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Introduction
Diseases caused by dominant, gain-of-function
mutations develop in heterozygotes bearing 1 mutant and 1
wild-type copy of the gene. The best-known examples of this
class are common neurodegenerative diseases, including
Alzheimer's disease, Huntington's disease, Parkinson's
disease, and amyotrophic lateral sclerosis (ALS or Lou
Gehrig's disease). In these diseases, the exact pathways
where the mutant proteins cause cell degeneration are not
clear, but the origin of the cellular toxicity is known to be the
mutant protein[1]. Thus, the design of compounds that
selectively lower or eliminate the mutant protein is a key step in
developing effective therapies.
Mutations in human CuZn superoxide dismutase (SOD)
1 cause motor neuron degeneration that leads to ALS because
of some toxic property acquired by the mutant
protein[2]. Although the molecular mechanism underlying this toxic
property that triggers motor neuron degeneration is not fully
understood, the artificial expression of mutant SOD1 in mice
causes ALS. Nonetheless, the wild-type SOD1 gene is
important for cells to function properly. SOD1 knockout mice
demonstrate a variety of abnormalities, including reduced
fertility[3], motor
axonopathy[4], age-associated loss of
cochlear hair cells[5], and neuromuscular junction
synapses[6], and become vulnerable to noxious assaults, such as axonal
injury[7], ischemia[8,9], hemolysate
exposure[10], and
irradiation[11]. Given the functional importance of the wild-type
allele and the unwanted effect of the mutant protein,
selectively blocking the expression of the mutant allele while
keeping the expression of the wild-type protein intact would be
an ideal therapy for the fraction of ALS caused by mutant
SOD1.
Sequence-specific gene silencing has been achieved in
eukaryotes by RNA interference
(RNAi)[12_14]. Subsequent studies of the RNAi
pathway[15_18] have been extended to cultured mammalian
cells[19,20]. In this approach, 21 nt,
double-stranded synthetic siRNA are used to destroy a target mRNA
containing the corresponding siRNA sequence. This
newly-developed technology holds a potential to cure diseases
caused by dominant, gain-of-function gene mutations, such
as ALS.
Previously, we showed that RNAi could selectively
inhibit the expression of the mutant SOD1 genes that cause
ALS[21]. In this study, we systematically tested a siRNA
design strategy in converting a naturally symmetric siRNA
to a favorable asymmetric siRNA that targets an
ALS-causing SOD1 mutant allele in human cells. We demonstrate that
this strategy can not only successfully achieve the
conversion of an siRNA that is originally favored to the ant-sense
of the mutant allele to the one that is favored to the sense
strand of the gene, but also enhances the selective silencing
of the target gene.
Materials and methods
siRNA preparation Chemically-synthesized single-strand
RNA were purchased from Dharmacon Research (Chicago,
USA), deprotected according to the manufac-turer's
instructions, and annealed as described
previously[21].
Cell culture and transfection Human embryonic kidney
293 cells were cultured in Dulbeccos's Modified Eagle's
Medium (Invitrogen, Carlsbad, USA) supplemented with 10%
fetal bovine serum plus 100 U/mL penicillin and 100 µg/mL
streptomycin. Twenty-four hours before the experiments,
the cells were detached with trypsin-EDTA (0.05% trypsin,
0.53 mmol/L EDTA·4Na) at 70%_90% confluency and
transferred into 96-well plates at approximately 30% cell density.
Transfection was performed using Lipofectamine 2000
(Invitrogen, Carlsbad, USA) following the manufacturer's
instructions on the following day. The concentrations of
the constructs and siRNA used in the transfection were 2.0
µg/mL firefly luciferase (pGL2 control vector, Promega, San
Luis Obispo, USA) with target sequence inserted into the 3'
untranslated region (UTR), 0.1 µg/mL of Renilla luciferase
vector (pRL-TK, Promega, San Luis Obispo, USA), and 2
nmol/L siRNA. The siRNA concentrations used for the
dose-effect study (Figure 1) were 0.002_31.25 nmol/L. Data are
presented as mean±SD (n=4).
Dual luciferase assay The dual luciferase system
(Promega, San Luis Obispo, USA) was used with
modifications to quantify RNAi efficiency in the cell culture. Two
restriction sites (Nde l and Spe I) were first engineered into
the 3' UTR of the firefly luciferase vector followed by
the insertion of a 39 nt fragment of the SOD1 gene (sense
strand5'AGGCATGTTGGAGACTTGGGCAATGTGACTGCTGA-CAAA-3', anti-sense strand
5'-tttgtcagcagtcaca-ttgcccaagtctccaacatgcct-3') between the
Nde I and Spe I sites. The modified firefly luciferase vector with
either the partial SOD1 sense (sense target) or antisense
sequence (antisense target) was cotransfected with the
Renilla luciferase vector plus siRNA into HEK293 cells in
quadruplicate. Twenty-four hours after the transfection, the
cells were lysed in 96-well plates with 20 µL passive lysis
buffer (Promega, San Luis Obispo, USA). In total, 10 µL
lysate was transferred into a well in a Microlite strip (Thermo
Labsystems, Waltham, USA), and the luminescence
intensity was measured with a Veritas microplate luminometer
(Turner Biosystems, Sunnyvale USA). The relative ratio of
the firefly/Renilla luciferase was used for calculating the RNAi
efficacy. All measures were normalized to the luciferase
vectors only with vectors plus siRNA against green florescent
protein (GFP) mRNA as an irrelevant control. The results
were plotted using mean±SD (n=4; Figures 2_4).
Results
The siRNA that we started with was an siRNA targeting
the ALS-causing mutant G85R SOD1 gene, which has been
previously shown to be inefficient in silencing the mutant
allele and has almost no effect on the gene expression of
wild-type SOD1[21]. This natural siRNA has the exact same
base pairing at the first 4 positions of both ends, therefore is
symmetric (Figure 1A). To quantify the effects of the siRNA,
we transfected the sense or antisense targets with different
doses of the siRNA. This original symmetric siRNA
simultaneously silenced the sense and antisense targets. However,
the maximal silencing effect (relative) for the antisense target
was 81%, with the IC50 (50% inhibitory
concentration) at 0.1 nmol/L, while the efficiency for the cleavage of the sense
target, the ALS-causing sequence, was only 60% with the
IC50 at 5 nmol/L (Figure 1B).
To convert the naturally symmetric siRNA that is favored
to the antisense stand of ALS-causing gene to the effective
siRNA that targets the disease-causing sequence itself, we
weakened the base pairing at the 5' of the antisense strand of
the siRNA (right end, R) by placing a mismatch at that end
(R1_R4; Figure 2A). Although the symmetric siRNA
naturally favored the antisense target (Figure 1B), it was
converted to favor the sense target after weakening the base
pairing at the 5' of the antisense strand of the siRNA (R1_R4;
Figure 2B). The mismatch at position 1 (R1) completely
reversed the strand preference compared to the original
symmetric siRNA, while the efficacies of mismatches from
positions 2 to 4 (R2_R4) decreased as the position moving
towards the middle of the strand. In contrast, the weakening
of the base pairing at the 5' of the sense strand of the siRNA
(left end, L) accentuated the preference of silencing the
antisense target (L1_L4; Figure 2B). All 4 siRNAs with a
mismatch from positions 1 to 4 (L1_L4; Figure 2A) abolished
the silencing of the sense target. Meanwhile, they
effectively silenced the antisense target and the siRNA with a
mismatch at position 1 (L1) exhibited the maximal efficacy.
To further weaken the base pairing, we placed 2
mismatches in the symmetric siRNA (Figure 3A). When the
mismatches were placed at the 5' end of the sense strand of
the siRNA, the target preference of this double-mismatched
siRNA was only slightly increased (L1+L2, L1+L3, and L1+L4;
Figure 3B) compared to one of the single-mismatched siRNA,
mainly because placing the 1 mismatch at this end of the
siRNA almost maximized the siRNA asymmetry already (L1,
L2, L3, and L4; Figure 2B). However, if the mismatches were
inserted into the 5' end of the antisense strand of the siRNA
where placing 1 mismatch did not completely convert the
siRNA from symmetry to asymmetry; the modified siRNA
noticeably increased the efficacy of the sense target
cleavage and dramatically decreased the inhibition of the antisense
target (R1+R2, R1+R3, and R1+R4; Figure 3B). Thus,
double-mismatched siRNA in general improved the preference and
efficacy of target silencing compared to single-mismatched
siRNA.
Finally, we tested the silencing efficacies of siRNAs with
mismatches at both ends by placing 1 of the 2 mismatches at
each end of the siRNA at corresponding positions (L1+R1,
L2+R2, L3+R3, L4+R4; Figure 4A). These siRNAs obtained
a symmetric mismatched structure and exhibited a similar
pattern of target silencing to the one of the original
symmetric siRNA that prefers the inhibition of the anti-sense target
expression, although the extent of target cleavage was slightly
different among the modified symmetric siRNAs with
double-mismatches (Figure 4B).
Discussion
RNAi has become a powerful tool in reverse genetics for
the investigation of gene functions in recent years. More
importantly, it has been increasingly applied as therapeutic
strategies in cells, animals, and potentially in
humans[22]. The silencing of mutant genes that cause ALS is one of the
leading applications of gene therapy by RNAi. The design of
effective siRNAs is the key for the success of RNAi therapy.
Poor siRNA efficacy caused by the inaccessibility of the
target region[23,24] and the unfavorable strand asymmetry of
the siRNA[25,26] could limit the therapeutic use of RNAi.
According to the asymmetric rule of RNAi, only 1 strand of
each siRNA will be loaded into the RNA-induced silencing
complex (RISC) and cleave its target mRNA. The other strand
will be degraded without executing RNAi. The
thermodynamic stability of base pairing at the 2 ends of the siRNA
determines which strand will function. The strand with a
less stable 5' base pairing than its 3' base pairing is more
likely to enter the RISC. For a symmetric siRNA, the stability
of base pairing at the 2 ends is the same so that both strands
enter the RISC equally and mediate RNAi with similar
potencies. Apparently, an unfavorable asymmetric siRNA
that has stability of its end base pairing favoring the sense
strand to enter the RISC will silence the complementary strand
of the target mRNA (antisense target) and therefore has poor
RNAi efficacy on the intended target (sense target).
Previous designs of siRNA have focused on searching
naturally asymmetric siRNAs[7,27]. This approach may be
limited in situations, such as ALS-causing mutant SOD1
genes, where the target region is confined by the location of
a mutation and within this confined region no favorable
asymmetric siRNAs can be obtained. Alternatively, weakening
base pairing by the incorporation of mismatches at the 5' of
the antisense strand of an siRNA can create strand
asymmetry favoring the silencing of an intended
gene[22,26].
In the present study, we tested the strategy to covert the
symmetric siRNA to the asymmetric one to increase the
capacity of siRNA in the selective silencing of target genes.
Consistent with previous findings[26], our study demonstrated
that a single mismatch at one end of the siRNA targeting a
mutant SOD1 gene (G85R) successfully disrupted its
original symmetry that led to the non-selective cleavage of both
sense and antisense targets and turned the siRNA into
asymmetric siRNA, thus selectively silencing either sense or
antisense targets (Figure 2). To further modify the
asymmetric design for generating more effective siRNAs, we
systematically modified the symmetric siRNA with double
mismatches. The new siRNAs with double mismatches at
one end demonstrated an increased asymmetry compared to
their single-mismatched counterparts (Figure 3). In addition,
double mismatched siRNAs that had 1 mismatch at each end
of the original symmetric siRNA remained in symmetry as
expected (Figure 4). These results suggest the
effectiveness of converting a symmetric siRNA to an asymmetric one
by introducing mismatches into its structure, and the
superiority of double-mismatched siRNA to single-mismatched
siRNA in producing effective gene silencing due to the
disruption of siRNA symmetry. More importantly, by placing
double mismatches at the 5' end of the antisense of the siRNA
(R1+R2, R1+R3, R1+R4; Figure 3), the modified siRNA
enhanced the silencing of mutant SOD1 gene expression while
abolishing the potential harmful cleavage of the antisense
sequence of this mutant allele. Therefore, we can achieve a
gene silencing of the ALS-causing mutant SOD1 and avoid
unwanted blockage of other functionally important genes
with partial homology to the antisense sequence of the
mutant gene (off-target effect). Although we can not completely
exclude the possibility that the decrease of target cleavage
might partially result from a single nucleotide mismatch
between either the sense or antisense strand of siRNA and its
target, it is unlikely to be a major contributor of the pattern
change in the efficacy or specificity of RNAi observed in our
experiments because the majority of these mismatches falls
into the first 3 positions at the end of siRNA in which a
single nucleotide mismatch has demonstrated inability of
substantially affecting the selectivity of
siRNA[28]. Overall, the findings from this study not only confirm the prediction
of asymmetric rule in the function of siRNA, but also
provide convincing evidence for a better strategy in the design
of effective siRNAs for gene therapy.
ALS is a lethal neurodegenerative disease without a cure.
The establishment of a connection between mutations in the
SOD1 gene and familiar ALS has provided an entry point to
elucidate the underlying mechanism and find an effective
treatment for the disease. Our previous study showed a
feasibility of allele-specific silencing of ALS-causing mutant
alleles by RNAi[21]. Here, we further demonstrated an
improved strategy in the design of effective siRNAs for the
gene therapy for the ALS type that is caused by a mutant
SOD1 (G85R). Since there are more than 100 mutations in
SOD1 that can lead to ALS, the success of gene silencing by
RNAi in these mutant genes may prevent many new cases
from occurring. Nevertheless, more tests need to be
performed to ensure the generalizability of this strategy to
different SOD1 mutant alleles and to other diseases caused by
dominant, gain-of-function gene mutations.
Acknowledgements
We thank Dr Zuo-shang XU, Dr Dianne SCHWARZ, and
Dr Phillip ZAMORE for advice and sharing reagents. This
work was supported by grants from the ALSAssociation,
the NIH/NINDS (No R01NS048145) and the Robert Pachard
Center for ALS Research at Johns Hopkins to Zuo-shang
XU). The contents of this report are solely the
responsibility of the authors and do not necessarily represent the
official views of the NINDS.
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