Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA

Article abstract of DOI:10.1016/j.omtn.2017.06.012

Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.

Full text of DOI:10.1016/j.omtn.2017.06.012

Original Article
Locked Nucleic Acid Gapmers and Conjugates
Potently Silence ADAM33, an Asthma-Associated
Metalloprotease with Nuclear-Localized mRNA
1
,2,8
3,4
1,2,3,4
1,2,3,4
Hannah M. Pendergraff,
Pranathi Meda Krishnamurthy, Alexandre J. Debacker,
Michael P. Moazami,
Vivek K. Sharma,1,2,3,4 Liisa Niitsoo,1,2 Yong Yu, Yen Nee Tan, Hans Michael Haitchi,
and Jonathan K. Watts1,2,3,4
5
5
2,6,7
1
3
2
Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK; Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK;
4
RNA Therapeutics Institute, UMass Medical School, Worcester, MA 01605, USA; Department of Biochemistry and Molecular Pharmacology, UMass Medical School,
5
6
Worcester, MA 01605, USA; Institute of Materials Research and Engineering, A*STAR, Singapore 138634, Singapore; Clinical and Experimental Sciences, Faculty of
7
Medicine, University of Southampton, Southampton SO16 6YD, UK; NIHR Southampton Respiratory Biomedical Research Unit at University Hospital Southampton
NHS Foundation Trust, Southampton, Southampton SO16 6YD, UK
Two mechanisms dominate the clinical pipeline for oligonucle-
otide-based gene silencing, namely, the antisense approach
that recruits RNase H to cleave target RNA and the RNAi
approach that recruits the RISC complex to cleave target
RNA. Multiple chemical designs can be used to elicit each
pathway. We compare the silencing of the asthma susceptibility
gene ADAM33 in MRC-5 lung fibroblasts using four classes of
gene silencing agents, two that use each mechanism: traditional
duplex small interfering RNAs (siRNAs), single-stranded small
interfering RNAs (ss-siRNAs), locked nucleic acid (LNA)
gapmer antisense oligonucleotides (ASOs), and novel hexade-
cyloxypropyl conjugates of the ASOs. Of these designs,
the gapmer ASOs emerged as lead compounds for silencing
ADAM33 expression: several gapmer ASOs showed subnano-
molar potency when transfected with cationic lipid and low
micromolar potency with no toxicity when delivered gymnoti-
cally. The preferential susceptibility of ADAM33 mRNA to
silencing by RNase H may be related to the high degree of
nuclear retention observed for this mRNA. Dynamic light
scattering data showed that the hexadecyloxypropyl ASO con-
jugates self-assemble into clusters. These conjugates showed
reduced potency relative to unconjugated ASOs unless the lipo-
philic tail was conjugated to the ASO using a biocleavable link-
age. Finally, based on the lead ASOs from (human) MRC-5
cells, we developed a series of homologous ASOs targeting
mouse Adam33 with excellent activity. Our work confirms
that ASO-based gene silencing of ADAM33 is a useful tool
for asthma research and therapy.
Single-nucleotide polymorphisms in ADAM33 have been linked
4,5
to asthma and bronchial hyperresponsiveness. ADAM33 encodes
a membrane-anchored metalloprotease, but a soluble metallopro-
tease-containing form of ADAM33 (sADAM33) is increased in bron-
6,7
choalveolar lavage fluid of asthmatic patients. Furthermore, in
human embryonic lung explant culture, treatment with sADAM33
8
7
induces angiogenesis and myogenesis, pathological features of
airway remodeling in asthma. Expression of human sADAM33 in
transgenic mice causes pathological airway remodeling and makes
airways more susceptible to allergen-induced inflammatory re-
7
sponses. Promisingly, when induction of human sADAM33 is ar-
rested, this leads to reversal of the remodeling and reduced sensitivity
7
to inflammatory responses. Furthermore, in Adam33-null mice chal-
lenged with allergen, both airway remodeling and inflammation are
suppressed. Therefore, inhibition of ADAM33 represents an attractive
disease-modifying therapeutic strategy for treating the root cause of
asthma in many patients.
It is thus crucial to develop therapeutic agents capable of silencing of
ADAM33 expression. Because small molecule metalloprotease inhib-
9
itors have shown a poor specificity profile, chemically optimized
oligonucleotide-based inhibitors could serve as an excellent alterna-
10–12
tive.
In this study, we compared four types of oligonucleotide-
based gene silencing agents for inhibition of ADAM33/Adam33 (Fig-
ure 1): (1) duplex small interfering RNAs (siRNAs), (2) chemically
Received 5 March 2017; accepted 16 June 2017;
http://dx.doi.org/10.1016/j.omtn.2017.06.012.
8
Present address: Roche Innovation Center Copenhagen A/S, 2970 Hørsholm,
INTRODUCTION
Denmark
Asthma is a chronic respiratory disease involving airway inflamma-
tion and structural changes in the form of remodeling of the con-
Correspondence: Hans Michael Haitchi, Institute for Life Sciences, University of
Southampton, Southampton SO17 1BJ, UK.
E-mail: h.m.haitchi@soton.ac.uk
Correspondence: Jonathan K. Watts, RNA Therapeutics Institute, UMass Medical
School, Worcester, MA 01605, USA.
E-mail: jonathan.watts@umassmed.edu
1
,2
ducting airways. It causes more than 345,000 deaths annually and
3
affects more than 340 million people worldwide. ADAM33 is the first
4
asthma susceptibility gene to be identified by positional cloning.
1
58
Molecular Therapy: Nucleic Acids Vol. 8 September 2017 ª 2017 The Author(s).
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

www.moleculartherapy.org
Figure 1. Types of Gene Silencing Oligonucleotides and Chemical Modifications Used in This Study
Types of gene silencing oligonucleotides (left) and chemical modifications (right) used in this study. p, phosphate.
modified single-stranded small interfering RNAs (ss-siRNAs) that
operate by the RNA-induced silencing complex (RISC) pathway,
neither siRNAs nor ss-siRNAs were able to surpass 70% silencing
of the ADAM33 transcript under any of the conditions we tested.
13,14
(
3) locked nucleic acid (LNA) gapmer ASOs operating via the RNase
1
5–17
H mechanism,
and (4) novel biocleavable lipid conjugates of the
To ensure that the relatively limited silencing we observed was not an
artifact of either of the preceding designs, we synthesized and tested a
third option for siRNA design—i.e., fully chemically modified duplex
potent ASOs (including biocleavable conjugates). The LNA gapmers,
1
8
delivered using cationic lipid or by gymnosis, provided significantly
increased potency for silencing ADAM33 relative to the siRNAs or
ss-siRNAs.
0
0
siRNAs. We applied an alternating pattern of 2 OMe-RNA and 2 -flu-
0
0
oro-RNA (2 F-RNA) to the HMH-1 sequence, using a 5 -phosphor-
ylated 21-mer guide strand and a 15-nt sense strand (Figure S1).
This and similar designs have been widely used and show excellent
RESULTS
ADAM33 Silencing by siRNAs and ss-siRNAs
22–26
results both in vitro and in vivo.
The fully modified duplex was
We designed and synthesized a panel of 13 duplex siRNAs targeting
different regions of the ADAM33 transcript (Figure 2A). We then
tested the efficacy of these siRNAs in MRC-5 human lung fibroblast
cells, transfecting the siRNAs with a lipid transfection reagent and
measuring silencing by qPCR. We found that most of the sequences
were inactive, while the most active duplexes were able to attain about
transfected into MRC-5 cells, and its efficacy was comparable to the
parent unmodified siRNA duplex (Figure S1).
LNA Gapmers Show High Potency when Transfected with a
Cationic Lipid
Another major class of single-stranded gene silencing oligonucleo-
27–29
70% silencing (Figure 2B).
tides consists of gapmer ASOs.
Gapmer ASOs operate through
a mechanism different from the preceding siRNAs and ss-siRNAs:
3
0–32
Single-stranded oligonucleotides can be recognized by the RNAi ma-
chinery and serve as guide strands, if the oligomer is stabilized to
they recruit RNase H to cleave target mRNAs.
The “wings” of
0
gapmers consist of sugar-modified nucleotides such as 2 -O-methox-
0
0
33
34–36
resist nuclease cleavage and contains a 5 -phosphate or phospho-
yethyl-RNA (2 -O-MOE-RNA) or LNA,
which increase bind-
1
3,14,19,20
nate.
To explore whether changing the biophysical properties
ing affinity and nuclease stability, while the central “gap” is phosphor-
othioate (PS) DNA, which elicits RNase H cleavage of the target. We
designed and synthesized 12 PS LNA gapmers, using a 3-9-3 pattern
of sugars and targeting various regions of ADAM33 mRNA (Fig-
ure 3A). We initially tested the LNA gapmers at a 50 nM concentra-
tion in MRC-5 fibroblasts, using Lipofectamine RNAiMAX as a
transfection agent. LNAs 33-G, 33-N, 33-O, 33-P, 33-Q, and 33-R
all achieved >80% silencing of ADAM33 when normalized to a scram-
bled siRNA duplex control (Figure 3B). This very high hit rate and
maximal efficacy contrast sharply with the low hit rate and maximal
efficacy observed when using RISC-dependent oligonucleotides
(siRNAs and ss-siRNAs) for this target. Furthermore, a dose-response
analysis shows potent, dose-dependent ADAM33 inhibition for 33-O
and 33-R to concentrations < 0.16 nM (Figure 3C).
of the RNAi trigger could improve activity, we synthesized and tested
ss-siRNA analogs of three siRNA sequences: highly active sequence
HMH-1, moderately active sequence HP-2, and inactive sequence
HP-3. The ss-siRNA analogs showed reduced efficacy (HMH-1 and
HP-2) or maintained inactivity (HP-3) (Figure 2C). For the most
active siRNA sequence, HMH-1, we carried out a chemical optimiza-
tion of the ss-siRNAs and found one analog with activity that was
comparable, but not superior, to the parent duplex RNA (Figure 2D).
0
0
Only the ss-siRNA with 2 -O-methyl-RNA (2 OMe-RNA) modifica-
0
tions at the 3 terminus was able to approach the potency of the
duplex siRNA (ssi-HMH-1c) (Figure 2D). This result was followed
up in the context of other sequences and targets, and this exploration
2
1
of the medicinal chemistry of ss-siRNAs was published. Overall,
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Molecular Therapy: Nucleic Acids
Figure 2. ADAM33 Gene Expression Can Be Inhibited by siRNA and ss-siRNA Oligonucleotides, but the Maximal Extent of Inhibition Is Modest
0
(
A) Oligonucleotide sequences: tt represents a 3 terminal overhang of two deoxy thymidines; duplex siRNAs are otherwise unmodified RNA. The ss-siRNA sequence
0
0
0
0
modifications are subscript s, phosphorothioate linkage; red, 2 F-RNA; blue, 2 OMe-RNA; purple, 2 -O-MOE-RNA; green, LNA; p, 5 -phosphate. For siRNA duplexes,
passenger strands are listed on top and guide strands underneath. (B) qRT-PCR results from a screen of siRNA duplexes showing that few siRNA sequences were active
against this target. (C) qRT-PCR results showing the gene silencing efficacy of ss-siRNA analogs of the three most potent duplex siRNAs, in comparison with the parent
duplexes. (D) qRT-PCR results comparing potencies of different chemical modification schemes on ss-siRNA activity. Error bars represent the SD of biological replicates. All
oligonucleotides were transfected at 50 nM into MRC-5 lung fibroblasts using Lipofectamine RNAiMAX. All results are normalized to a scrambled siRNA duplex control.
Gymnotic Delivery of LNA Gapmers
The gymnotic approach was adopted to test (1) whether the LNA
gapmers could enter the MRC-5 cells without the use of transfection
agents, (2) how the potency of the LNA gapmers delivered via gym-
notic delivery compared to the transfections using a cationic lipid,
and (3) whether the gymnotically delivered LNA gapmers were toxic
to cultured cells.
Although the LNA gapmers were highly potent in cultured cells,
transfection efficiency in vitro does not always correlate well with
in vivo studies. Ex vivo lung tissue and in vivo lung experiments
also require oligonucleotides with the ability to exert potent silencing
0
without the aid of toxic transfection agents. Naked LNA or 2 -fluo-
0
roarabinonucleic acid (2 F-ANA) ASOs can be taken up by most
1
8,37,38
types of dividing cells in culture in a process termed gymnosis.
A time course experiment was performed, gymnotically delivering
33-O into MRC-5 cells at a 1 mM dose to determine the optimal
day to harvest the cells after treatment, between day 4 and day 9.
Our results indicate that day 7 post-treatment was an appropriate
time to harvest cells (Figure S2). We then selected LNA gapmers
Gymnotic delivery does not require serum additives or transfection
reagents but is a slower process compared to lipofection. It tends to
show low toxicity and shows an improved correlation between
1
8
in vitro and in vivo results.
1
60
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Figure 3. LNA Gapmers Are Highly Potent when Transfected with a Cationic Lipid
(A) LNA gapmer sequence modifications are subscript s, phosphorothioate linkage; green uppercase, LNA; lowercase, DNA. (B) qRT-PCR results showing gene silencing by
LNA gapmers at 50 nM. All results are normalized to a scrambled siRNA duplex control. Error bars represent the SD of biological replicates. (C) qRT-PCR results showing
dose-response analysis of 33-O and 33-R. Results are normalized to a scrambled LNA gapmer control. Error bars for the dose responses represent the SD of technical
replicates.
3
3
3-G, 33-N, 33-O, 33-P, and 33-R for further testing. Oligomers
3-N, 33-O, and 33-P achieved >80% reduction of ADAM33 tran-
mide with sodium hydride followed by addition of hexadecyl bromide
and catalytic potassium iodide gave 1-O-hexadecyl-1,3,-propanediol
45
script levels, while 33-R showed 60% ADAM33 silencing at a 3 mM
dose (Figure 4).
in a single step as previously observed; recrystallization with hexane
yielded white crystals of excellent purity. The phosphoramidite was
synthesized under standard conditions using 2-cyanoethyloxy(N,N-
diisopropylamino)phosphonamidic chloride. The phosphoramidite
The LNA gapmers are able to efficiently silence ADAM33 expression
without the aid of transfection agents. Although the potency of the
gymnotically delivered oligonucleotides was not as high as the cationic
lipid-mediated LNAdelivery, thegymnotically deliveredLNAs showed
no toxicity to cultured cells (based on phenotype and cell confluence).
0
was then conjugated to the 5 end of LNA gapmers 33-N, 33-O,
and 33-P via solid-phase synthesis (Figure 5A).
Besides direct recognition of cell-surface receptors by conjugated
small molecules, conjugation approaches may change the biophysical
properties of the oligonucleotide, which may affect their cell uptake in
a more indirect way. For example, a variety of amphiphilic oligonu-
cleotide conjugates have been shown to assemble into micelle-type
structures. Previous work has described very long polymer conjugates
Hexadecyloxypropyl Conjugates for Cellular Uptake
One of the major limitations to oligonucleotides as therapeutic agents is
their relatively poor uptake into most cells. One promising solution is
the covalent attachment of a ligand that allows recognition by cell-sur-
3
9
4
0–42
46
face receptors.
We sought to improve uptake by attaching a lipid
that induce self-assembly of oligonucleotides into clusters, as well as
moiety based on 1-O-hexa-decyloxy-1,3-propanediol, which has been
previously shown to increase small molecule uptake by MRC-5 fibro-
blast cells and improve the oral bioavailability of nucleoside drugs.
oligonucleotides with shorter hydrophobic tails that assemble around
a liposomal core.
47
4
3
44
Therefore, we tested whether our conjugates self-assemble using dy-
namic light scattering (DLS). We found that conjugation of a hexade-
cyloxypropyl tail clearly induced assembly of oligonucleotides into
A 1-O-hexadecylpropanediol phosphoramidite was synthesized in
two steps (Figure 5A). Treatment of propanediol in dimethylforma-
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Molecular Therapy: Nucleic Acids
Two of our active ASOs targeting the human ADAM33 transcript also
had significant sequence homology to the mouse transcript (G and N)
0
(
Table 1). Although the sequence conservation in the 3 UTR was
poor, we did an analysis of the conservation of structural features us-
51
ing T-Coffee and designed targets to regions of conserved structure
0
(
P and Q, near the end of the ORF, and R, in the 3 UTR) (Table 1).
These five sequences were synthesized as 3-9-3 LNA gapmers and
tested for their ability to inhibit Adam33 expression in mouse embry-
onic fibroblasts (Figure 6).
The most active ASOs in terms of silencing mouse Adam33 expres-
0
0
sion were those targeting the 3 end of the ORF or the 3 UTR (Fig-
ure 6), which were also the three sequences that were less well
conserved relative to the human hits. In future studies, we will test
these sequences in mouse models to further explore the role of
ADAM33/Adam33 in asthma and the potential for oligonucleotides
to play a therapeutic role in its treatment.
Figure 4. Gymnotically Delivered Gapmers Are Potent Inhibitors of ADAM33
Gene silencing measured by qRT-PCR after gymnotic delivery. Oligonucleotides are
delivered to MRC-5 cells at a 3 mM dose and normalized to a non-treated control
(NT). Error bars represent the SD of biological replicates.
clusters (Figure 5B). Our work demonstrates that even a simple hy-
drophobic conjugate may be sufficient to induce efficient self-assem-
bly into clusters or micelles.
DISCUSSION
Single-stranded oligonucleotides are amenable to simple delivery ap-
proaches (including free uptake by simply delivering naked oligonu-
cleotides into the blood or the CNS), which has led to a significant
Clustering or assembly of oligonucleotides often correlates with
enhanced uptake and activity, across multiple size scales and likely
4
8,49
13,14
via multiple mechanisms.
Nevertheless, in our case, despite the
drive to develop single-stranded oligonucleotides, including fully
23,24
efficient self-assembly observed for the lipid tail conjugates, the bio-
stable hexadecyloxypropyl conjugates showed reduced silencing ef-
ficacy in cells (Figure 5C). A highly lipophilic tail might lead to
stronger association with cell membranes but fail to release active
or partly
single-stranded oligomers that can engage the RNAi ma-
chinery (RISC). These are appropriate for gene silencing applications
in which recruitment of RISC is required—for example, in the context
of allele-selective silencing of mutant Huntingtin, RISC-dependent
5
0
13,52,53
oligonucleotide into the cytoplasm. We therefore generated bio-
cleavable analogs based on a section of phosphodiester (PO)-linked
DNA between the lipid tail and the PS gapmer ASO (Figure 5A).
Nuclease cleavage at the PO linkages would allow release of the
gapmer from the lipid tail inside endosomes (M.W. Lindholm
et al., 2016, Oligonucleotide Therapeutics Society, conference). We
found that treatment of cells with these biocleavable constructs
produced similar potency silencing of ADAM33 relative to the anal-
ogous unconjugated gapmers but did not provide a potency advan-
tage in vitro (Figure 5C). However, hydrophobic conjugates of
gapmer oligonucleotides may provide an advantage for tissue
distribution or other in vivo properties, and our data point to the
importance of making hydrophobic conjugates in a biocleavable
manner, to avoid any membrane entrapment and associated po-
tency reduction.
approaches showed far greater selectivity (>30-fold)
with simpler steric blocker ASOs (ꢀ3- to 6-fold).
compared
5
4,55
However, for silencing nuclear-localized transcripts, recruitment of
RISC may not be ideal. ADAM33 mRNA is ꢀ90% nuclear localized
56
even though it is a protein-coding transcript. RNase H is predom-
57
inantly localized in the nucleus and plays a role in genome defense,
58
including elimination of R loops. After gapmer-induced RNase H
0
cleavage of a target RNA, the 3 fragment is predominantly degraded
0
0
59
by the nuclear enzyme 5 -3 exoribonuclease 2 (XRN2). Nuclear
RNAs including intronic sequences are vulnerable to cleavage
6
0–62
induced by gapmer ASOs,
and in the context of noncoding
RNAs, it was observed that ASOs are less sensitive to subcellular local-
ization of their targets, while siRNAs tend to be more effective at
63
silencing ncRNAs that are predominantly cytoplasmic. This empir-
ical tendency exists even though a cytoplasmic mechanism has also
64
ASOs Designed to Homologous Regions of the Mouse Adam33
Transcript Are Also Active
been described for gapmer ASO action and RNAi factors are present
65
and active in human cell nuclei. Our current study suggests that it is
important to consider subcellular localization of the target when se-
lecting a preferred oligonucleotide class for silencing, even if the target
is a protein-coding mRNA.
To explore the biological role of ADAM33/Adam33 in asthma
7
progression, we have developed a series of mouse models. We
therefore set out to identify analogs of the human lead ASOs that
could silence mouse Adam33 expression. The mouse and human
ADAM33/Adam33 transcripts are relatively well conserved in the
Our results should not be taken as a statement about the relative value
or potency of ASO and siRNA silencing in general. For many targets,
particularly in vitro, it is easier to find potent siRNAs than potent
ASOs. The siRNA-Argonaute and ASO-RNase H approaches should
be considered complementary rather than redundant.
0
coding region but diverge substantially toward the 3 end of the
0
open reading frame (ORF) and in their 3 UTR sequences (for
example, the mouse transcript has a much shorter UTR sequence
than the human transcript).
1
62
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Figure 5. Lipid-Conjugated LNA Gapmers Show Reduced Potency Unless the Lipid Is Joined to the ASO via a Biocleavable Linkage
i
(A) Synthesis and structure of hexadecyloxypropyl LNA conjugates. Reagents and conditions: (i) NaH, CH
3
(CH
2
)
15Br, cat. KI; (ii) Pr
2
NP(O(CH
2
)
2
CN)Cl, DIPEA, CH
2
Cl
2
; (iii)
standard oligonucleotide synthesis conditions. (B) Dynamic light scattering shows that addition of a lipid tail causes self-assembly of ASOs into larger structures (comparing
3-O, red bars, with its biostable conjugate, black bars). (C) qRT-PCR results showing dose-response analysis of A33-N, A33-O, and A33-P and both types of lipid
3
conjugates after gymnotic delivery to MRC-5 cells at concentrations from 3 to 0.11 mM. Results are normalized to a non-treated control sample (NT). Error bars represent the
SD of biological replicates.
2
In this project, multiple RNase H-dependent LNA gapmers were
highly effective silencing agents for ADAM33 mRNA, while siRNAs
failed to yield potent silencing. Because gymnotically delivered oligo-
nucleotides showed no toxicity and high efficiency in cultured cells,
they are ideal for further ADAM33 studies. We are exploring the
delivery of anti-Adam33 oligonucleotides to mouse airways and
the therapeutic potential of ASO-mediated inhibition of ADAM33
for reversing pathological airway remodeling in asthma and other
chronic lung diseases, including chronic obstructive pulmonary dis-
including a UPC Waters HSS C18 SB column (100 mm ꢁ
3.0 mm ꢁ 1.8 mm) gradient 90% CO
2
:10% methanol modifier
:40% methanol modifier
(25 mM ammonium acetate) to 60% CO
2
(25 mM ammonium acetate) in 3 min at a flow rate of 1.5 mL/min.
The makeup flow (methanol/1% formic acid) was pumped at a flow
rate of 0.45 mL/min into the mass spectrometer. Mass spectra were
recorded using positive ion electrospray ionization.
Oligonucleotides
6
6,67
68
ease (COPD)
and sarcoidosis.
siRNAs were designed using the Whitehead siRNA selection server
(
http://sirna.wi.mit.edu). ASOs were manually designed using the
69
MATERIALS AND METHODS
General Methods
predicted secondary structure of the mRNA (from mFold ), the pre-
dicted self-structure of the oligonucleotide, and the predicted speci-
ficity (from NCBI BLAST).
1
13
31
H-, C-, and P-NMR spectra were recorded on Bruker DPX or
Bruker AV NMR spectrometers operating at 400, 101, and 162
MHz, respectively. Small molecules were analyzed using a Waters
TQD mass spectrometer equipped with a triple quadrupole analyzer.
Samples were introduced to the mass spectrometer via an Acquity
The first set of siRNAs for sequence screening was purchased
from Integrated DNA Technologies (IDT). All other oligonucleotides
were synthesized in-house at a 1-mmol scale on Applied Bio-
systems 394 DNA/RNA synthesizers with Unylinker (ChemGenes)
2
UltraPerformance Convergence Chromatography (UPC ) system,
Molecular Therapy: Nucleic Acids Vol. 8 September 2017
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Molecular Therapy: Nucleic Acids
Table 1. Design of Mouse Analogs of Lead ASO Sequences
Figure 6. Identification of ASOs that Provide Highly Effective Silencing of
Mouse Adam33 Expression in Mouse Embryonic Fibroblasts
Oligonucleotides were delivered at 50 nM with Lipofectamine RNAiMAX. Error bars
represent the SD of biological replicates. Mouse-targeted sequences are shown in
Table 1 and were made as fully phosphorothioate (PS) 3-9-3 LNA gapmers (the
same chemical architecture as the human sequences). The sequence of LNA-scr2,
0
0
also a PS 3-9-3 gapmer, is 5 -AACacgtctataCGC-3 . 33-O is the human sequence
from Figure 3, included as an additional negative control.
thylsilyl protecting group was removed with a 4:1 DMSO/triethyl-
ꢂ
amine trihydrofluoride (TEA$3HF) solution at 65 C for 3 hr. The re-
action was cooled to RT and then precipitated by the addition of 3 M
Small numbers refer to the base numbering within the target transcripts (in each case,
human on top and mouse below). Gray highlighting in the mouse ASOs represents non-
sequence-conserved residues relative to the human ASOs. While P, Q, and R showed
only modest sequence conservation, they showed a greater degree of structural conser-
vation as predicted by T-Coffee. RefSeq accession numbers of the sequences used for
alignments were as follows: human, NM_025220.3 (3,573 nt total length, where the
ORF ends at 2,569), and mouse, NM_033615.2 (3,165 nt total length, where the ORF
ends at 2,694).
NaOAc (25 mL) and BuOH (1 mL). The mixture was centrifuged at
ꢂ
4 C for 5 min at 8,000 rpm, washed with 70% EtOH, and air dried,
0
and the pellet was resuspended in RNase-free water. 2 -modified
RNA, LNA, and DNA were deprotected with concentrated NH
at 55 C overnight.
4
OH
ꢂ
Oligonucleotides were evaporated to dryness by rotary evaporation
and then resuspended in 1 mL RNase-free water. If liquid chro-
matography-mass spectrometry (LC-MS) and analytical PAGE
(described later) indicated that the oligomer was sufficiently
pure, it was desalted with a Nap-10 column (GE Healthcare) and
used directly. All oligonucleotides were characterized on Bruker
MicrOTOF Ultimate 3000 or Agilent Q-TOF LC-MS systems with
electrospray ionization and time-of-flight analysis, using negative
ionization mode. All sequences and mass spectrometric data are pro-
vided in Tables S1–S5.
or nucleoside-loaded CpG supports and standard detritylation and
capping reagents. Activation was achieved with 5-benzylthio-1H-tet-
razole (BTT, 0.3 M in acetonitrile). Oxidation was achieved using
0.02 M iodine in a mixture of tetrahydrofuran (THF), water, and
pyridine. Sulfurization was accomplished with the 1,2,4-dithiazolines
EDITH (Link Technologies) or DDTT (0.1 M, ChemGenes). RNA,
0
0
2
F-RNA, and 2 OMe-RNA phosphoramidites (ChemGenes) were
dissolved to a concentration of 0.15 M in anhydrous acetonitrile
immediately before use. LNA and MOE phosphoramidites were
3
3,70,71
0
synthesized by standard methods
from 3 -hydroxyl pre-
cursors (Rasayan) using 2-cyanoethyloxy(N,N-diisopropylamino)
phosphonamidic chloride and were used at 0.1 or 0.15 M in
acetonitrile, with the exception of LNA 5-MeC (LNA-C is actually
LNA 5-Me-C), which was dissolved in a 3:1 mixture of THF:acetoni-
trile. Coupling times for all modified phosphoramidites were 10 min.
Coupling yields for all nucleoside phosphoramidites were >98%.
Coupling yields for the hexadecyloxypropyl phosphoramidite were
good as long as the quality of the phosphoramidite was excellent.
During one experiment, we used a batch of hexadecyloxypropyl
phosphoramidite with somewhat lower purity and the coupling
yield dropped to 50%.
20 mM working stocks of siRNAs were prepared by annealing the
sense and antisense strands in a final 2.5ꢁ PBS buffer. The solutions
ꢂ
ꢂ
were heated at 95 C for 10 min and then cooled to RT at a rate of 1 C
per minute.
Oligonucleotide Purification and Electrophoresis
Approximately 20 A260 units (preparative gel) or 0.1 A260 units
(analytical gel) was loaded into a 20% polyacrylamide gel containing
7 M urea and run at 400 V for ꢀ3 hr. Analytical gels were visualized
using Stains-All (Sigma). For preparative gels, the product band
was briefly visualized by UV shadowing, excised from the gel, and
incubated in RNase-free water overnight. The aqueous solution
was then concentrated by rotary evaporator, resuspended in RNase-
free water, and desalted via a Nap-25 column (GE Healthcare). The
Unmodified RNA was deprotected using a 3:1 ratio of NH OH/EtOH
4
0
for 48 hr at room temperature (RT). The RNA 2 OH tert-butyldime-
1
64
Molecular Therapy: Nucleic Acids Vol. 8 September 2017

www.moleculartherapy.org
desalted oligonucleotide was evaporated to dryness again and resus-
pended in a small volume of RNase-free water.
qRT-PCR was performed using iTaq Supermix (Bio-Rad) on a Bio-
Rad CFX96 real-time system. Data were normalized relative to levels
of GAPDH mRNA. A primer and probe assay (IDT) specific for the
0
Hexadecyloxypropyl-conjugated oligonucleotides were purified by
ADAM33 3 untranslated region was used (unless otherwise stated):
0
0
2
0% denaturing PAGE as earlier or by ion exchange chromatography
using Agilent 1200 series high-performance liquid chromatography
HPLC), an Agilent PL-SAX column, and eluents containing 30%
forward primer, 5 -GGCCTCTGCAAACAAACATAATT-3 ; reverse
0
0
0
primer, 5 -GGGCTCAGGAACCACCTAGG-3 ; probe, 5 -CTTCC
0
(
TGTTTCTTCCCACCCTGTCTTCTCT-3 .
probe assay (IDT) was also used: forward primer, 5 -TGGT
CCAGGGGTCTTACT-3 ; reverse primer, 5 -CCTCAACGAC-
CACTTTGT-3 ; probe, 5 -CTCATTTCCTGGTATGACAACGAA
A
GAPDH primer-
0
aqueous acetonitrile with increasing sodium perchlorate. Either
method allowed us to remove all unconjugated oligonucleotide and
obtain the pure conjugate.
0
0
0
0
0
TTTGGC-3 . For mouse Adam33 mRNA quantitation, we used
Cell Culture and Transfection
TaqMan (Thermo Fisher Scientific) probe set Mm00459697_g1. All
ꢂ
MRC-5 embryonic fibroblasts were maintained at 37 C and 5% CO
2
qRT-PCR experiments were performed in technical replicates. The
ꢂ
ꢂ
in DMEM supplemented with 10% fetal bovine serum (FBS), 2%
L-glutamine, 1% non-essential amino acid (NEAA), and 1% sodium
pyruvate (all from Sigma). Cells were plated in 6-well plates at 150,000
cells/well (cationic lipid) or 25,000 cells/well (gymnotic) 24 hr before
transfection, unless otherwise stated. Mouse embryonic fibroblasts
qRT-PCR cycle was as follows: 95 C for 7 min (95 C for 15 s and
60 C for 30 s) ꢁ 40 cycles.
ꢂ
DLS
The lyophilized gapmer ASO (33-O) and its hexadecyloxypropyl con-
jugate (33-O biostable conjugate) were dissolved in RNase-free water
at a concentration of 20 nM and filtered with a 0.2-mm filter before
measurement. The filtered samples were then transferred to a 384-
well microplate and placed into a DynaPro PlateReader-II system
(Wyatt Technology) for DLS measurement. The data were collected
by averaging ten measurements (ꢀ5 s each) for each sample.
(MEFs) were maintained in DMEM supplemented with 10% FBS
and were plated in 6-well plates at 100,000 cells/well 24 hr before
transfection.
Oligonucleotides were transfected at a 50 nM concentration for a sin-
gle dose or decreasing doses for dose responses. Cells were transfected
using RNAiMAX (Life Technologies) using 0.75 mL of lipid per 1 mL
of oligonucleotide (siRNA) or 0.67 mL of lipid per 1 mL of oligonucle-
otide (LNA) in Opti-MEM (Life Technologies). Gymnotic delivery
was achieved using a 1 or 3 mM oligonucleotide concentration
in full cell culture media. Cells were harvested for RNA analysis
2-Cyanoethyl (3-(Hexadecyloxy)Propyl)
Diisopropylphosphoramidite
3-(hexadecyloxy)propan-1-ol was synthesized in one step according
45
to the method of Yamano et al. and recrystallized from hexane.
The crystalline product (200 mg, 0.67 mmol) was dissolved in dry
CH Cl and diisopropylethylamine (0.70 mL, 2.68 mmol, 4 equiv)
3
days after transfection (lipid) or 7 days after treatment (gymnotic)
unless otherwise stated.
2
2
was added while stirring at RT. 2-cyanoethyloxy(N,N-diisopropyla-
mino) phosphonamidic chloride (0.18 mL, 0.8 mmol, 1.2 equiv)
was added dropwise, and the solution was stirred for 45 min. After
RNA Harvest and Real-Time qPCR
Total RNA from cells was harvested 3 days post-transfection (lipid
transfection) or 7 days post-transfection (gymnotic) unless otherwise
stated. After washing each well with 1 mL of PBS, 1 mL of RiboZol
completion, 15 mL of CH
was washed with saturated aqueous NaHCO
MgSO . The crude product was purified on a silica column with
50:50:1 Hex:EtOAc:NEt as eluent to afford the title compound as a
2
Cl
2
were added and the organic phase
3
and dried over
(Amresco) was added to each well, incubated for 2 min at RT, and
4
transferred to a 1.5-mL microcentrifuge tube. Chloroform (200 mL)
3
was added to each tube, and the mixture was shaken vigorously for
colorless liquid (175 mg, 52% yield). R in EtOAc = 0.28. MS (ESI):
f
1
min and then incubated at RT for 10 min. The mixture was centri-
found 501 (M+H); mass expected for (C H N O P + H = 501.4).
H NMR (400 MHz, CDCl ) d 0.89 (t, J = 6.85 Hz, 3H, CH CH )
3 3 2
28
57 2 3
1
fuged at 13,000 rpm for 20 min, and then the clear aqueous layer was
transferred to a new 1.5-mL tube, avoiding any interphase. 2-propa-
nol (600 mL) was added to the aqueous layer, followed by a 1-min
1.19 (dd, J = 6.72, 3.42 Hz, 12H, 2 (CH ) CHN) 1.26 (s, 26 H, 13
3
2
(CH
J = 6.30 Hz, 2H, POCH
CH CN) 3.40 (t, J = 6.66 Hz, 2H, OCH
6.30 Hz, 2H, POCH CH CH O) 3.54–3.65 (m, 2H, 2 CH) 3.65–
3.79 (m, 2H, POCH CH CH O) 3.79–3.93 ppm (m, 2H,
CN). C NMR (101 MHz, CDCl ) d 14.1 (s, 1C,
) 20.3 (d, J = 6.60 Hz, 1C, CH CN) 22.7 (s, 1C, CH CH CH
4.5 and 24.63 (2 d, J = 7.70 Hz, 2x2C, (CH ) CHN) 26.2 (s, 1C,
2
)
n
) 1.56 (quin, J = 6.94 Hz, 2H, OCH
CH CH O) 2.64 (t, J = 6.60 Hz, 2H,
CH CH ) 3.50 (t, J =
2 2 2
CH CH ) 1.88 (quin,
ꢂ
vigorous shake and then a 20-min incubation at ꢃ20 C, and followed
2
2
2
ꢂ
by a 15-min centrifugation at 14,000 rpm at 4 C. The resulting pellet
2
2
2
2
was washed with ice-cold 70% ethanol, re-centrifuged at 8,000 rpm
for 10 min at 4 C, and then briefly allowed to air dry. The pellet
was resuspended in RNase-free water, heated to 55 C for 5 min,
and then quantitated by UV spectroscopy.
2
2
2
ꢂ
2
2
2
ꢂ
13
POCH
2
CH
2
3
CH CH
3
2
2
2
2
3
)
2
3
2
1
mg of RNA was treated with 2 U of DNase I (Worthington Biochem-
OCH CH CH ) 29.3 (s, 1CH ) 29.5 (s, 1CH ) 29.6 (s, 2CH )
2 2 2 2n 2n 2n
29.6 (s, 1CH ) 29.7 (s, 5CH ) 29.8 (s, 1CH ) 31.5 (d, J =
2n 2n 2n
ꢂ
ꢂ
ical) for 10 min at 37 C, followed by 10 min at 75 C. RNA was reverse
transcribed using a High-Capacity cDNA Reverse Transcription Kit
7.34 Hz, 1C, POCH CH CH O) 31.9 (s, 1CH ) 43.0 (d, J =
2
2
2
2n
(Life Technologies) per the manufacturer’s protocol.
11.74 Hz, 2C, 2 CH) 58.3 (d, J = 19.07 Hz, 1C, POCH
2
CH
2
CN)
Molecular Therapy: Nucleic Acids Vol. 8 September 2017
165

Molecular Therapy: Nucleic Acids
6
CH
0.7 (d, J = 17.61 Hz, 1C, POCH CH CH O) 67.3 (s, 1C, POCH CH
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2
2
2
2
2
31
apies of clinical utility. Nat. Biotechnol. 35, 238–248.
2
O) 71.1 (s, 1C, OCH
2
CH
2
2
CH ), 117.6 ppm (s, 1C, CN). P NMR
1
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1
2. Deleavey, G.F., and Damha, M.J. (2012). Designing chemically modified oligonucle-
otides for targeted gene silencing. Chem. Biol. 19, 937–954.
SUPPLEMENTAL INFORMATION
Supplemental Information includes two figures and five tables and
can be found with this article online at http://dx.doi.org/10.1016/j.
omtn.2017.06.012.
3. Yu, D., Pendergraff, H., Liu, J., Kordasiewicz, H.B., Cleveland, D.W., Swayze, E.E.,
Lima, W.F., Crooke, S.T., Prakash, T.P., and Corey, D.R. (2012). Single-stranded
RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expres-
sion. Cell 150, 895–908.
1
4. Lima, W.F., Prakash, T.P., Murray, H.M., Kinberger, G.A., Li, W., Chappell, A.E., Li,
C.S., Murray, S.F., Gaus, H., Seth, P.P., et al. (2012). Single-stranded siRNAs activate
RNAi in animals. Cell 150, 883–894.
AUTHOR CONTRIBUTIONS
Conceptualization: H.M.H. and J.K.W.; Methodology: H.M.P.,
Y.N.T., H.M.H., and J.K.W.; Validation: H.M.P., Y.N.T., H.M.H.,
and J.K.W.; Investigation: H.M.P., P.M.K., and Y.Y.; Resources
1
5. Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hökfelt, T., Broberger, C.,
Porreca, F., Lai, J., Ren, K., et al. (2000). Potent and nontoxic antisense oligonucleo-
tides containing locked nucleic acids. Proc. Natl. Acad. Sci. USA 97, 5633–5638.
(Oligonucleotide and conjugate synthesis): H.M.P., P.M.K., A.J.D.,
M.P.M., L.N., and V.K.S.; Writing (draft): H.M.P. and J.K.W.; Review
and Editing: all authors; Visualization: H.M.P. and J.K.W.; Supervi-
sion: H.M.H. and J.K.W.; Funding Acquisition: H.M.H. and J.K.W.
16. Kurreck, J., Wyszko, E., Gillen, C., and Erdmann, V.A. (2002). Design of antisense
oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 30, 1911–1918.
1
7. Fluiter, K., Mook, O.R.F., Vreijling, J., Langkjaer, N., Højland, T., Wengel, J., and
Baas, F. (2009). Filling the gap in LNA antisense oligo gapmers: the effects of un-
locked nucleic acid (UNA) and 4 -C-hydroxymethyl-DNA modifications on RNase
0
ACKNOWLEDGMENTS
H recruitment and efficacy of an LNA gapmer. Mol. Biosyst. 5, 838–843.
3
1
We thank Dr. Neil Wells for help establishing P NMR protocols. We
are also grateful to Dr. Dorcas Brown and ATDBio for support with
oligonucleotide synthesis while we were at the University of South-
ampton. This work was supported by a pilot project grant by the Insti-
tute for Life Sciences (University of Southampton) to J.K.W. and
H.M.H., UMass Medical School startup funds to J.K.W., and the En-
gineering and Physical Sciences Research Council (EP/M003973/1 to
J.K.W.). H.M.H. was supported by Medical Research Council (MRC)
Clinician Scientist Fellowship G0802804.
1
8. Stein, C.A., Hansen, J.B., Lai, J., Wu, S., Voskresenskiy, A., Høg, A., Worm, J.,
Hedtjärn, M., Souleimanian, N., Miller, P., et al. (2010). Efficient gene silencing by
delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection
reagents. Nucleic Acids Res. 38, e3.
1
9. Kini, H.K., and Walton, S.P. (2007). In vitro binding of single-stranded RNA by hu-
man Dicer. FEBS Lett. 581, 5611–5616.
2
0. Holen, T., Amarzguioui, M., Babaie, E., and Prydz, H. (2003). Similar behaviour
of single-strand and double-strand siRNAs suggests they act through a common
RNAi pathway. Nucleic Acids Res. 31, 2401–2407.
21. Pendergraff, H.M., Debacker, A.J., and Watts, J.K. (2016). Single-stranded silencing
RNAs: hit rate and chemical modification. Nucleic Acid Ther. 26, 216–222.
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