
have
identified
a
lead
siRNA
specific
to
the
TGFBI
-Arg
124
Cys
mutant
allele
associated
with
LCDI
.
Silencing
of
exogenous
TGFBI
was
observed
at
mRNA
and
protein
levels
,
and
in
an
ex
vivo
model
of
LCDI
with
an
efficient
suppression
of
the
endogenous
mutant
allele
.
This
result
indicates
the
potential
of
siRNA
treatment
as
a
personalized
medicine
approach
for
the
management
of
heritable
TGFBI
-associated
corneal
dystrophies
.
