Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs

Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
Nature Biotechnology (Impact Factor: 41.51). 06/2009; 27(5):478-84. DOI: 10.1038/nbt.1539
Source: PubMed
ABSTRACT
Expanded trinucleotide repeats cause many neurological diseases. These include Machado-Joseph disease (MJD) and Huntington's disease (HD), which are caused by expanded CAG repeats within an allele of the ataxin-3 (ATXN3) and huntingtin (HTT) genes, respectively. Silencing expression of these genes is a promising therapeutic strategy, but indiscriminate inhibition of both the mutant and wild-type alleles may lead to toxicity, and allele-specific approaches have required polymorphisms that differ among individuals. We report that peptide nucleic acid and locked nucleic acid antisense oligomers that target CAG repeats can preferentially inhibit mutant ataxin-3 and HTT protein expression in cultured cells. Duplex RNAs were less selective than single-stranded oligomers. The activity of the peptide nucleic acids does not involve inhibition of transcription, and differences in mRNA secondary structure or the number of oligomer binding sites may be important. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length may offer new options for developing treatments for MJD, HD and related hereditary diseases.

Full-text

Available from: David Reid Corey, Sep 02, 2014
Allele-specific silencing of mutant huntingtin and
ataxin-3 genes by targeting expanded CAG repeats
in mRNAs
Jiaxin Hu
1,4
, Masayuki Matsui
1,4
, Keith T Gagnon
1
, Jacob C Schwartz
1
, Sylvie Gabillet
3
, Khalil Arar
3
,JunWu
2
,
Ilya Bezprozvanny
2
& David R Corey
1
Expanded trinucleotide repeats
1
cause many neurological
diseases. These include Machado-Joseph disease (MJD)
2
and
Huntington’s disease (HD)
3
, which are caused by expanded
CAG repeats within an allele of the ataxin-3 (ATXN3) and
huntingtin (HTT) genes, respectively. Silencing expression
of these genes is a promising therapeutic strategy, but
indiscriminate inhibition of both the mutant and wild-type
alleles may lead to toxicity, and allele-specific approaches
have required polymorphisms that differ among individuals.
We report that peptide nucleic acid and locked nucleic acid
antisense oligomers that target CAG repeats can preferentially
inhibit mutant ataxin-3 and HTT protein expression in cultured
cells. Duplex RNAs were less selective than single-stranded
oligomers. The activity of the peptide nucleic acids does not
involve inhibition of transcription, and differences in mRNA
secondary structure or the number of oligomer binding sites
may be important. Antisense oligomers that discriminate
between wild-type and mutant genes on the basis of repeat
length may offer new options for developing treatments for
MJD, HD and related hereditary diseases.
Expanded trinucleotide repeats have been implicated in at least 19
inherited diseases
1
, including MJD
2
and HD
3
. These diseases are
autosomal dominant disorders with most patients expressing both
mutant and wild-type alleles. Toxicity of the mutant protein is usually
attributed to aggregation of the mutant protein or alteration of native
protein-protein interactions. MJD, one of the most common ataxias
with an incidence as high as 1 in 4,000 in some ethnicities
2
,remains
incurable. It is usually first diagnosed in adults, with patients even-
tually becoming wheelchair bound or bedridden, and is caused by
expanded CAG repeats (12–39 repeats are normal, 445 repeats
indicates disease) within the AT XN3 gene. HD has an incidence of
5–10 per 100,000 individuals in Europe and North America
3,4
.
Unaffected individuals have up to 35 repeats, whereas HD patients
can have from 36 to 4100 repeats
5
. The disease is characterized by
adult onset and progressive neurodegeneration. Like MJD, there is no
cure. HD is caused by the expansion of CAG trinucleotide repeats
within the first exon of the HTT gene, leading to disruption of protein
function and neurodegeneration.
Antisense oligonucleotides
6
or double-stranded RNAs
7–16
have been
proposed as a therapeutic alternative to small-molecule drugs, which
cannot disrupt the many toxic interactions formed between proteins
and mutant HTT or ataxin-3 (ref. 17). Although small interfering
(si)RNAs can inhibit HTT expression after infusion into the central
nervous system
10
, most double-stranded or antisense oligonucleotides
tested to date inhibit expression of the mutant and wild-type protein
indiscriminately
6–10
. HTT is known to play an essential role in
embryogenesis, neurogenesis and normal adult function
18,19
, raising
concerns that agents inhibiting both mutant and wild-type HTT may
induce serious side effects in HD patients, especially if chronic
administration is necessary. One approach to distinguish mutant
from wild-type alleles in HD and other neurological diseases involves
siRNAs that target single-nucleotide or deletion polymorphisms
11–16
.
But differences in polymorphisms between patients complicate rou-
tine clinical application of allele-specific RNA interference (RNAi).
To identify agents that block the neurodegenerative effects of the
mutant gene while preserving the normal biological function of the
wild-type allele, we tested whether single-stranded oligomers might
discriminate between wild-type and mutant alleles based on differ-
ences in the lengths of the expanded mRNA sequences. Our rationale,
based on the ability of triplet-repeat sequences within RNA to form
hairpin structures (Supplementary Fig. 1 online)
20
, was that because
the structures formed by wild-type and mutant mRNAs possess
different energies and stabilities, they might enable selective recogni-
tion of the mutant allele and subsequent selective inhibition of mutant
protein expression. These differences are likely to be subtle, but even
small differences may be enough to achieve useful levels of discrimi-
nation. Alternatively, the expanded repeats create additional target
sequence and more potential binding sites. For example, an allele with
a wild-type repeat number of 20 would accommodate a maximum of
three 20-base oligomers, whereas a mutant allele with 40 repeats
would be large enough to accommodate six 20-base oligomers.
To test this hypothesis, we synthesized 19-base peptide nucleic acid
(PNA)–peptide conjugates targeting HTT mRNA (Supplementary
Received 18 February; accepted 3 April; published online 3 May 2009; doi:10.1038/nbt.1539
1
Departments of Pharmacology and Biochemistry and
2
Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
3
SIGMA-Aldrich
Genopole Campus, Evry Cedex, France.
4
These authors contributed equally to this work. Correspondence should be addressed to D.R.C. (david.corey@utsouthwestern.edu).
478 VOLUME 27 NUMBER 5 MAY 2009 NATURE BIOT ECHNOLOGY
LETTERS
© 2009 Nature America, Inc. All rights reserved.
Page 1
Tab le 1 online and Fig. 1a,b) and examined PNA-mediated inhibition
of HTT expression in GM04281 patient-derived fibroblast cells
(wild-type HTT allele contains 17 repeats and mutant HTT allele
contains 69 repeats) (Fig. 1c). PNAs are a class of DNA/RNA mimics
with an uncharged amide backbone that facilitates recognition of
target sequences within RNA structure
21
. Unless otherwise noted,
PNA conjugates were synthesized to contain a cationic peptide
D-Lys
8
at the C terminus to promote the import of PNAs into cells
22
.
We targeted the CAG repeat region (REP19) and also the 5¢ junction
(5J/HTT) and the 3¢ junction (3J/HTT) because complementarity to
mRNA sequence outside the CAG repeat may further enhance the
specificity for targeting mutant HTT mRNA relative to other cellular
mRNAs. PNA conjugates REP19 (PNA REP19) and 3J/HTT (PNA 3J/
HTT) inhibited expression of HTT protein (Fig. 1d) and were chosen
for further analysis. Inhibition of mutant HTT expression by PNA
REP19 and PNA 3J/HTT was characterized by IC
50
values of 0.34 mM
and 0.96 mM, respectively (Fig. 1e,f and Supplementary
Fig. 2 and Supplementary Table 2 online) and 3.5-fold and fivefold
selectivities (wild-type IC
50
/mutant IC
50
), respectively, 4 d after
transfection. Inhibition decreased as we progressively adjusted the
PNA target site downstream of that of HTT/3J, thereby gradually
decreasing complementarity to the CAG repeat (Supplementary
Fig. 3 online). PNA REP19 selectively inhibited expression of mutant
HTT relative to wild-type HTT for up to 22 d after a one-time
addition to cells (Fig. 1g). Noncomplementary control PNAs CTL1
and –CTL2 did not inhibit HTT expression.
Many genes contain CAG repeats, including some that are essential
for cellular function. At concentrations sufficient for selective inhibi-
tion of mutant HTT, addition of PNA REP19 did not affect expression
of representative genes containing CAG repeats, including TATA box
binding protein (TBP) (19 CAG repeats), ATN1 (15 CAG repeats),
FOXP2 (40 consecutive glutamines encoded by mixed CAG and
CAA codons), AAK1 (6 CAG repeats) and POU3F2 (6 CAG repeats)
(Fig. 1h and Supplementary Fig. 4a online) and did not cause cellular
toxicity or affect rates of cell proliferation (Supplementary Fig. 4b).
Cells began to exhibit toxic effects when PNA REP19 was
added at concentrations Z 2 mM, a concentration almost fourfold
greater than that required for allele-selective inhibition (Fig. 1i and
Supplementary Fig. 4c).
To test the consequences of selectively inhibiting expression of
mutant HTT protein on phenotypes related to HD, we added PNA
REP19 to primary neuronal cell medium spiny neurons (MSN)
cultures derived from YAC128 transgenic mice (Fig. 1j and Supple-
mentary Fig. 5 online)
23,24
. In this model, full-length human HTT
mRNA containing 128 CAG repeats is expressed under control of its
endogenous promoter in mice that also express wild-type murine
HTT. MSN cells expressing mutant HTT protein are more susceptible
to apoptosis upon addition of glutamate
24
. Addition of PNA REP19 to
striatal cultures was neuroprotective against glutamate-induced toxi-
city, reducing the percentage of apoptotic YAC128 cells to B30%,
similar to levels seen in wild-type MSN. Although not a perfect mimic
of the situation in vivo because it is an engineered transgene model
NH
2
NH
Base
Base
OR
O
O
O
O
OR
5 3
+CTL
+CTL
Inhib/mu (%) 50
45 98 43 50
100 43 99 0
140
120
Wild type
REP19
–CTL1
Fold-change caspase 3
activity
REP19
Mutant
Wild type
Mutant
3J/HTT
100
Percent exp. of HTT
80
60
40
7
*
***
6
5
4
3
2
1
0
0
0.25 0.5
Concentration of PNA (µM)
241
[PNA-Peptide]/
µM
20
0
021
0
ni
niInhib/wt (%)
REP19
PNA (5 µM)
5J/HTT
3J/HTT
–CTL1
–CTL2
5J/HTT
LNA/5J
REP19
REP16
REP13
LNA/REP
Mutant
(69 repeats)
12
140
a
fg h
j
i
b
d
e
c
Percent exp. of HTT
120
100
80
60
40
20
0
024
[PNA-Peptide]/µM
6
Wild type
(17 repeats)
3J/HTT
LNA/3J
CAG repeat
PNA LNA
PNA
Days
HTT
Actin
Inhib/mu % 0
0
100
4
–CTL1 –CTL1
–CTL1
PNA conc.
(µM)
HTT
REP19
0110.5
AT N1
FOXP2
Actin
TBP
100
WT
YAC128
Percent apoptosis
80
***
***
***
n.s.
60
40
20
0
No
treatment
Glutamate (0 µm) Glutamate (250 µm)
REP19
0.25 µM
REP19
0.5 µM
–CTL1
0.5 µ
M
REP19
0.25 µM
REP19
0.5 µM
–CTL1
0.5
µM
No
treatment
REP19
4 7 10 13 16 19 22 22
99 94 91 79 54 47
24223035373734
ni
niInhib/wt %
HTT mRNA
O
O
P
O
O
O
O
O
O
OH
Base
Base
N
N
Figure 1 PNAs, LNAs and inhibition of HTT expression by PNA-peptide conjugates.
Unless otherwise noted, experiments use GM04281 fibroblast cells that are heterozygous
for mutant HTT expression and graphs are quantifications of triplicate independent
experiments. (a) Chemical structures of PNA and LNA. (b) Target sites for PNA and LNA
oligomers within HTT mRNA. (c) Western analysis showing that wild-type and mutant HTT
protein can be separated by gel electrophoresis. Lane 1 shows HTT from GM04795 cells,
a fibroblast cell line that is homozygous for wild-type HTT. Lane 2 shows HTT from
GM04281 cells, a fibroblast cell line that is heterozygous for mutant HTT. (d) Effect on
HTT expression of adding 5 mM PNA-peptide conjugates. mu, mutant; wt, wild type (e,f).
Effect on HTT expression of adding PNA REP19 (e)or3J/HTT(f). For e and f, examples
of western gels used for quantification are shown in Supplementary Figure 2.(g) Time
course of inhibition of HTT expression by PNA REP19 (1 mM). (h) Effect of PNA REP19 on expression of other proteins with mRNAs that contain CAG
repeats. (i) Effect of adding PNA REP19 on cell toxicity measured by monitoring levels of caspase 3. *P o 0.05, ***P o 0.001 relative to negative control
PNA/–CTL1. n ¼ 3. (j) Glutamate-induced apoptosis of WT and YAC128 MSN. The fraction of TUNEL-positive is shown for WT (open bars) and YAC128
(filled bars) MSN. Experiments were repeated five times and data from a blinded cell count is shown. Data were evaluated using one-way analysis of
variance. Statistical difference was considered to be significant if P r 0.05. n.s., not statistically significant.
NATURE B IOTECHNOLOGY VOLUME 27 NUMBER 5 MAY 2009 479
LETTERS
© 2009 Nature America, Inc. All rights reserved.
Page 2
with mutant human HTT expression along with two wild-type mouse
htt alleles, this experiment offers a first indication that the strategy
may exert a protective effect in neuronal cells.
The PNAs are complementary to both mRNA and chromosomal
DNA and could, in theory, block transcription by binding to HTT
DNA. It is known that the binding of PNAs to mRNA does not reduce
mRNA levels
25
. By contrast, the binding of PNAs to DNA blocks
transcription and reduces mRNA levels
26
. We observed that addition
of PNA REP19 did not decrease levels of HTT mRNA (monitored by
quantitative PCR) or alter levels of RNA polymerase 2 at the HTT
promoter (monitored by chromatin immunoprecipitation) (Supple-
mentary Fig. 6 and Supplementary Methods online). Indeed, effi-
cient inhibition of both HTT alleles increased levels of HTT mRNA by
a mechanism that does not involve enhancing the stability of HTT
mRNA. These data are consistent with a mechanism of our allele-
selective PNAs binding to mRNA and blocking translation rather than
binding to DNA and inhibiting transcription.
Given the many options for improving the activity of antisense
oligonucleotides through changing their lengths or introducing
chemical modifications, we attempted to optimize allele-selective
inhibition by varying PNA length and peptide conjugation. PNA-
peptide conjugates that were 16 and 13 bases in length (PNA REP16
and PNA REP13, respectively) were potent and selective inhibitors
with IC
50
values of 0.39 mM and 0.47 mM, respectively (Fig. 2a,b and
Supplementary Fig. 2). PNA REP13 did not affect expression of other
proteins when used at 1 mM(Supplementary Fig. 7 online). These
results suggest that shorter PNAs can achieve potent and selective
inhibition and broaden the options for designing effective agents.
A simple chemical modification to PNAs is replacement of
D-arginine for D-lysine in the attached peptide. PNA REP19Arg was
as potent an inhibitor (0.33 mM) as PNA REP19 attached to
D-Lys
8
(0.34 mM), but selectivity for the mutant allele relative to the wild-type
allele was reduced (1.9-fold for PNA REP19Arg versus 3.5-fold for
PNA REP19) (Fig. 2c, Supplementary Fig. 2 and Supplementary
Tab le 2). This demonstrates that the composition of the peptide
sequence affects selectivity for inhibition of mutant versus wild-type
protein. The importance of cationic peptide sequence for recognition
is not surprising; we have previously documented examples where
replacement of an attached peptide that contains lysine with an
analogous peptide that contains arginine substantially alters recogni-
tion of complementary nucleic acids
27
.
Another straightforward modification is attachment of the peptide
domain to the N rather than the C terminus of the PNA to afford PNA
REP19N. In contrast to results showing lower selectivity with PNA
REP19Arg relative to PNA REP19, PNA REP19N showed greater
selectivity than PNA REP19. The IC
50
value for inhibiting mutant
HTT expression was 2.1 mM, with relatively no obvious inhibition of
wild-type HTT at concentrations as high as 16 mM(Fig. 2d and
Supplementary Fig. 2). This experiment demonstrates that improved
selectivity can be achieved by varying conjugate design. We observed
no inhibition of other proteins and only mild toxicity (Supplemen-
tary Fig. 7).
In contrast to PNAs, which have a neutral amide backbone,
oligonucleotides have negatively charged phosphodiester backbones.
Because of this basic difference in their chemical properties relative to
PNAs, oligonucleotides will have a much different potential for
developing antisense oligomers for therapy. Oligonucleotides are
approved drugs and are being used in several clinical trials, and this
clinical experience may offer practical advantages for their develop-
ment for treating HD.
To explore whether this difference in chemical properties might
influence the capacity for allele-selective inhibition, we tested single-
stranded oligonucleotides that contain locked nucleic acid (LNA)
bases
28
. LNA is an RNA analog that contains a methylene bridge
between the 2¢-O and 4¢-C of the ribose (Fig. 1a). LNA bases can be
placed at any position and allow the thermal stability of oligonucleo-
tides to be precisely tailored for any application. In contrast to the
neutral amide backbone of PNAs, LNAs have a negatively charged
phosphodiester backbone, allowing us to use cationic lipid to introduce
LNAs into cells. LNA oligomers are being tested in clinical trials
28
.
140
abcd
ef g
h
Wild type
Mutant
REP16
Wild type
Mutant
REP13
Wild type
Mutant
REP19Arg
120
100
80
60
40
20
0
01
[PNA-Peptide]/µM
Percent exp. of HTT
2
140
120
100
80
60
40
20
0
01
[PNA-Peptide]/µM
Percent exp. of HTT
2
140
120
100
80
60
40
20
0
01
[PNA-Peptide]/µM
Percent exp. of HTT
2
Wild type
Mutant
REP19N
140
120
100
80
60
40
20
0
048 16
[PNA-Peptide]/µM
Percent exp. of HTT
12
Wild type
Mutant
LNA/REP
Wild type
Mutant
LNA/3J
Wild type
Mutant
siRNA/REP
Wild type
Mutant
siRNA/5J
140
120
100
80
60
40
20
0
0 20406080 120
[LNA]/nM
Percent exp. of HTT
100
140
120
100
80
60
40
20
0
0 20406080 120
[LNA]/nM
Percent exp. of HTT
100
140
120
100
80
60
40
20
0
0 20406080 120
[siRNA]/nM
Percent exp. of HTT
100
140
120
100
80
60
40
20
0
0 20406080 120
[siRNA]/nM
Percent exp. of HTT
100
Figure 2 Effect of PNA modification, LNAs and siRNAs on selectivity. All quantification of western analysis of protein levels in GM04281 fibroblast cells
is derived from at least three independent experiments. Examples of immunoblots used for quantification are shown in Supplementary Figure 2.
(ah) Effect on HTT expression of adding increasing concentrations of PNA REP16 (a), PNA REP13 (b), PNA REP19Arg (c), PNA REP19N (d),
LNA/REP (e), LNA/3J (f), siRNA/REP (g) and siRNA/5J (h).
480 VOLUME 27 NUMBER 5 MAY 2009 NATURE BIOTECHNOLOGY
LETTERS
© 2009 Nature America, Inc. All rights reserved.
Page 3
We observed allele-selective inhibition of mutant HTTexpression by
LNA/REP or LNA/3J (Fig. 2e,f and Supplementary Fig. 8a online).
Inhibition by LNA/REP and LNA/3J was characterized by IC
50
values
of 0.017 and 0.086 mM, respectively (Supplementary Fig. 2 and
Supplementary Table 2). These IC
50
values are lower than those
achieved using PNA-peptide conjugates, but because the transfection
protocols differ, it is impossible to draw direct conclusions regarding
relative potencies. Inhibition of wild-type HTT was r30% at 100 nM,
the maximum concentration tested. Concentrations of LNA that
selectively blocked expression of mutant HTT did not affect other
genes that contain CAG repeats (Supplementary Fig. 8b). LNA/REP
caused a modest decrease in levels of HTT mRNA relative to
noncomplementary LNA controls (Supplementary Fig. 8c). In con-
trast to PNAs, LNAs that contain LNA bases spread throughout a
DNA backbone form DNA:RNA hybrids upon binding to mRNA and
can recruit RNase H
29
. The resultant cleavage may explain the
observed lower levels of mRNA.
The potency and widespread use of duplex RNAs (siRNAs)
30
make them a good benchmark for evaluating the effectiveness of
PNAs and LNAs. To test whether siRNAs would also achieve selective
inhibition of mutant HTT, we introduced duplex RNAs analogous
in sequence to PNA REP19, PNA 5J/HTT and PNA 3J/HTT into
GM04281 fibroblast cells. Like LNAs, and unlike PNA-peptide
conjugates, siRNAs have a phosphodiester backbone and we used
cationic lipid to assist their entry into cells. We observed inhibition
of HTT expression by siRNA/REP and siRNA/5J (Fig. 2g,h,
Supplementary Fig. 2)characterizedbyIC
50
values of 0.005 and
0.018, respectively (Supplementary Table 2). siRNA/REP revealed a
narrow window for selectivity with relatively low statistical significance
(Fig. 2g), whereas siRNA/5J exhibited a selectivity of approxi-
mately threefold (Fig. 2h and Supplementary Table 2). Our
RNAs were not chemically modified and duplex RNAs with
well-chosen modifications might achieve
better selectivity.
Most HD patients have mutant mRNAs
with 40–50 CAG repeats
5
.Weextendedour
studies to patient-derived fibroblast cell lines
GM04869 (wild-type allele, 15 repeats;
mutant allele, 47 repeats), GM04719 (wild-
type allele, 15 repeats; mutant allele, 44
repeats) and GM04717 (wild-type allele, 20
repeats; mutant allele, 41 repeats) (Fig. 3).
Upon addition of PNA REP19 to cells, we
observed inhibition of mutant HTT in GM04869 (Fig. 3b), GM04719
(Fig. 3d) and GM04717 (Fig. 3f) cells with selectivities (wild-type IC
50
value/mutant IC
50
value) of 2.1, 1.8 and 1.2, respectively (Supple-
mentary Fig. 9 and Supplementary Table 2 online), values reduced
relative to the 3.5-fold selectivity achieved in GM04281 cells (Fig. 1e).
We observed slight decreases in the potency of inhibition of mutant
HTT as the number of mutant repeats was decreased from 47 to 44
and 41. The observation that the most potent inhibition of wild-type
HTT occurred in the cell line (GM04717) with the most repeats in the
wild-type allele is consistent with a correlation between the number of
repeats and susceptibility to inhibition by PNA REP19. However,
additional experiments are needed to better evaluate this hypothesis.
Because we had previously observed that attachment of the
D-Lys
8
peptide to the PNA N terminus improves selectivity in GM04281 cells
(Fig. 2d), we tested N-linked conjugate PNA REP19N in the cell lines
that possess mutant alleles with fewer CAG repeats. We observed
inhibition of mutant HTT in GM04869 (Fig. 3c), GM04719 (Fig. 3e)
and GM04717 (Fig. 3g) cells with selectivities (wild-type IC
50
value/mutant IC
50
value) of 43.5, 41.8 and 41.5, respectively
(Supplementary Fig. 9 and Supplementary Table 2). These data
suggest that simple chemical modifications could potentially extend
allele-selective inhibition of HTT protein expression to a broader
subset of HD patients.
We se lec ted ATXN3 , the gene in which a CAG repeat expan-
sion causes MJD
2
, as a target to test whether allele-selective inhibi-
tion could be achieved with other genes containing repeats. To
examine the potential for allele-specific inhibition in MJD cells,
we obtained patient-derived cell line GM06151, which is hetero-
zygous for an expanded CAG repeat (wild-type allele, 24 repeats;
mutant allele, 74 repeats). The 74 repeats of the mutant allele fall
within the middle of the range of repeat numbers found in
patient samples
31
.
140
bdf
ceg
Wild type
Mutant
REP19
120
100
Percent exp. of HTT
80
60
40
20
0
01
[PNA-Peptide]/µM
2
140
Wild type
Mutant
REP19N
120
100
Percent exp. of HTT
80
60
40
20
0
04
[PNA-Peptide]/µM
628
140
Wild type
Mutant
REP19N
120
100
Percent exp. of HTT
80
60
40
20
0
04
[PNA-Peptide]/µM
628
140
Wild type
Mutant
REP19N
120
100
Percent exp. of HTT
80
60
40
20
0
04
[PNA-Peptide]/µM
628
140
Wild type
Mutant
REP19
Wild type
Mutant
REP19
120
100
Percent exp. of HTT
80
60
40
20
0
01
[PNA-Peptide]/µM
2
140
120
100
Percent exp. of HTT
80
60
40
20
0
01
[PNA-Peptide]/µM
2
a
Mutant HTT
CAG repeat
number
17/15 41/20 44/15 47/15 69/17
wt fibroblast
GM04717
GM04719
GM04869
GM04281
Wild-type HTT
Figure 3 Selectivity is affected by the number
of repeats in mutant HTT. All quantification of
protein levels in fibroblast cells by western
analysis is derived from at least three
independent experiments. Examples of
immunoblots used for quantification are shown
in Supplementary Figure 9.(a) Separation by
SDS-PAGE of wild-type and mutant HTT protein
variants in four different patient-derived cell
lines. The first number is the number of repeats
in the mutant allele, the second number is the
number of repeats in the wild-type allele.
(bg) Effect on HTT protein expression of adding
REP19 to GM04869 cells (b), REP19N to
GM04869 cells (c), REP19 to GM04719 cells
(d), REP19N to GM04719 cells (e), REP19 to
GM04717 cells (f) and REP19N to GM04717
cells (g).
NATURE B IOTECHNOLOGY VOLUME 27 NUMBER 5 MAY 2009 481
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© 2009 Nature America, Inc. All rights reserved.
Page 4
We tested PNA conjugates that targeted the CAG repeat region
(PNA REP19), the 5¢ junction (PNA 5J/ATX), and the 3¢ junction
(PNA 3J/ATX) (Fig. 4a, Supplementary Fig. 10 and Supplementary
Tab le 2 online). PNA REP19 selectively inhibited expression of mutant
ataxin-3 with an IC
50
value of 0.36 mM(Fig. 4b). Conjugates that
target the 5¢ and 3¢ junctions also caused selective inhibition, with IC
50
values of 0.7 mM and 2.2 mM, respectively (Fig. 4c,d). These data
suggest that our strategy can be extended beyond HTT to other
therapeutic targets. We also tested siRNA/REP. Similar to our observa-
tions for inhibition of HTT protein expression (Fig. 2), this siRNA was
an inhibitor of ataxin-3 expression but yielded less-selective reduction
of the mutant protein than did PNA REP19 (Fig. 4e). A failure of a
repeat-targeted siRNA to selectively inhibit ataxin-3 expression had
been reported previously
11
.
One obstacle to therapy for HD or MJD will be delivery to the
central nervous system because oligonucleotides are not distributed to
the brain after intravenous or oral administration. Single-stranded
oligonucleotides can be delivered directly to the central nervous
system and block gene expression
32
. Although LNAs have not
been used in animal models of HD or MJD, they have been
administered into rodent brains by several methods (intrathecal,
intracerebroventricular and intrastriatal)
33,34
. Toxicity is minimal
and potent inhibitory effects were observed for targeting deltorphin
II
33
and miRNA-21 (ref. 34).
Much attention has been focused on allele-selective inhibition using
siRNAs complementary to sequences that have single-nucleotide or
deletion polymorphisms
11–16
. Although an advantage of this approach
is that it involves familiar RNAi technology, the diversity of
polymorphisms within patient populations would necessitate
developing multiple siRNAs. Even then, this approach, based on
single-nucleotide polymorphisms, is unlikely to benefit patients like
the one from whom our benchmark patient-derived cell line
GM04281, which has none of the most commonly identified poly-
morphisms
14
, was derived. We were able to perform experiments in
GM09197 cells that have a deletion polymorphism and have been used
to observe siRNA-mediated allele-selective inhibition
16
.LNA/REP
offered better selectivity than the best allele-selective RNA (Supple-
mentary Fig. 11 online).
Patient populations are heterogeneous and approaches that target
polymorphisms, triplet repeats or make no attempt at allele-selectivity
may benefit different groups of patients. One advantage is that our
approach uses one oligomer strand rather than the two needed for
siRNAs, simplifying issues associated with clinical development such
as compound synthesis and the potential for off-target effects. Con-
versely, although we demonstrate that optimizing the structure of the
oligomer can lead to better selectivity over a wide range of repeats, it
may be difficult, using our strategy, to achieve adequate selectivity for
patients with shorter repeats.
Another important issue is the minimum efficacy sufficient for
successful application in vivo. Complete inhibition of mutant protein
expression might not be necessary to achieve beneficial therapeutic
effects; even partial reduction of mutant protein levels may be
adequate. Conversely, partial inhibition of wild-type protein may
not have adverse consequences because the remaining wild-type
protein may be sufficient for function. The window for therapy may
be relatively broad, requiring less than complete inhibition of mutant
protein expression and tolerating partial reduction in wild-type
protein levels. Such information will help set the therapeutic window
for drug development.
Given that the allele-selective inhibition of HTTand ataxin-3 occurs
intracellularly, it is difficult to assign an exact molecular mechanism to
the effects we have observed. Clearly, some complementarity to the
CAG repeat is essential, because potency is lost when downstream
regions are targeted (Supplementary Fig. 3). Although the number of
oligomers bound to the mutant and wild-type expanded repeats may
contribute to selectivity, our evidence involving different PNAs, LNAs
and siRNAs is consistent with a mechanism that at least partially
involves an ability of PNAs and LNAs to sense structural differences
between extended repeats in the cell. Addition of PNAs does not
reduce levels of mRNA or recruitment of RNA polymerase 2 to the
HTT promoter (Supplementary Fig. 6), consistent with a steric
blocking mechanism in which the PNA binds to mRNA rather than
DNA. This ‘steric blocking mode of action for antisense PNAs
was expected based on previous studies of PNAs complementary
to mRNA
25
.
Our findings offer two lessons. The first is that single-stranded
oligomers can discriminate among sequences inside cells on the
basis of context—in this case the length of a polyglutamine tract
and the potential of different repeat lengths to form energetically
different structures—rather than base differences. The second is
that the potential for developing single-stranded oligomers as allele-
selective treatment for genetic disease is greater than had been
appreciated previously. There is broad potential for optimizing
the selectivity and potency of our most promising candidates. Anti-
sense oligomers for other diseases are making good progress in
clinical trials
35
and appear to offer near-term potential for wider
therapeutic application. Exploiting the ability of antisense oligomers
to selectively recognize mutant nucleic acid sequences offers a
promising strategy for developing therapies for HD, MJD and other
triplet-repeat disorders.
140
bc
Wild type
Mutant
REP19
120
100
Percent exp. of ataxin-3
80
60
40
20
0
01
[PNA-Peptide]/µM
2
Percent exp. of ataxin-3
140
Wild type
Mutant
3J/ATX
120
100
80
60
40
20
0
024
[PNA-Peptide]/µM
6
de
Wild type
Mutant
5J/ATX
140
120
100
Percent exp. of ataxin-3
80
60
40
20
0
024
[PNA-Peptide]/µM
6
Percent exp. of ataxin-3
Wild type
Mutant
siRNA/REP
140
120
100
80
60
40
20
0
0 20406080
[siRNA]/nM
100 120
Ataxin-3 mRNA
a
5 3
CAG repeat
5J/ATX REP19 3J/ATX
Figure 4 Potent and selective inhibition of mutant ataxin-3 in GM06151
fibroblasts. Examples of immunoblots used for quantification are shown in
Supplementary Figure 10.(a) Target sites for PNAs within ataxin-3 mRNA.
(be) Inhibition of ataxin-3 expression by (b) PNA REP19, (c) PNA 3J/ATX,
(d) PNA 5J/ATX and (e) siRNA/REP.
482 VOLUME 27 NUMBER 5 MAY 2009 NATURE BIOTECHNOLOGY
LETTERS
© 2009 Nature America, Inc. All rights reserved.
Page 5
METHODS
Cell culture and transfection. PNA-peptide conjugates were synthesized and
purified as desccribed
22,26
. LNA oligonucleotides were provided by Sigma-
Aldrich. siRNAs were purchased from Integrated DNA Technologies (IDT).
Patient-derived fibroblast cell lines GM04281, GM06151, GM04719, GM04869,
GM04717, GM04795 and GM09197 were obtained from the Coriell Institute.
Cells were maintained at 37 1Cand5%CO
2
in MEM (Sigma) supplemented
with 10% heat inactivated FBS (Sigma) and 0.5% MEM nonessential amino
acids (Sigma). Cells were plated in 6-well plates at 60,000 cells/well in
supplemented MEM 2 d before transfection. Stock solutions of PNA-peptide
conjugates were heated at 65 1C for 5 min before use to dissolve any aggregates
that may have formed. PNA-peptide conjugates were diluted to the appropriate
concentration using OptiMEM (Invitrogen) and then added to cells. After 24 h,
the media containing PNA-peptides were removed and replaced by fresh
supplemented MEM. Unless indicated otherwise, cells were harvested 4 d after
transfection for protein assay. siRNAs or LNAs were transfected to cells using
RNAiMAX (Invitrogen) according to the manufacturers instructions. The
appropriate amount of the lipid (3 ml for 100 nM oligonucleotides) was added
to OptiMEM containing oligonucleotides and the oligonucleotide-lipid mix-
ture (250 ml) was incubated for 20 min. OptiMEM was added to the mixture to
a final volume of 1.25 ml and then added to cells. The media were exchanged
24 h later with fresh supplemented MEM.
Analysis of HTT and ataxin-3 expression. Cells were harvested with trypsin-
EDTA solution (Invitrogen) and lysed. The protein concentration in each
sample was quantified with bicinchoninic acid (BCA) assay (Thermo Scien-
tific). SDS-PAGE (separating gel: 5% acrylamide-bisacrylamide/34.7:1, 450 mM
Tris-acetate pH 8.8; stacking gel: 4% acrylamide-bisacrylamide/34.7:1, 150 mM
Tris-acetate pH 6.8) (XT Tricine Running Buffer, Bio-Rad) was used to separate
WT and mutant HTT proteins (Supplementary Figs. 12 and 13 online). Gels
were run at 70 V for 15 min followed by 100 V for 4 h. For separation of HTT
variants containing shorter CAG repeats, gels were run at 70 V for 15 min, then
110 V for 6 h. The electrophoresis apparatus was placed in an ice-water bath to
prevent overheating of the running buffer. We monitored expression of actin
protein to ensure even loading on protein in each lane. In parallel with analysis
for HTT expression, portions of each protein lysate sample were analyzed for
b-actin expression by SDS-PAGE (7.5% acrylamide pre-cast gels; Bio-Rad).
These gels were run at 70 V for 15 min followed by 100 V for 1 h. After gel
electrophoresis, proteins were transferred to membrane (Hybond-C Extra; GE
Healthcare Bio-Sciences). Ataxin-3 and b-actin were analyzed by SDS-PAGE
(7.5% acrylamide pre-cast gels; Bio-Rad). Primary antibodies specific for each
protein were obtained and used at the indicated dilution ratio: anti-HTT
antibody (MAB2166; 1:10,000; Chemicon), anti-ataxin-3 antibody (MAB5360;
1:10,000; Chemicon), and anti-b-actin antibody (1:10,000; Sigma).
HRP conjugate anti-mouse or anti-rabbit secondary antibody (1:10,000
and 1:5,000, respectively; Jackson ImmunoResearch Laboratories) was used
for visualizing proteins using SuperSignal West Pico Chemiluminescent
Substrate (Thermo Scientific). Protein bands were quantified using ImageJ
software. The percentage of inhibition was calculated as a relative value to a
control sample.
Neuronal cell glutamate susceptibility assay
23,24
. YAC128 mice (FVBN/NJ
background strain) were obtained from Jackson Labs. The male YAC128 mice
were crossed to WT female FVBN/NJ mice and P1-P2 pups were collected and
genotyped by PCR. The primary cultures of striatal MSN were established from
YAC128 and control WT pups. Striata were dissected, diced and digested with
trypsin. After dissociation, neurons were plated on poly-
L-lysine (Sigma)-
coated 12-mm round coverslips (Assistent) in Neurobasal-A medium supple-
mented with 2% B27, 1 mM glutamine and penicillin-streptomycin (all from
Invitrogen) and kept at 37 1C in a 5% CO
2
environment. PNA was added to the
9-days-in-vitro (DIV) MSN. The 13-DIV MSN were exposed for 7 h to 250 mM
glutamate in Neurobasal-A added to the culture medium. Immediately after the
treatment with glutamate, neurons were fixed for 30 min in 4% paraformalde-
hyde plus 4% sucrose in PBS (pH 7.4), permeabilized for 5 min in 0.25%
Triton-X-100 and stained by using the DeadEnd fluorometric TUNEL System
(Promega). Nuclei were counterstained with 5 mM propidium iodine (PI)
(Molecular Probes). Coverslips were extensively washed with PBS and mounted
in Mowiol 4-88 (Polysciences). For quantification six to eight randomly
chosen microscopic fields, each containing 100–300 MSN, were counted for
YAC128 and WT cultures. The number of TUNEL-positive neuronal nuclei
was calculated as a fraction of PI-positive neuronal nuclei in each micro-
scopic field. The fractions of TUNEL-positive nuclei determined
for each microscopic field were averaged and the results are presented as
means ± s.e.m. (n ¼ number of fields counted). MSN cells were supported in
culture by surrounding glial cells, but only MSN cells were counted during the
neuroprotection assay.
PNA REP19 was added at the concentration of 0.25 mM and 0.5 mM, 4 d
before the glutamate application. Noncomplementary PNA –CTL1 was added
at 0.5 mM. MSN were exposed to 250 mM glutamate at 13-DIV for 7 h, fixed,
permeabilized and analyzed by TUNEL staining and PI counterstaining. The
data are presented as mean ± s.e.m. (n ¼ 6–8 microscopic fields, 100–300 MSN
per field).
For protein assays, after the addition of PNA REP19 at 9-DIV, the 13-DIV
MSN were washed with ice-cold PBS and solubilized for 30 min at 4 1Cin
extraction buffer A (1% CHAPS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na
2
HPO
4
, 1.4 mM KH
2
PO
4
(pH 7.2), 5 mM EDTA, 5 mM EGTA and
protease inhibitors). Extracts were clarified by centrifugation for 20 min at
10,000g at 4 1C and the upper solutions were collected for SDS-PAGE.
For selectively harvesting MSN, cells were incubated with cell dissociation
solution (Sigma) for 4 min at 37 1C and neuron basal-A medium with 10%
FBS were added. MSN were detached from plate and most of the glial cells
were not harvested. The combined solution was collected and lysed as
previously described
23,24
.
Caspase-3 activity assay. Caspase-3 activity was measured by hydrolysis of
acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) according to the
manufacturer’s instruction in a colorimetric assay kit (Sigma). Fibroblast cells
in 6-well plate were harvested as previously described. The cell pellet was
resuspended in 15 mlof1 lysis buffer without protease inhibitor and
incubated for 30 min on ice. Protein concentration was determined by
BCA assay (Thermo Scientific). We mixed 50 mg of cell extract with 2 mM
Ac-DEVD-pNA substrate to a total volume of 100 mlof1 assay buffer in
a 96-well plate and incubated at 37 1C overnight. The absorbance was read
at 405 nm.
Analysis of TBP, AAK1, ATN1, FOXP2 and POU3F2. The number of CAG
repeats was estimated according to the published mRNA sequence in GenBank.
TATA box binding protein (TBP)(B19 CAG repeats, NM_003194), AAK1 (6
CAG repeats, NM_014911), ATN1 (15 CAG repeats, NM_001940), FOXP2
(B40 CAACAG repeats, NM_148899) and POU3F2 (B6CAGrepeats,
NM_005604). Protein lysates were analyzed by SDS-PAGE (7.5% acrylamide
pre-cast gels; Bio-Rad) followed by western blotting with anti-TBP antibody
(1:2,000; Sigma), anti-AAK1 antibody (1:1,000; Abcam), anti-ATN1 antibody
(1:300; Affinity Bioreagents), anti-FOXP2 antibody (1:1,000; Abcam) or anti-
POU3F2 antibody (1:1,000; Abnova) (Supplementary Fig. 13).
Statistical analysis and curve fitting. Student t-test was performed for
evaluating statistical significance between two study groups. Each data plot
from dose response experiments for inhibition of HTTor ataxin-3 was fit to the
following model equation, y ¼ 100(1-x
m
/(n
m
+x
m
)), where y is percent inhibi-
tion of protein and x is concentration of synthetic oligomers. m and n are
fitting parameters, where n is taken as the IC
50
value.
Note: Supplementary information is available on the Nature Biotechnology website.
ACKNOWLEDGMENTS
This work was supported by the High-Q Foundation, the US National Institutes
of Health (NIGMS 60642 and 73042 to D.R.C.; NINDS RO1NS056224 to
I.B.; NIBIB EB 05556 to J.C.S.), and the Robert A. Welch Foundation
(I-1244 and I-1336) and Ataxia MJD research project. We thank
B. Janowski for helpful comments and Y. Li for help maintaining the
YAC128 mouse colony.
AUTHOR CONTRIBUTIONS
J.H. and M.M. designed and performed experiments in patient-derived
fibroblast cells. J.W. and J.H. designed and performed experiments in MSN cells.
NATURE B IOTECHNOLOGY VOLUME 27 NUMBER 5 MAY 2009 483
LETTERS
© 2009 Nature America, Inc. All rights reserved.
Page 6
K.T.G. and J.C.S assisted with experiments. K.A. and S.G. supplied LNAs.
D.R.C. and I.B. supervised experiments.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/naturebiotechnology/
Published online at http://www.nature.com/naturebiotechnology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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484 VOLUME 27 NUMBER 5 MAY 2009 NATURE BIOTECHNOLOGY
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Page 7
  • Source
    • "Allele-selective silencing of different genes is described for some neurodegenerative diseases inherited in a dominant manner, including Huntington disease, different types of spinocerebellar ataxias, Alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS)394041424344. For that purpose, siRNAs [43,4546474849 and ASOs5051525354 are commonly used. "
    [Show abstract] [Hide abstract] ABSTRACT: Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.
    Full-text · Article · Nov 2015 · PLoS ONE
  • Source
    • "Nonallele-selective RNaseH ASOs [32] and siRNA [33] , which target homologous sequence tracts in mu HTT and wt HTT mRNA, partially lower both allelic variants and produce therapeutic benefits in murine HD disease models. Allele selectivity can be achieved by targeting the CAG tract directly [34] or by targeting SNPs tightly linked to the CAG expansion [35]. ASOs targeting SNP rs7685686_A in intron 42 of the HTT gene can selectively suppress mutant HTT mRNA and HTT protein in human patient fibroblasts [12,36]. "
    [Show abstract] [Hide abstract] ABSTRACT: We report the effect of introducing a single incorporation of 2-thio-deoxythymidine (2S-dT) or C5-Triazolylphenyl-deoxythymidine (5-TrPh-dT) at four positions within the gap region of RNase H gapmer antisense oligonucleotides (ASOs) for reducing wild-type and mutant huntingtin mRNA in human patient fibroblasts. We show that these modifications can modulate processing of the ASO/RNA heteroduplexes by recombinant human RNase H1 in a position-dependent manner. We also created a structural model of the catalytic domain of human RNase H bound to ASO/RNA heteroduplexes to rationalize the activity and selectivity observations in cells and in the biochemical assays. Our results highlight the ability of chemical modifications in the gap region to produce profound changes in ASO behavior.
    Full-text · Article · Jul 2015 · Nucleic Acid Therapeutics
  • Source
    • "Выключение гена, кодирующего мутантный хантингтин, с помощью РНК-интерференции показало улучшение в поведенческих тестах и при нейропатологических расстройствах у мышей с болезнью Хантингтона [25]. Была продемонстрирована возможность снижения количества мРНК, кодирующей мутантный хантингтин, с использованием антисмысловых олигомеров пептидной нуклеиновой кислоты, содержащей CAG-повторы, и заблокированных нуклеиновых кислот (locked nucleic acids) [26]. Еще одна потенциальная стратегия может заключаться в блокировании возможности образования хантингтином токсичных агрегатов. "
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