Nucleobase and ribose modifications control immunostimulation by a MicroRNA-122-mimetic RNA

Article abstract of DOI:10.1021/ja202492e

Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2′-ribose modifications. The

Full text of DOI:10.1021/ja202492e

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Nucleobase and Ribose Modifications Control Immunostimulation
by a MicroRNA-122-mimetic RNA
Hayden Peacock,† Raymond V. Fucini,‡ Prasanna Jayalath,† Josꢀe M. Ibarra-Soza,† Henry J. Haringsma,‡
W. Michael Flanagan,‡ Aarron Willingham,*,‡ and Peter A. Beal*,†
†Department of Chemistry, University of California—Davis, One Shields Avenue, Davis, California 95616, United States
‡Sirna Therapeutics, a wholly owned subsidiary of Merck and Co., 1700 Owens Street, fourth Floor, San Francisco, California 94158,
United States
S Supporting Information
b
ABSTRACT: Immune stimulation is a significant hurdle in
the development of effective and safe RNA interference
therapeutics. Here, we address this problem in the context of
a mimic of microRNA-122 by employing novel nucleobase
and known 20-ribose modifications. The nucleobase mod-
ifications are analogues of adenosine and guanosine that
contain cyclopentyl and propyl minor-groove projections.
Via a site-by-site chemical modification analysis, we identify
several immunostimulatory ‘hot spots’ within the miRNA
guide strand at which single base modifications significantly
reduce immune stimulation. A duplex containing one base
modification on each strand proved to be most effective in
preventing immune stimulation.
icroRNAs (miRNAs) are endogenous ∼22 nucleotide
M
RNAs that regulate gene expression by inhibiting the
translation of mRNAs (mRNAs) via the RNA interference
(RNAi) pathway. MicroRNA-122 (miR-122) is of particular
therapeutic interest, being specifically and abundantly expressed
in healthy liver cells but substantially down-regulated in hepato-
cellular carcinomas (HCC).1 Mimics of miR-122 represent an
attractive therapy for treating HCC, as evidenced by recent
reports indicating that restoration of miR-122 expression in
non-miR122 expressing HCC cells reverses their tumorigenic
properties2,3 and that small molecule activation of miR-122
selectively induces apoptosis in an HCC cell line.4 In addition,
the biodistribution of miR-122 is complementary to the delivery
profile of current liposomal delivery vehicles for nucleic acids,
which deliver the majority of the dose to the liver.5,6
Figure 1. (A) Structure of the microRNA-122-mimetic RNA used in
this study. Modification sites are indicated by numbered nucleotides,
and putative immunostimulatory regions are highlighted in red. (B) Base
modifications with minor groove-directed substituents replace selected
adenosines and guanosines.
We created a miR-122 mimic by synthesizing a 22 nt duplex
RNA (Figure 1a). Such a microRNA mimic is functionally
equivalent to the native miRNA. When we administered the
miR-122 mimic in a lipid-nanoparticle formulation to peripheral
blood mononuclear cells (PBMCs), we observed production of
the cytokine tumor necrosis factor-R (TNF-R), indicative of
immune stimulation (Figure 2). Immunostimulatory activity is a
significant and undesirable off-target effect of RNA therapeutics
due to the toxicity of induced cytokine production. The miR122
guide strand contains two occurrences of an alternating U/G
motif, and such motifs have been implicated in contributing
to immune stimulation.7 The guide strand of a mature miRNA
confers a unique spectrum of activity across a network of genes
and must be conserved. Consequently, immunostimulatory pro-
perties cannot be tempered by sequence changes but, rather,
must be addressed by chemical modification.
It is known that modification of the ribose 20-position can
inhibit the immunostimulatory properties of single-stranded
RNAs and short duplex RNAs;8ꢀ11 however, little is known
about the effect of base modifications or the position dependence
of modifications on immune stimulation. MiRNA mimics, like
other short duplex RNAs, cause immunostimulation via pattern
Received: March 18, 2011
Published: May 25, 2011
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2011 American Chemical Society
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recognition receptors of the innate immune system, primarily the
toll-like receptors (TLRs) 3,12,13 7, and 8.14,15 The recognition of
U and UG-rich motifs is consistent with the involvement of TLRs
7 and 8, whose activation is sequence-specific.16ꢀ18 The mechan-
ism of TLR 7/8 activation is poorly characterized; however, it
stands to reason that since the oligonucleotide sequence is a
criterion for activation of the innate immune system, the nucleo-
bases (in addition to riboses) are being recognized. Therefore,
we hypothesized that creating structural modifications to the
nucleobases that alter their shape while maintaining their ability
to engage in Watson/Crick base pairing could destroy interac-
tions between the microRNA mimic and immune receptors.
It has previously been shown that certain modifications in the
major groove of a short interfering RNA (siRNA) are ineffective
at blocking immune stimulation;9 thus, we decided to investigate
the immunosuppressant effect of minor groove-directed base
modifications. In addition, minor groove modifications block
double-stranded RNA binding motif (dsRBM) proteins impli-
cated in sequence-independent RNAi off-target effects (such as
the RNA-dependent protein kinase (PKR) and adenosine dea-
minase that acts on RNA 1 (ADAR1)).19 Such modifications
could potentially shield microRNA mimics from both TLR-
mediated immunostimulatory effects and dsRBM-mediated off-
target effects.
We synthesized a novel guanosine analogue containing an
N2-cyclopentyl group as an O6-p-nitrophenethyl, 50-DMTrO,
20-TBDMSO, 30-β-cyanoethyl phosphoramidite (cPent-G, Figure 1b)
and incorporated it at selected positions, replacing guanosines, in
the guide strand of the miR-122 mimic. We made substitu-
tions within putative immunostimulatory motifs (50-GUGU-30
sections) and within the seed region (nucleotides 2ꢀ8). Thermal
melting experiments indicated that cPent-G is well tolerated
in a base pair with C (see Supporting Information). Within the
passenger strand, we incorporated adenosine replacements at
two positions opposite the 50-most putative immunostimulatory
motif of the guide strand. We used 2-aminopurines containing
N2-propyl and N2-cyclopentyl substituents20 (Pr-AP and cPent-
AP, respectively, Figure 1b), which have previously been shown
to be excellent analogues of A in base pairs with U.20
To test RNAi activity, we measured the knockdown of two
native miR-122 targets21,22 (Necap2, Slc7a1) after 24 h at 10 nM
in Hepa1ꢀ6 cell culture (Figures 2, 3). The guide strand
modification analysis revealed position-dependent effects on
miR-122 activity as a result of cPent-G incorporation. The base
modification was tolerated at positions 3 and 5, both of which are
within the seed region, and at positions 9, 16, and 18. However, it
was not tolerated at positions 2 and 7, also within the seed region
(Figure 2a). The observation of a position-sensitive minor
groove modification in the guide strand is consistent with our
previous work,19 which also demonstrated that position 2 is
sensitive to minor-groove modification. The cause of this posi-
tion dependence is most likely variation in the distance between
protein components of the RNA-induced silencing complex
(RISC) and the minor-groove edge of the loaded guide strand.
For instance, modeling of the Ago crystal structure and a loaded
oligonucleotide highlights a steric hindrance for the nucleotide
base at position 2 relative to several amino acid residues.19,23
RNAi activity was retained with multiple guide strand mod-
ifications, such as the incorporation of cPent-G at positions
9 and 16.
Figure 2. Target knockdown and cytokine levels for chemical mod-
ifications made to the guide strand of the microRNA mimic. (A)
N2-Cyclopentyl-G (cPent-G) modification at selected positions. Back-
ground shading indicates the positions that are investigated in second
half of graph. (B) N2-Cyclopentyl-G (cPent-G) and 20-ribose modifica-
tions at positions 5 and 9.
individually and simultaneously. We also prepared a duplex
containing a single cyclopentyl modification in both strands
(cPent-G at guide 9 and cPent-AP at passenger 13), for reasons
addressed below, and this retained native RNAi activity. Finally,
to allow for the comparison of base and ribose modifications, we
prepared RNA duplexes containing 20-methoxy (20-OMe), 20-
fluoro (20-F), and 20-deoxy (20-H) modifications (at either A or
G, as appropriate) in a few of the modification patterns. Con-
sistent with the base modifications, the ribose modifications
retained activity at positions 5 and 9 of the guide strand
(Figure 2b) and at positions 13 and 15 of the passenger strand
(see Supporting Information).
Within the passenger strand, base modifications (Pr-AP and
cPent-AP) were tolerated at positions 13 and 15 (Figure 3), both
Next, we determined the immunostimulatory properties of the
modified miRNA mimics by analyzing TNF-R production in
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strand and the occurrence of several immunostimulatory sites,
such as those identified here.
For the passenger strand, modification at either position 13
or 15 with either Pr-AP or cPent-AP reduced immune stimula-
tion. cPent-AP modification at position 13 reduced TNF-R levels
5-fold and was as effective as the most effective ribose modifica-
tion at this site (20-F; see Supporting Information). Based on the
results of the guide and passenger strand modifications, we inves-
tigated the effect of miR-122 containing dual modifications. When
two effective modification sites were combined in either the guide
or passenger strands, the resulting duplex did not further decrease
TNF-R levels (i.e., dual cPent-G modifications at positions 9 and
16 of the guide strand and dual cPent-AP modifications and
positions 13 and 15 of the passenger strand) (Figures 2, 3).
Since additional modification within the same strand was
ineffective, we tried combining two singly modified strands.
Modifications at position 13 of the passenger strand and position
9 of the guide strand are both highly effective at suppressing
cytokine levels, equaling or improving upon the corresponding
ribose-modified RNAs, and were thus combined to form a doubly
modified duplex. These two sites are located adjacent to each other
and within opposite strands of the duplex. The resulting miRNA
mimic reduced TNF-R below the levels recorded for singly
modified duplexes containing each of the component strands
(Figure 3). This modification strategy was the most effective out
of the base-modification strategies evaluated in this work. At this
time we cannot predict the generality of the effect of this modifica-
tion pattern. However, the occurrence of localized ‘hot spots’ within
the miR-122 sequence where modification has a greater effect than
at other sites prompts the investigation of additional immunos-
timulatory sequences incorporating these base modifications.
In summary, we present a new approach for blocking immune
stimulation in miRNA mimics by means of minor-groove loca-
lized base projections. Incorporation of single modifications at
sites throughout the guide strand of a miR-122 mimic revealed
several positions to be immunostimulatory ‘hot spots’, critical to
cytokine stimulation. The position-dependent nature of immune
stimulation was mirrored by 20-ribose modifications at two sites.
A duplex containing a single base modification on each strand
was superior in inhibiting immune stimulation while retaining
RNAi activity. This work shows that minor-groove base mod-
ifications inhibit immune stimulation in short duplex RNAs and
thereby provides an additional route to generating nonimmu-
nostimulatory RNAi therapeutics.
Figure 3. Target knockdown and cytokine levels for chemical mod-
ifications made to the passenger strand and to both passenger and guide
strands.
PBMCs (Figures 2, 3) 16ꢀ20 h post-transfection. The effect of
base modifications on TNF-R stimulation was highly position-
specific and did not exclusively correlate with the locations of the
putative immunostimulatory domains outlined above. cPent-G
incorporation at positions 3, 9, and 16 on the guide strand was
most effective at preventing immune stimulation (Figure 2a). All
of these modified miR-122 mimics displayed TNF-R levels that
were at least 5-fold lower than those of the unmodified duplex.
Both positions 9 and 16 are within a 50-GUGUG-30 motif;
however, position 3 is outside of a putative immunostimulatory
region. In contrast, cPent-G modification at positions 5 and 7
were minimally effective at reducing TNF-R levels, and both of
these sites are within a putative immunostimulatory region.
These observations query the validity of our initial assumption
that the UG-rich motifs define the immunostimulatory regions.
Rather, the site-by-site analysis identified several positions that
are immunostimulatory ‘hot spots’, at which single base mod-
ifications inhibit the recognition of the siRNA, or siRNA-lipid
complex, by the immune receptors.
’ ASSOCIATED CONTENT
We then investigated the generality of the position-specific
nature of TNF-R stimulation by testing ribose modifications at
positions 5 and 9 of the guide strand (Figure 2b). These two sites
were chosen based on their contrasting effects on TNF-R
production with cPent-G modifications. We found that the effect
of 20-F and 20-OMe modifications mirrors the effect of the cPent-
G modifications, in that both of these ribose modifications were
ineffective at blocking immune stimulation at position 5 but
effective at position 9. 20-H modifications were not highly
effective at either of these sites. Interestingly, at position 9 the
effectiveness of immunosuppression correlates with the size of
the modification at the 20-position. Previous reports indicate
that as few as two 20-OMe-G residues in the guide strand of
an immunostimulatory siRNA are required to inhibit cytokine
induction;24 however no information exists on the position
dependence of modifications throughout a microRNA guide
S
Supporting Information. Procedure for the synthesis of
b
N2-cyclopentyl guanosine phosphoramidite and details of RNA
synthesis and purification. Procedures for the RNAi activity and
cytokine assays, and additional assay data. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
pabeal@ucdavis.edu; aarron_willingham@merck.com
’ ACKNOWLEDGMENT
P.A.B. acknowledges support from the National Institutes of
Health in the form of Grant R01 GM080784.
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