U.S. patent application number 13/006099 was filed with the patent office on 2012-04-05 for pharmaceutical composition.
This patent application is currently assigned to Santaris Pharma A/S. Invention is credited to Joacim Elmen, Sakari Kauppinen, Phil Kearney.
Application Number | 20120083596 13/006099 |
Document ID | / |
Family ID | 38564007 |
Filed Date | 2012-04-05 |
United States Patent
Application |
20120083596 |
Kind Code |
A1 |
Elmen; Joacim ; et
al. |
April 5, 2012 |
Pharmaceutical Composition
Abstract
The invention provides pharmaceutical compositions comprising
short single stranded oligonucleotides, of length of between 8 and
17 nucleobases which are complementary to human microRNAs. The
short oligonucleotides are particularly effective at alleviating
miRNA repression in vivo. It is found that the incorporation of
high affinity nucleotide analogues into the oligonucleotides
results in highly effective anti-microRNA molecules which appear to
function via the formation of almost irreversible duplexes with the
miRNA target, rather than RNA cleavage based mechanisms, such as
mechanisms associated with RNaseH or RISC.
Inventors: |
Elmen; Joacim; (Malmo,
SE) ; Kearney; Phil; (Picton, AU) ; Kauppinen;
Sakari; (Smorum, DK) |
Assignee: |
Santaris Pharma A/S
Horsholm
DK
|
Family ID: |
38564007 |
Appl. No.: |
13/006099 |
Filed: |
January 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12296084 |
Sep 10, 2009 |
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PCT/DK2007/000169 |
Mar 30, 2007 |
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13006099 |
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60788995 |
Apr 3, 2006 |
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60796813 |
May 1, 2006 |
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60838710 |
Aug 18, 2006 |
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Current U.S.
Class: |
536/24.5 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/321 20130101; A61P 35/00 20180101; A61P 1/16 20180101;
A61P 35/02 20180101; C12N 2310/322 20130101; A61P 3/06 20180101;
A61P 3/10 20180101; A61P 25/00 20180101; A61P 31/04 20180101; C12N
2310/3515 20130101; A61P 29/00 20180101; A61P 31/12 20180101; A61P
37/02 20180101; A61P 43/00 20180101; C12N 2310/113 20130101; A61P
9/10 20180101; A61P 15/00 20180101; A61P 11/00 20180101; C12N
2310/3231 20130101; A61P 21/00 20180101; A61P 3/00 20180101; A61P
31/14 20180101; C12N 2310/321 20130101; C12N 2310/3521 20130101;
C12N 2310/322 20130101; C12N 2310/3533 20130101 |
Class at
Publication: |
536/24.5 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 3, 2006 |
DK |
PA 2006 00478 |
May 1, 2006 |
DK |
PA 2006 00615 |
Oct 30, 2006 |
DK |
PA 2006 01401 |
Claims
1-95. (canceled)
96. An oligonucleotide 8-22 nucleotides in length, for inhibition
of a microRNA target in a cell, wherein the oligonucleotide
comprises Locked Nucleic Acid (LNA) nucleobase units; wherein at
least 30% of the nucleobases are LNA units; wherein the
oligonucleotide does not comprise a region of more than 5
consecutive 2' deoxyribose nucleoside units; wherein the
oligonucleotide does not comprise a region of more than 2
consecutive LNA units; and wherein said oligonucleotide comprises
at least one phosphorothioate internucleoside linkage group.
97. The oligonucleotide according to claim 96, wherein all the
internucleoside linkage groups are phosphorothioate linkages.
98. The oligonucleotide according to claim 96, wherein the LNA
units are beta D oxy-LNA units.
99. The oligonucleotide according to claim 98 wherein the first
nucleobase of the oligonucleotide, counting from the 3' end, is an
LNA unit.
100. The oligonucleotide according to 99 wherein the second
nucleobase of the oligonucleotide, counting from the 3' end, is an
LNA unit.
101. The oligonucleotide according to claim 96, wherein the
oligonucleotide comprises at least one region of two consecutive
LNA units.
102. The oligonucleotide according to claim 96, wherein the
oligonucleotide comprises an LNA unit at the 5' end.
103. The oligonucleotide according to claim 96, wherein at least
50% of the nucleobase units of the oligonucleotide are LNA
nucleobase units.
104. The oligonucleotide according to claim 96 wherein the
oligonucleotide comprises at least one further non-LNA nucleotide
analogue unit.
105. The oligonucleotide according to claim 104 wherein said at
least one further non-LNA nucleotide analogue unit is a DNA
nucleotide analogue where the 2' hydrogen group of the DNA
nucleotide has been substituted with a group selected from the
group consisting of --O--CH.sub.3,
O--CH.sub.2--CH.sub.2--O--CH.sub.3,
--O--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2,
O--CH.sub.2--CH.sub.2--CH.sub.2--OH and --F.
106. The oligonucleotide according to claim 104, wherein the
non-LNA nucleotide analogue units are 2'-MOE nucleotide
analogues.
107. The oligonucleotide according to claim 104, wherein the
non-LNA nucleotide analogue unit or units are independently
selected from 2'-OMe RNA units and 2'-fluoro DNA units.
108. The oligonucleotide according to claim 96 wherein each
nucleobase unit is independently selected from the group consisting
of LNA nucleobase units and DNA nucleobase units.
109. The oligonucleotide according to claim 96, wherein the
oligonucleotide comprises 5 LNA units.
110. The oligonucleotide according to claim 96, wherein at least 3
of the LNA nucleobases are either cytosine or guanine.
111. The oligonucleotide according to claim 96, wherein the
oligonucleotide comprises both LNA nucleotide analogue units and
non-LNA nucleotide analogue units and all the nucleobase units of
the oligonucleotide are nucleotide analogues.
112. The oligonucleotide according to 96, wherein the
oligonucleotide has a length of 10 to 16 nucleobases.
113. The oligonucleotide according to 96, wherein the
oligonucleotide has a length of 15 or 16 nucleobases.
114. The oligonucleotide according to claim 96, wherein the
oligonucleotide has a length of 15 nucleotides and the substitution
pattern, starting from the 3' end, is 3' XXxXxXxxXXxxXxX 5',
wherein "X" denotes an LNA unit and "x" denotes a DNA unit.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns pharmaceutical compositions
comprising LNA-containing single stranded oligonucleotides capable
of inhibiting disease-inducing microRNAs.
BACKGROUND OF THE INVENTION
MicroRNAs--Novel Regulators of Gene Expression
[0002] MicroRNAs (miRNAs) are an abundant class of short endogenous
RNAs that act as post-transcriptional regulators of gene expression
by base-pairing with their target mRNAs. The mature miRNAs are
processed sequentially from longer hairpin transcripts by the RNAse
III ribonucleases Drosha (Lee et al. 2003) and Dicer (Hutvagner et
al. 2001, Ketting et al. 2001). To date more than 3400 miRNAs have
been annotated in vertebrates, invertebrates and plants according
to the miRBase microRNA database release 7.1 in October 2005
(Griffith-Jones 2004, Griffith-Jones et al. 2006), and many miRNAs
that correspond to putative genes have also been identified.
[0003] Most animal miRNAs recognize their target sites located in
3'-UTRs by incomplete base-pairing, resulting in translational
repression of the target genes (Bartel 2004). An increasing body of
research shows that animal miRNAs play fundamental biological roles
in cell growth and apoptosis (Brennecke et al. 2003), hematopoietic
lineage differentiation (Chen et al. 2004), life-span regulation
(Boehm and Slack 2005), photoreceptor differentiation (Li and
Carthew 2005), homeobox gene regulation (Yekta et al. 2004,
Hornstein et al. 2005), neuronal asymmetry (Johnston et al. 2004),
insulin secretion (Poy et al. 2004), brain morphogenesis (Giraldez
et al. 2005), muscle proliferation and differentiation (Chen,
Mandel et al. 2005, Kwon et al. 2005, Sokol and Ambros 2005),
cardiogenesis (Zhao et al. 2005) and late embryonic development in
vertebrates (Wienholds et al. 2005).
MicroRNAs in Human Diseases
[0004] miRNAs are involved in a wide variety of human diseases. One
is spinal muscular atrophy (SMA), a paediatric neurodegenerative
disease caused by reduced protein levels or loss-of-function
mutations of the survival of motor neurons (SMN) gene (Paushkin et
al. 2002). A mutation in the target site of miR-189 in the human
SLITRK1 gene was recently shown to be associated with Tourette's
syndrome (Abelson et al. 2005), while another recent study reported
that the hepatitis C virus (HCV) RNA genome interacts with a
host-cell microRNA, the liver-specific miR-122a, to facilitate its
replication in the host (Jopling et al. 2005). Other diseases in
which miRNAs or their processing machinery have been implicated,
include frag-ile X mental retardation (FXMR) caused by absence of
the fragile X mental retardation protein (FMRP) (Nelson et al.
2003, Jin et al. 2004) and DiGeorge syndrome (Landthaler et al.
2004).
[0005] In addition, perturbed miRNA expression patterns have been
reported in many human cancers. For example, the human miRNA genes
miR15a and miR16-1 are deleted or down-regulated in the majority of
B-cell chronic lymphocytic leukemia (CLL) cases, where a unique
signature of 13 miRNA genes was recently shown to associate with
prognosis and progression (Calin et al. 2002, Calin et al. 2005).
The role of miRNAs in cancer is further supported by the fact that
more than 50% of the human miRNA genes are located in
cancer-associated genomic regions or at fragile sites (Calin et al.
2004). Recently, systematic expression analysis of a diversity of
human cancers revealed a general down-regulation of miRNAs in
tumors compared to normal tissues (Lu et al. 2005). Interestingly,
miRNA-based classification of poorly differentiated tumors was
successful, whereas mRNA profiles were highly inaccurate when
applied to the same samples. miRNAs have also been shown to be
deregulated in breast cancer (Iorio et al. 2005), lung cancer
(Johnson et al. 2005) and colon cancer (Michael et al. 2004), while
the miR-17-92 cluster, which is amplified in human B-cell lymphomas
and miR-155 which is upregulated in Burkitt's lymphoma have been
reported as the first human miRNA oncogenes (E is et al. 2005, He
et al. 2005). Thus, human miRNAs would not only be highly useful as
biomarkers for future cancer diagnostics, but are rapidly emerging
as attractive targets for disease intervention by oligonucleotide
technologies.
Inhibition of microRNAs Using Single Stranded Oligonucleotides
[0006] Several oligonucleotide approaches have been reported for
inhibition of miRNAs.
[0007] WO03/029459 (Tuschl) claims oligonucleotides which encode
microRNAs and their complements of between 18-25 nucleotides in
length which may comprise nucleotide analogues. LNA is suggested as
a possible nucleotide analogue, although no LNA containing
oligonucleotides are disclosed. Tuschl claims that miRNA
oligonucleotides may be used in therapy.
[0008] US2005/0182005 discloses a 24mer 2'OMe RNA
oligoribonucleotide complementary to the longest form of miR 21
which was found to reduce miR 21 induced repression, whereas an
equivalent DNA containing oligonucleotide did not. The term
2'OMe-RNA refers to an RNA analogue where there is a substitution
to methyl at the 2' position (2'OMethyl).
[0009] US2005/0227934 (Tuschl) refers to antimir molecules with up
to 50% DNA residues. It also reports that antimirs containing 2'
OMe RNA were used against pancreatic microRNAs but it appears that
no actual oligonucleotide structures are disclosed.
[0010] US20050261218 (ISIS) claims an oligomeric compound
comprising a first region and a second region, wherein at least one
region comprises a modification and a portion of the oligomeric
compound is targeted to a small non-coding RNA target nucleic acid,
wherein the small non-coding RNA target nucleic acid is a miRNA.
Oligomeric compounds of between 17 and 25 nucleotides in length are
claimed. The examples refer to entirely 2' OMe PS compounds, 21mers
and 20mer and 2'OMe gapmer oligonucleotides targeted against a
range of pre-miRNA and mature miRNA targets.
[0011] Boutla et al. 2003 (Nucleic Acids Research 31: 4973-4980)
describe the use of DNA antisense oligonucleotides complementary to
11 different miRNAs in Drosophila as well as their use to
inactivate the miRNAs by injecting the DNA oligonucleotides into
fly embryos. Of the 11 DNA antisense oligonucleotides, only 4
constructs showed severe interference with normal development,
while the remaining 7 oligonucleotides didn't show any phenotypes
presumably due to their inability to inhibit the miRNA in
question.
[0012] An alternative approach to this has been reported by
Hutvagner et al. (2004) and Leaman et al. (2005), in which
2'-O-methyl antisense oligonucleotides, complementary to the mature
miRNA could be used as potent and irreversible inhibitors of short
interfering RNA (sRNA) and miRNA function in vitro and in vivo in
Drosophila and C. elegans, thereby inducing a loss-of-function
phenotype. A drawback of this method is the need of high
2'-O-methyl oligonucleotide concentrations (100 micromolar) in
transfection and injection experiments, which may be toxic to the
animal. This method was recently applied to mice studies, by
conjugating 2'-O-methyl antisense oligonucleotides complementary to
four different miRNAs with cholesterol for silencing miRNAs in vivo
(Krutzfedt et al. 2005). These so-called antagomirs were
administered to mice by intravenous injections. Although these
experiments resulted in effective silencing of endogenous miRNAs in
vivo, which was found to be specific, efficient and long-lasting, a
major drawback was the need of high dosage (80 mg/kg) of 2'-O-Me
antagomir for efficient silencing.
[0013] Inhibition of microRNAs using LNA-modified oligonucleotides
have previously been described by Chan et al. Cancer Research 2005,
65 (14) 6029-6033, Lecellier et al. Science 2005, 308, 557-560,
Naguibneva et al. Nature Cell Biology 2006 8 (3), 278-84 and Orum
et al. Gene 2006, (Available online 24 Feb. 2006). In all cases,
the LNA-modified anti-mir oligonucleotides were complementary to
the entire mature microRNA, i.e. 20-23 nucleotides in length, which
hampers efficient in vivo uptake and wide biodistribution of the
molecules.
[0014] Naguibneva (Naguibneva et al. Nature Cell Biology 2006 8
describes the use of mixmer DNA-LNA-DNA antisense oligonucleotide
anti-mir to inhibit microRNA miR-181 function in vitro, in which a
block of 8 LNA nucleotides is located at the center of the molecule
flanked by 6 DNA nucleotides at the 5' end, and 9 DNA nucleotides
at the 3' end, respectively. A major drawback of this antisense
design is low in vivo stability due to low nuclease resistance of
the flanking DNA ends.
[0015] While Chan et al. (Chan et al. Cancer Research 2005, 65 (14)
6029-6033), and Orum et al. (Orum et al. Gene 2006, (Available
online 24 Feb. 2006) do not disclose the design of the LNA-modified
anti-mir molecules used in their study, Lecellier et al. (Lecellier
et al. Science 2005, 308, 557-560) describes the use of gapmer
LNA-DNA-LNA antisense oligonucleotide anti-mir to inhibit microRNA
function, in which a block of 4 LNA nucleotides is located both at
the 5' end, and at the 3' end, respectively, with a window of 13
DNA nucleotides at the center of the molecule. A major drawback of
this antisense design is low in vivo uptake, as well as low in vivo
stability due to the 13 nucleotide DNA stretch in the anti-mir
oligonucleotide.
[0016] Thus, there is a need in the field for improved
oligonucleotides capable of inhibiting microRNAs.
SUMMARY OF THE INVENTION
[0017] The present invention is based upon the discovery that the
use of short oligonucleotides designed to bind with high affinity
to miRNA targets are highly effective in alleviating the repression
of mRNA by microRNAs in vivo.
[0018] Whilst not wishing to be bound to any specific theory, the
evidence disclosed herein indicates that the highly efficient
targeting of miRNAs in vivo is achieved by designing
oligonucleotides with the aim of forming a highly stable duplex
with the miRNA target in vivo. This is achieved by the use of high
affinity nucleotide analogues such as at least one LNA units and
suitably further high affinity nucleotide analogues, such as LNA,
2'-MOE RNA of 2'-Fluoro nucleotide analogues, in a short, such as
10-17 or 10-16 nucleobase oligonucleotides. In one aspect the aim
is to generate an oligonucleotide of a length which is unlikely to
form a siRNA complex (i.e. a short oligonucleotide), and with
sufficient loading of high affinity nucleotide analogues that the
oligonucleotide sticks almost permanently to its miRNA target,
effectively forming a stable and non-functional duplex with the
miRNA target. We have found that such designs are considerably more
effective than the prior art oligonucleotides, particularly gapmer
and blockmer designs and oligonucleotides which have a long length,
e.g. 20-23mers. The term 2' fluor-DNA refers to an DNA analogue
where the is a substitution to fluor at the 2' position (2'F).
[0019] The invention provides a pharmaceutical composition
comprising a single stranded oligonucleotide having a length of
between 8 and 17, such as 10 and 17, such as 8-16 or 10-16
nucleobase units, a pharmaceutically acceptable diluent, carrier,
or adjuvant, wherein at least one of the nucleobase units of the
single stranded oligonucleotide is a high affinity nucleotide
analogue, such as a Locked Nucleic Acid (LNA) nucleobase unit, and
wherein the single stranded oligonucleotide is complementary to a
human microRNA sequence.
[0020] The high affinity nucleotide analogues are nucleotide
analogues which result in oligonucleotide which has a higher
thermal duplex stability with a complementary RNA nucleotide than
the binding affinity of an equivalent DNA nucleotide. This is
typically determined by measuring the T.sub.m.
[0021] We have not identified any significant off-target effects
when using these short, high affinity oligonucleotides targeted
against specific miRNAs. Indeed, the evidence provided herein
indicates the effects on mRNA expression are either due to the
presence of a complementary sequence to the targeted miRNA (primary
mRNA targets) within the mRNA or secondary effects on mRNAs which
are regulated by primary mRNA targets (secondary mRNA targets). No
toxicity effects were identified indicating no significant
detrimental off-target effects.
[0022] The invention further provides a pharmaceutical composition
comprising a single stranded oligonucleotide having a length of
between 8 and 17 nucleobase units, such as between 10 and 17
nucleobase units, such as between 10 and 16 nucleobase units, and a
pharmaceutically acceptable diluent, carrier, or adjuvant, wherein
at least one of the nucleobase units of the single stranded
oligonucleotide is a Locked Nucleic Acid (LNA) nucleobase unit, and
wherein the single stranded oligonucleotide is complementary to a
human microRNA sequence.
[0023] The invention further provides for the use of an
oligonucleotide according to the invention, such as those which may
form part of the pharmaceutical composition, for the manufacture of
a medicament for the treatment of a disease or medical disorder
associated with the presence or over-expression (upregulation) of
the microRNA.
[0024] The invention further provides for a method for the
treatment of a disease or medical disorder associated with the
presence or over-expression of the microRNA, comprising the step of
administering a composition (such as the pharmaceutical
composition) according to the invention to a person in need of
treatment.
[0025] The invention further provides for a method for reducing the
effective amount of a miRNA in a cell or an organism, comprising
administering a composition (such as the pharmaceutical
composition) according to the invention or a single stranded
oligonucleotide according to the invention to the cell or the
organism. Reducing the effective amount in this context refers to
the reduction of functional miRNA present in the cell or organism.
It is recognised that the preferred oligonucleotides according to
the invention may not always significantly reduce the actual amount
of miRNA in the cell or organism as they typically form very stable
duplexes with their miRNA targets.
[0026] The invention further provides for a method for
de-repression of a target mRNA of a miRNA in a cell or an organism,
comprising administering a composition (such as the pharmaceutical
composition) or a single stranded oligonucleotide according to the
invention to the cell or the organism.
[0027] The invention further provides for the use of a single
stranded oligonucleotide of between 8-16 such as 10-16 nucleobases
in length, for the manufacture of a medicament for the treatment of
a disease or medical disorder associated with the presence or
over-expression of the microRNA.
[0028] The invention further provides for a method for the
treatment of a disease or medical disorder associated with the
presence or over-expression of the microRNA, comprising the step of
administering a composition (such as the pharmaceutical
composition) comprising a single stranded oligonucleotide of
between 8-16 such as between 10-16 nucleobases in length to a
person in need of treatment.
[0029] The invention further provides for a method for reducing the
effective amount of a miRNA target (i.e. `available` miRNA) in a
cell or an organism, comprising administering a composition (such
as the pharmaceutical composition) comprising a single stranded
oligonucleotide of between 8-16 such as between 10-16 nucleobases
to the cell or the organism.
[0030] The invention further provides for a method for
de-repression of a target mRNA of a miRNA in a cell or an organism,
comprising a single stranded oligonucleotide of between 8-16 such
as between 10-16 nucleobases or (or a composition comprising said
oligonucleotide) to the cell or the organism.
[0031] The invention further provides for a method for the
synthesis of a single stranded oligonucleotide targeted against a
human microRNA, such as a single stranded oligonucleotide described
herein, said method comprising the steps of: [0032] a. Optionally
selecting a first nucleobase, counting from the 3' end, which is a
nucleotide analogue, such as an LNA nucleobase. [0033] b.
Optionally selecting a second nucleobase, counting from the 3' end,
which is an nucleotide analogue, such as an LNA nucleobase. [0034]
c. Selecting a region of the single stranded oligonucleotide which
corresponds to the miRNA seed region, wherein said region is as
defined herein. [0035] d. Optionally selecting a seventh and eight
nucleobase is as defined herein. [0036] e. Optionally selecting a
5' region of the single stranded oligonucleotide is as defined
herein. [0037] f. Optionally selecting a 5' terminal of the single
stranded oligonucleotide is as defined herein.
[0038] Wherein the synthesis is performed by sequential synthesis
of the regions defined in steps a-f, wherein said synthesis may be
performed in either the 3'-5' (a to f) or 5'-3' (f to a) direction,
and wherein said single stranded oligonucleotide is complementary
to a sequence of the miRNA target.
[0039] In one embodiment the oligonucleotide of the invention is
designed not to be recruited by RISC or to mediate RISC directed
cleavage of the miRNA target. It has been considered that by using
long oligonucleotides, e.g. 21 or 22mers, particularly RNA
oligonucleotides, or RNA `analogue` oligonucleotide which are
complementary to the miRNA target, the oligonucleotide can compete
against the target mRNA in terms of RISC complex association, and
thereby alleviate miRNA repression of miRNA target mRNAs via the
introduction of an oligonucleotide which competes as a substrate
for the miRNA.
[0040] However, the present invention seeks to prevent such
undesirable target mRNA cleavage or translational inhibition by
providing oligonucleotides capable of complementary, and apparently
in some cases almost irreversible binding to the mature microRNA.
This appears to result in a form of protection against degradation
or cleavage (e.g. by RISC or RNAseH or other endo or
exo-nucleases), which may not result in substantial or even
significant reduction of the miRNA (e.g. as detected by northern
blot using LNA probes) within a cell, but ensures that the
effective amount of the miRNA, as measured by de-repression
analysis is reduced considerably. Therefore, in one aspect, the
invention provides oligonucleotides which are purposefully designed
not to be compatible with the RISC complex, but to remove miRNA by
titration by the oligonucleotide. Although not wishing to be bound
to a specific theory of why the oligonucleotides of the present
invention are so effective, in analogy with the RNA based
oligonucleotides (or complete 2'OMe oligonucleotides), it appears
that the oligonucleotides according to the present invention work
through non-competitive inhibition of miRNA function as they
effectively remove the available miRNA from the cytoplasm, where as
the prior art oligonucleotides provide an alternative miRNA
substrate, which may act as a competitor inhibitor, the
effectiveness of which would be far more dependant upon the
concentration of the oligonucleotide in the cytoplasm, as well as
the concentration of the target mRNA and miRNA.
[0041] Again, whilst not wishing to be bound to any specific
theory, one further possibility that may exist with the use of
oligonucleotides of approximately similar length to the miRNA
targets, is that the oligonucleotides could form a siRNA like
duplex with the miRNA target, a situation which would reduce the
effectiveness of the oligonucleotide. It is also possible that the
oligonucleotides themselves could be used as the guiding strand
within the RISC complex, thereby generating the possibility of RISC
directed degradation of non-specific targets which just happen to
have sufficient complementarity to the oligonucleotide guide.
[0042] By selecting short oligonucleotides for targeting miRNA
sequences, such problems are avoided.
[0043] Short oligonucleotides which incorporate LNA are known from
the reagents area, such as the LNA (see for example WO2005/098029
and WO 2006/069584). However the molecules designed for diagnostic
or reagent use are very different in design than those for
pharmaceutical use. For example, the terminal nucleobases of the
reagent oligos are typically not LNA, but DNA, and the
internucleoside linkages are typically other than phosphorothioate,
the preferred linkage for use in the oligonucleotides of the
present invention. The invention therefore provides for a novel
class of oligonucleotide per se.
[0044] The invention further provides for a (single stranded)
oligonucleotide as described in the context of the pharmaceutical
composition of the invention, wherein said oligonucleotide
comprises either [0045] i) at least one phosphorothioate linkage
and/or [0046] ii) at least one 3' terminal LNA unit, and/or [0047]
iii) at least one 5' terminal LNA unit.
[0048] It is preferable for most therapeutic uses that the
oligonucleotide is fully phosphorothiolated--the exception being
for therapeutic oligonucleotides for use in the CNS, such as in the
brain or spine where phosphorothioation can be toxic, and due to
the absence of nucleases, phosphodiester bonds may be used, even
between consecutive DNA units. As referred to herein, other
preferred aspects of the oligonucleotide according to the invention
is that the second 3' nucleobase, and/or the 9.sup.th and 10.sup.th
(from the 3' end), may also be LNA.
[0049] The inventors have found that other methods of avoiding RNA
cleavage (such as exo-nuclease degradation in blood serum, or RISC
associated cleavage of the oligonucleotide according to the
invention are possible, and as such the invention also provides for
a single stranded oligonucleotide which comprises of either: [0050]
a. an LNA unit at position 1 and 2 counting from the 3' end and/or
[0051] b. an LNA unit at position 9 and/or 10, also counting from
the 3' end, and/or [0052] c. either one or two 5' LNA units.
[0053] Whilst the benefits of these other aspects may be seen with
longer oligonucleotides, such as nucleotide of up to 26 nucleobase
units in length, it is considered these features may also be used
with the shorter oligonucleotides referred to herein, such as the
oligonucleotides of between 10-17 or 10-16 nucleobases described
herein. It is highly preferably that the oligonucleotides comprise
high affinity nucleotide analogues, such as those referred to
herein, most preferably LNA units.
[0054] The inventors have therefore surprisingly found that
carefully designed single stranded oligonucleotides comprising
locked nucleic acid (LNA) units in a particular order show
significant silencing of microRNAs, resulting in reduced microRNA
levels. It was found that tight binding of said oligonucleotides to
the so-called seed sequence, nucleotides 2 to 8 or 2-7, counting
from the 5' end, of the target microRNAs was important. Nucleotide
1 of the target microRNAs is a non-pairing base and is most likely
hidden in a binding pocket in the Ago 2 protein. Whilst not wishing
to be bound to a specific theory, the present inventors consider
that by selecting the seed region sequences, particularly with
oligonucleotides that comprise LNA, preferably LNA units in the
region which is complementary to the seed region, the duplex
between miRNA and oligonucleotide is particularly effective in
targeting miRNAs, avoiding off target effects, and possibly
providing a further feature which prevents RISC directed miRNA
function.
[0055] The inventors have surprisingly found that microRNA
silencing is even more enhanced when LNA-modified single stranded
oligonucleotides do not contain a nucleotide at the 3' end
corresponding to this non-paired nucleotide 1. It was further found
that two LNA units in the 3' end of the oligonucleotides according
to the present invention made said oligonucleotides highly nuclease
resistant.
[0056] It was further found that the oligonucleotides of the
invention which have at least one nucleotide analogue, such as an
LNA nucleotide in the positions corresponding to positions 10 and
11, counting from the 5' end, of the target microRNA may prevent
cleavage of the oligonucleotides of the invention
[0057] Accordingly, in one aspect of the invention relates to an
oligonucleotide having a length of from 12 to 26 nucleotides,
wherein [0058] i) the first nucleotide, counting from the 3' end,
is a locked nucleic acid (LNA) unit; [0059] ii) the second
nucleotide, counting from the 3' end, is an LNA unit; and [0060]
iii) the ninth and/or the tenth nucleotide, counting from the 3'
end, is an LNA unit.
[0061] The invention further provides for the oligonucleotides as
defined herein for use as a medicament.
[0062] The invention further relates to compositions comprising the
oligonucleotides defined herein and a pharmaceutically acceptable
carrier.
[0063] As mentioned above, microRNAs are related to a number of
diseases. Hence, a fourth aspect of the invention relates to the
use of an oligonucleotide as defined herein for the manufacture of
a medicament for the treatment of a disease associated with the
expression of microRNAs selected from the group consisting of
spinal muscular atrophy, Tourette's syndrome, hepatitis C virus,
fragile X mental retardation, DiGeorge syndrome and cancer, such as
chronic lymphocytic leukemia, breast cancer, lung cancer and colon
cancer, in particular cancer.
[0064] A further aspect of the invention is a method to reduce the
levels of target microRNA by contacting the target microRNA to an
oligonucleotide as defined herein, wherein the oligonucleotide
[0065] 1. is complementary to the target microRNA [0066] 2. does
not contain a nucleotide at the 3' end that corresponds to the
first 5' end nucleotide of the target microRNA.
[0067] The invention further provides for an oligonucleotide
comprising a nucleobase sequence selected from the group consisting
of SEQ IDs NO 1-534, SEQ ID NOs 539-544, SEQ ID NOs 549-554, SEQ ID
NOs 559-564, SEQ ID NOs 569-574 and SEQ ID NOs 594-598, and SEQ ID
NOs 579-584, or a pharmaceutical composition comprising said
oligonucleotide. In one embodiment, the oligonucleotide may have a
nucleobase sequence of between 1-17 nucleobases, such as 8, 9, 10,
11, 12, 13, 14, 15, 16 or 17 nucleobases, and as such the
oligonucleobase in such an embodiment may be a contiguous
subsequence within the oligonucleotides disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1. The effect of treatment with different LNA anti-miR
oligonucleotides on target nucleic acid expression in the miR-122a
expressing cell line Huh-7. Shown are amounts of miR-122a
(arbitrary units) derived from miR-122a specific qRT-PCR as
compared to untreated cells (mock). The LNA anti-miR
oligonucleotides were used at two concentrations, 1 and 100 nM,
respectively. Included is also a mismatch control (SPC3350) to
SPC3349 (also referred to herein as SPC3549).
[0069] FIG. 2. Assessment of LNA anti-miR-122a knock-down
dose-response for SPC3548 and SPC3549 in comparison with SPC3372 in
vivo in mice livers using miR-122a real-time RT-PCR.
[0070] FIG. 2b miR-122 levels in the mouse liver after treatment
with different LNA-antimiRs. The LNA-antimiR molecules SPC3372 and
SPC3649 were administered into normal mice by three i.p. injections
on every second day over a six-day-period at indicated doses and
sacrificed 48 hours after last dose. Total RNA was extracted from
the mice livers and miR-122 was measured by miR-122 specific
qPCR.
[0071] FIG. 3. Assessment of plasma cholesterol levels in
LNA-antimiR-122a treated mice compared to the control mice that
received saline.
[0072] FIG. 4a. Assessment of relative Bckdk mRNA levels in LNA
antimiR-122a treated mice in comparison with saline control mice
using real-time quantitative RT-PCR.
[0073] FIG. 4b. Assessment of relative aldolase A mRNA levels in
LNA antimiR-122a treated mice in comparison with saline control
mice using real-time quantitative RT-PCR.
[0074] FIG. 4c. Assessment of GAPDH mRNA levels in LNA antimiR-122a
treated mice (animals 4-30) in comparison with saline control mice
(animals 1-3) using real-time quantitative RT-PCR.
[0075] FIG. 5. Assessment of LNA-antimiR.TM.-122a knock-down
dose-response in vivo in mice livers using miR-122a real-time
RT-PCR. Six groups of animals (5 mice per group) were treated in
the following manner. Group 1 animals were injected with 0.2 ml
saline by i.v. on 3 successive days, Group 2 received 2.5 mg/kg
SPC3372, Group 3 received 6.25 mg/kg, Group 4 received 12.5 mg/kg
and Group 5 received 25 mg/kg, while Group 6 received 25 mg/kg SPC
3373 (mismatch LNA-antimiR.TM. oligonucleotide), all in the same
manner. The experiment was repeated (therefore n=10) and the
combined results are shown.
[0076] FIG. 6. Northern blot comparing SPC3649 with SPC3372. Total
RNA from one mouse in each group were subjected to miR-122 specific
northern blot. Mature miR-122 and the duplex (blocked microRNA)
formed between the LNA-antimiR and miR-122 is indicated.
[0077] FIG. 7. Mice were treated with 25 mg/kg/day LNA-antimiR.TM.
or saline for three consecutive days and sacrificed 1, 2 or 3 weeks
after last dose. Included are also the values from the animals
sacrificed 24 hours after last dose (example 11 "old design").
miR-122 levels were assessed by qPCR and normalized to the mean of
the saline group at each individual time point. Included are also
the values from the animals sacrificed 24 hours after last dose
(shown mean and SD, n=7, 24 h n=10). Sacrifice day 9, 16 or 23
corresponds to sacrifice 1, 2 or 3 weeks after last dose.).
[0078] FIG. 8. Mice were treated with 25 mg/kg/day LNA-antimiR.TM.
or saline for three consecutive days and sacrificed 1, 2 or 3 weeks
after last dose. Included are also the values from the animals
sacrificed 24 hours after last dose (example 11 "old design").
Plasma cholesterol was measured and normalized to the saline group
at each time point (shown mean and SD, n=7, 24 h n=10).
[0079] FIG. 9. Dose dependent miR-122a target mRNA induction by
SPC3372 inhibition of miR-122a. Mice were treated with different
SPC3372 doses for three consecutive days, as described above and
sacrificed 24 hours after last dose. Total RNA extracted from liver
was subjected to qPCR. Genes with predicted miR-122 target site and
observed to be upregulated by microarray analysis were investigated
for dose-dependent induction by increasing SPC3372 doses using
qPCR. Total liver RNA from 2 to 3 mice per group sacrificed 24
hours after last dose were subjected to qPCR for the indicated
genes. Shown in FIG. 9 is mRNA levels relative to Saline group,
n=2-3 (2.5-12.5 mg/kg/day: n=2, no SD). Shown is also the mismatch
control (m, SPC3373)
[0080] FIG. 10. Transient induction of miR-122a target mRNAs
following SPC3372 treatment. NMRI female mice were treated with 25
mg/kg/day SPC3372 along with saline control for three consecutive
days and sacrificed 1, 2 or 3 weeks after last dose, respectively.
RNA was extracted from livers and mRNA levels of predicted miR-122a
target mRNAs, selected by microarray data were investigated by
qPCR. Three animals from each group were analysed.
[0081] FIG. 11. Induction of Vldlr in liver by SPC3372 treatment.
The same liver RNA samples as in previous example (FIG. 10) were
investigated for Vldlr induction.
[0082] FIG. 12. Stability of miR-122a/SPC3372 duplex in mouse
plasma. Stability of SPC3372 and SPC3372/miR-122a duplex were
tested in mouse plasma at 37.degree. C. over 96 hours. Shown in
FIG. 12 is a SYBR-Gold stained PAGE.
[0083] FIG. 13. Sequestering of mature miR-122a by SPC3372 leads to
duplex formation. Shown in FIG. 13 is a membrane probed with a
miR-122a specific probe (upper panel) and re-probed with a Let-7
specific probe (lower panel). With the miR-122 probe, two bands
could be detected, one corresponding to mature miR-122 and one
corresponding to a duplex between SPC3372 and miR-122.
[0084] FIG. 14. miR-122a sequestering by SPC3372 along with SPC3372
distribution assessed by in situ hybridization of liver sections.
Liver cryo-sections from treated animals were
[0085] FIG. 15. Liver gene expression in miR-122 LNA-antimiR
treated mice.
[0086] Saline and LNA-antimiR treated mice were compared by
genome-wide expression profiling using Affymetrix Mouse Genome 430
2.0 arrays. (a,1) Shown is number of probes displaying
differentially expression in liver samples of LNA-antimiR-122
treated and saline treated mice 24 hours post treatment. (b,2) The
occurrence of miR-122 seed sequence in differentially expressed
genes. The plot shows the percentage of transcripts with at least
one miR-122 seed recognition sequence in their 3' UTR. Random:
Random sequences were generated and searched for miR-122 seed
recognition sequences.
[0087] Temporal liver gene expression profiles in LNA-antimiR
treated mice. Mice were treated with 25 mg/kg/day LNA-antimiR or
saline for three consecutive days and sacrificed 1, 2 or 3 weeks
after last dose. Included are also the values from the animals
sacrificed 24 hours after last dose. (c,3) RNA samples from
different time points were also subjected to expression profiling.
Hierarchical cluster analysis of expression profiles of genes
identified as differentially expressed between LNA-antimiR and
saline treated mice 24 hours, one week or three weeks post
treatment. (d,4) Expression profiles of genes identified as
differentially expressed between LNA-antimiR and saline treated
mice 24 hours post treatment were followed over time. The
expression ratios of up- and down-regulated genes in LNA-antimiR
treated mice approach 1 over the time-course, indicating a
reversible effect of the LNA-antimiR treatment.
[0088] FIG. 16. The effect of treatment with SPC3372 and 3595 on
miR-122 levels in mice livers.
[0089] FIG. 17. The effect of treatment with SPC3372 and 3595 on
Aldolase A levels in mice livers.
[0090] FIG. 18. The effect of treatment with SPC3372 and 3595 on
Bckdk levels in mice livers.
[0091] FIG. 19. The effect of treatment with SPC3372 and 3595 on
CD320 levels in mice livers.
[0092] FIG. 20. The effect of treatment with SPC3372 and 3595 on
Ndrg3 levels in mice livers.
[0093] FIG. 21. The effect of long-term treatment with SPC3649 on
total plasma cholesterol in hypercholesterolemic and normal mice.
Weekly samples of blood plasma were obtained from the SPC3649
treated and saline control mice once weekly followed by assessment
of total plasma cholesterol. The mice were treated with 5 mg/kg
SPC3649, SPC3744 or saline twice weekly. Normal mice given were
treated in parallel.
[0094] FIG. 22. The effect of long-term treatment with SPC3649 on
miR-122 levels in hypercholesterolemic and normal mice.
[0095] FIG. 23. The effect of long-term treatment with SPC3649 on
Aldolase A levels in hypercholesterolemic and normal mice.
[0096] FIG. 24. The effect of long-term treatment with SPC3649 on
Bckdk levels in hypercholesterolemic and normal mice.
[0097] FIG. 25. The effect of long-term treatment with SPC3649 on
AST levels in hypercholesterolemic and normal mice.
[0098] FIG. 26. The effect of long-term treatment with SPC3649 on
ALT levels in hypercholesterolemic and normal mice.
[0099] FIG. 27. Functional de-repression of renilla luciferase with
miR-155 target by miR-155 blocking oligonucleotides in an
endogenously miR-155 expressing cell line, 518A2. "psiCheck2" is
the plasmid without miR-155 target, i.e. full expression and
"miR-155 target" is the corresponding plasmid with miR-155 target
but not co-transfected with oligo blocking miR-155 and hence
represent fully miR-155 repressed renilla luciferase
expression.
[0100] FIG. 28. Functional de-repression of renilla luciferase with
miR-19b target by miR-19b blocking oligonucleotides in an
endogenously miR-19b expressing cell line, HeLa. "miR-19b target"
is the plasmid with miR-19b target but not co-transfected with
oligo blocking miR-19b and hence represent fully miR-19b repressed
renilla luciferase expression.
[0101] FIG. 29. Functional de-repression of renilla luciferase with
miR-122 target by miR-122 blocking oligonucleotides in an
endogenously miR-122 expressing cell line, Huh-7. "miR-122 target"
is the corresponding plasmid with miR-122 target but not
co-transfected with oligo blocking miR-122 and hence represent
fully miR-122 repressed renilla luciferase expression.
[0102] FIG. 30. Diagram illustrating the alignment of an
oligonucleotide according to the invention and a microRNA
target.
DETAILED DESCRIPTION OF THE INVENTION
[0103] The invention provides pharmaceutical compositions
comprising short single stranded oligonucleotides, of length of
between 8 and 17 such as between 10 and 17 nucleobases which are
complementary to human microRNAs. The short oligonucleotides are
particularly effective at alleviating miRNA repression in vivo. It
is found that the incorporation of high affinity nucleotide
analogues into the oligonucleotides results in highly effective
anti-microRNA molecules which appear to function via the formation
of almost irreversible duplexes with the miRNA target, rather than
RNA cleavage based mechanisms, such as mechanisms associated with
RNaseH or RISC.
[0104] It is highly preferable that the single stranded
oligonucleotide according to the invention comprises a region of
contiguous nucleobase sequence which is 100% complementary to the
human microRNA seed region.
[0105] It is preferable that single stranded oligonucleotide
according to the invention is complementary to the mature human
microRNA sequence.
[0106] In one embodiment the single stranded oligonucleotide
according to the invention is complementary to a microRNA sequence,
such as a microRNA sequence selected from the group consisting of:
hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e,
hsa-let-7f, hsa-miR-15a, hsa-miR-16, hsa-miR-17-5p, hsa-miR-17-3p,
hsa-miR-18a, hsa-miR-19a, hsa-miR-19b, hsa-miR-20a, hsa-miR-21,
hsa-miR-22, hsa-miR-23a, hsa-miR-189, hsa-miR-24, hsa-miR-25,
hsa-miR-26a, hsa-miR-26b, hsa-miR-27a, hsa-miR-28, hsa-miR-29a,
hsa-miR-30a-5p, hsa-miR-30a-3p, hsa-miR-31, hsa-miR-32, hsa-miR-33,
hsa-miR-92, hsa-miR-93, hsa-miR-95, hsa-miR-96, hsa-miR-98,
hsa-miR-99a, hsa-miR-100, hsa-miR-101, hsa-miR-29b, hsa-miR-103,
hsa-miR-105, hsa-miR-106a, hsa-miR-107, hsa-miR-192, hsa-miR-196a,
hsa-miR-197, hsa-miR-198, hsa-miR-199a, hsa-miR-199a*, hsa-miR-208,
hsa-miR-129, hsa-miR-148a, hsa-miR-30c, hsa-miR-30d, hsa-miR-139,
hsa-miR-147, hsa-miR-7, hsa-miR-10a, hsa-miR-10b, hsa-miR-34a,
hsa-miR-181a, hsa-miR-181b, hsa-miR-181c, hsa-miR-182,
hsa-miR-182*, hsa-miR-183, hsa-miR-187, hsa-miR-199b, hsa-miR-203,
hsa-miR-204, hsa-miR-205, hsa-miR-210, hsa-miR-211, hsa-miR-212,
hsa-miR-181a*, hsa-miR-214, hsa-miR-215, hsa-miR-216, hsa-miR-217,
hsa-miR-218, hsa-miR-219, hsa-miR-220, hsa-miR-221, hsa-miR-222,
hsa-miR-223, hsa-miR-224, hsa-miR-200b, hsa-let-7g, hsa-let-71,
hsa-miR-1, hsa-miR-15b, hsa-miR-23b, hsa-miR-27b, hsa-miR-30b,
hsa-miR-122a, hsa-miR-124a, hsa-miR-125b, hsa-miR-128a,
hsa-miR-130a, hsa-miR-132, hsa-miR-133a, hsa-miR-135a, hsa-miR-137,
hsa-miR-138, hsa-miR-140, hsa-miR-141, hsa-miR-142-5p,
hsa-miR-142-3p, hsa-miR-143, hsa-miR-144, hsa-miR-145, hsa-miR-152,
hsa-miR-153, hsa-miR-191, hsa-miR-9, hsa-miR-9*, hsa-miR-125a,
hsa-miR-126*, hsa-miR-126, hsa-miR-127, hsa-miR-134, hsa-miR-136,
hsa-miR-146a, hsa-miR-149, hsa-miR-150, hsa-miR-154, hsa-miR-154*,
hsa-miR-184, hsa-miR-185, hsa-miR-186, hsa-miR-188, hsa-miR-190,
hsa-miR-193a, hsa-miR-194, hsa-miR-195, hsa-miR-206, hsa-miR-320,
hsa-miR-200c, hsa-miR-155, hsa-miR-128b, hsa-miR-106b, hsa-miR-29c,
hsa-miR-200a, hsa-miR-302a*, hsa-miR-302a, hsa-miR-34b,
hsa-miR-34c, hsa-miR-299-3p, hsa-miR-301, hsa-miR-99b, hsa-miR-296,
hsa-miR-130b, hsa-miR-30e-5p, hsa-miR-30e-3p, hsa-miR-361,
hsa-miR-362, hsa-miR-363, hsa-miR-365, hsa-miR-302b*, hsa-miR-302b,
hsa-miR-302c*, hsa-miR-302c, hsa-miR-302d, hsa-miR-367,
hsa-miR-368, hsa-miR-369-3p, hsa-miR-370, hsa-miR-371, hsa-miR-372,
hsa-miR-373*, hsa-miR-373, hsa-miR-374, hsa-miR-375, hsa-miR-376a,
hsa-miR-377, hsa-miR-378, hsa-miR-422b, hsa-miR-379,
hsa-miR-380-5p, hsa-miR-380-3p, hsa-miR-381, hsa-miR-382,
hsa-miR-383, hsa-miR-340, hsa-miR-330, hsa-miR-328, hsa-miR-342,
hsa-miR-337, hsa-miR-323, hsa-miR-326, hsa-miR-151, hsa-miR-135b,
hsa-miR-148b, hsa-miR-331, hsa-miR-324-5p, hsa-miR-324-3p,
hsa-miR-338, hsa-miR-339, hsa-miR-335, hsa-miR-133b, hsa-miR-325,
hsa-miR-345, hsa-miR-346, ebv-miR-BHRF1-1, ebv-miR-BHRF1-2*,
ebv-miR-BHRF1-2, ebv-miR-BHRF1-3, ebv-miR-BART1-5p, ebv-miR-BART2,
hsa-miR-384, hsa-miR-196b, hsa-miR-422a, hsa-miR-423, hsa-miR-424,
hsa-miR-425-3p, hsa-miR-18b, hsa-miR-20b, hsa-miR-448, hsa-miR-429,
hsa-miR-449, hsa-miR-450, hcmv-miR-UL22A, hcmv-miR-UL22A*,
hcmv-miR-UL36, hcmv-miR-UL112, hcmv-miR-UL148D, hcmv-miR-US5-1,
hcmv-miR-US5-2, hcmv-miR-US25-1, hcmv-miR-US25-2-5p,
hcmv-miR-US25-2-3p, hcmv-miR-US33, hsa-miR-191*, hsa-miR-200a*,
hsa-miR-369-5p, hsa-miR-431, hsa-miR-433, hsa-miR-329, hsa-miR-453,
hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-409-5p,
hsa-miR-409-3p, hsa-miR-412, hsa-miR-410, hsa-miR-376b,
hsa-miR-483, hsa-miR-484, hsa-miR-485-5p, hsa-miR-485-3p,
hsa-miR-486, hsa-miR-487a, kshv-miR-K12-10a, kshv-miR-K12-10b,
kshv-miR-K12-11, kshv-miR-K12-1, kshv-miR-K12-2, kshv-miR-K12-9*,
kshv-miR-K12-9, kshv-miR-K12-8, kshv-miR-K12-7, kshv-miR-K12-6-5p,
kshv-miR-K12-6-3p, kshv-miR-K12-5, kshv-miR-K12-4-5p,
kshv-miR-K12-4-3p, kshv-miR-K12-3, kshv-miR-K12-3*, hsa-miR-488,
hsa-miR-489, hsa-miR-490, hsa-miR-491, hsa-miR-511, hsa-miR-146b,
hsa-miR-202*, hsa-miR-202, hsa-miR-492, hsa-miR-493-5p,
hsa-miR-432, hsa-miR-432*, hsa-miR-494, hsa-miR-495, hsa-miR-496,
hsa-miR-193b, hsa-miR-497, hsa-miR-181d, hsa-miR-512-5p,
hsa-miR-512-3p, hsa-miR-498, hsa-miR-520e, hsa-miR-515-5p,
hsa-miR-515-3p, hsa-miR-519e*, hsa-miR-519e, hsa-miR-520f,
hsa-miR-526c, hsa-miR-519c, hsa-miR-520a*, hsa-miR-520a,
hsa-miR-526b, hsa-miR-526b*, hsa-miR-519b, hsa-miR-525,
hsa-miR-525*, hsa-miR-523, hsa-miR-518f*, hsa-miR-518f,
hsa-miR-520b, hsa-miR-518b, hsa-miR-526a, hsa-miR-520c,
hsa-miR-518c*, hsa-miR-518c, hsa-miR-524*, hsa-miR-524,
hsa-miR-517*, hsa-miR-517a, hsa-miR-519d, hsa-miR-521,
hsa-miR-520d*, hsa-miR-520d, hsa-miR-517b, hsa-miR-520g,
hsa-miR-516-5p, hsa-miR-516-3p, hsa-miR-518e, hsa-miR-527,
hsa-miR-518a, hsa-miR-518d, hsa-miR-517c, hsa-miR-520h,
hsa-miR-522, hsa-miR-519a, hsa-miR-499, hsa-miR-500, hsa-miR-501,
hsa-miR-502, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa-miR-513,
hsa-miR-506, hsa-miR-507, hsa-miR-508, hsa-miR-509, hsa-miR-510,
hsa-miR-514, hsa-miR-532, hsa-miR-299-5p, hsa-miR-18a*,
hsa-miR-455, hsa-miR-493-3p, hsa-miR-539, hsa-miR-544, hsa-miR-545,
hsa-miR-487b, hsa-miR-551a, hsa-miR-552, hsa-miR-553, hsa-miR-554,
hsa-miR-92b, hsa-miR-555, hsa-miR-556, hsa-miR-557, hsa-miR-558,
hsa-miR-559, hsa-miR-560, hsa-miR-561, hsa-miR-562, hsa-miR-563,
hsa-miR-564, hsa-miR-565, hsa-miR-566, hsa-miR-567, hsa-miR-568,
hsa-miR-551b, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572,
hsa-miR-573, hsa-miR-574, hsa-miR-575, hsa-miR-576, hsa-miR-577,
hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582,
hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-548a, hsa-miR-586,
hsa-miR-587, hsa-miR-548b, hsa-miR-588, hsa-miR-589, hsa-miR-550,
hsa-miR-590, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-595,
hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,
hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,
hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,
hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615,
hsa-miR-616, hsa-miR-548c, hsa-miR-617, hsa-miR-618, hsa-miR-619,
hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624,
hsa-miR-625, hsa-miR-626, hsa-miR-627, hsa-miR-628, hsa-miR-629,
hsa-miR-630, hsa-miR-631, hsa-miR-33b, hsa-miR-632, hsa-miR-633,
hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638,
hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643,
hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648,
hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-548d,
hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-449b, hsa-miR-653,
hsa-miR-411, hsa-miR-654, hsa-miR-655, hsa-miR-656, hsa-miR-549,
hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-421,
hsa-miR-542-5p, hcmv-miR-US4, hcmv-miR-UL70-5p, hcmv-miR-UL70-3p,
hsa-miR-363*, hsa-miR-376a*, hsa-miR-542-3p, ebv-miR-BART1-3p,
hsa-miR-425-5p, ebv-miR-BART3-5p, ebv-miR-BART3-3p, ebv-miR-BART4,
ebv-miR-BART5, ebv-miR-BART6-5p, ebv-miR-BART6-3p, ebv-miR-BART7,
ebv-miR-BART8-5p, ebv-miR-BART8-3p, ebv-miR-BART9, ebv-miR-BART10,
ebv-miR-BART11-5p, ebv-miR-BART11-3p, ebv-miR-BART12,
ebv-miR-BART13, ebv-miR-BART14-5p, ebv-miR-BART14-3p,
kshv-miR-K12-12, ebv-miR-BART15, ebv-miR-BART16, ebv-miR-BART17-5p,
ebv-miR-BART17-3p, ebv-miR-BART18, ebv-miR-BART19,
ebv-miR-BART20-5p, ebv-miR-BART20-3p, hsv1-miR-H1, hsa-miR-758,
hsa-miR-671, hsa-miR-668, hsa-miR-767-5p, hsa-miR-767-3p,
hsa-miR-454-5p, hsa-miR-454-3p, hsa-miR-769-5p, hsa-miR-769-3p,
hsa-miR-766, hsa-miR-765, hsa-miR-768-5p, hsa-miR-768-3p,
hsa-miR-770-5p, hsa-miR-802, hsa-miR-801, hsa-miR-675.
[0107] In one embodiment the single stranded oligonucleotide
according to the invention is complementary to a microRNA sequence,
such as a microRNA sequence selected from the group consisting of:
hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e,
hsa-let-7f, hsa-miR-15a, hsa-miR-16, hsa-miR-17-5p, hsa-miR-17-3p,
hsa-miR-18a, hsa-miR-19a, hsa-miR-20a, hsa-miR-22, hsa-miR-23a,
hsa-miR-189, hsa-miR-24, hsa-miR-25, hsa-miR-26a, hsa-miR-26b,
hsa-miR-27a, hsa-miR-28, hsa-miR-29a, hsa-miR-30a-5p,
hsa-miR-30a-3p, hsa-miR-31, hsa-miR-32, hsa-miR-33, hsa-miR-92,
hsa-miR-93, hsa-miR-95, hsa-miR-96, hsa-miR-98, hsa-miR-99a,
hsa-miR-100, hsa-miR-101, hsa-miR-29b, hsa-miR-103, hsa-miR-105,
hsa-miR-106a, hsa-miR-107, hsa-miR-192, hsa-miR-196a, hsa-miR-197,
hsa-miR-198, hsa-miR-199a, hsa-miR-199a*, hsa-miR-208, hsa-miR-129,
hsa-miR-148a, hsa-miR-30c, hsa-miR-30d, hsa-miR-139, hsa-miR-147,
hsa-miR-7, hsa-miR-10a, hsa-miR-10b, hsa-miR-34a, hsa-miR-181a,
hsa-miR-181b, hsa-miR-181c, hsa-miR-182, hsa-miR-182*, hsa-miR-183,
hsa-miR-187, hsa-miR-199b, hsa-miR-203, hsa-miR-204, hsa-miR-205,
hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa-miR-181a*, hsa-miR-214,
hsa-miR-215, hsa-miR-216, hsa-miR-217, hsa-miR-218, hsa-miR-219,
hsa-miR-220, hsa-miR-221, hsa-miR-222, hsa-miR-223, hsa-miR-224,
hsa-miR-200b, hsa-let-7g, hsa-let-7i, hsa-miR-1, hsa-miR-15b,
hsa-miR-23b, hsa-miR-27b, hsa-miR-30b, hsa-miR-124a, hsa-miR-125b,
hsa-miR-128a, hsa-miR-130a, hsa-miR-132, hsa-miR-133a,
hsa-miR-135a, hsa-miR-137, hsa-miR-138, hsa-miR-140, hsa-miR-141,
hsa-miR-142-5p, hsa-miR-142-3p, hsa-miR-143, hsa-miR-144,
hsa-miR-145, hsa-miR-152, hsa-miR-153, hsa-miR-191, hsa-miR-9,
hsa-miR-9*, hsa-miR-125a, hsa-miR-126*, hsa-miR-126, hsa-miR-127,
hsa-miR-134, hsa-miR-136, hsa-miR-146a, hsa-miR-149, hsa-miR-150,
hsa-miR-154, hsa-miR-154*, hsa-miR-184, hsa-miR-185, hsa-miR-186,
hsa-miR-188, hsa-miR-190, hsa-miR-193a, hsa-miR-194, hsa-miR-195,
hsa-miR-206, hsa-miR-320, hsa-miR-200c, hsa-miR-128b, hsa-miR-106b,
hsa-miR-29c, hsa-miR-200a, hsa-miR-302a*, hsa-miR-302a,
hsa-miR-34b, hsa-miR-34c, hsa-miR-299-3p, hsa-miR-301, hsa-miR-99b,
hsa-miR-296, hsa-miR-130b, hsa-miR-30e-5p, hsa-miR-30e-3p,
hsa-miR-361, hsa-miR-362, hsa-miR-363, hsa-miR-365, hsa-miR-302b*,
hsa-miR-302b, hsa-miR-302c*, hsa-miR-302c, hsa-miR-302d,
hsa-miR-367, hsa-miR-368, hsa-miR-369-3p, hsa-miR-370, hsa-miR-371,
hsa-miR-372, hsa-miR-373*, hsa-miR-373, hsa-miR-374, hsa-miR-376a,
hsa-miR-377, hsa-miR-378, hsa-miR-422b, hsa-miR-379,
hsa-miR-380-5p, hsa-miR-380-3p, hsa-miR-381, hsa-miR-382,
hsa-miR-383, hsa-miR-340, hsa-miR-330, hsa-miR-328, hsa-miR-342,
hsa-miR-337, hsa-miR-323, hsa-miR-326, hsa-miR-151, hsa-miR-135b,
hsa-miR-148b, hsa-miR-331, hsa-miR-324-5p, hsa-miR-324-3p,
hsa-miR-338, hsa-miR-339, hsa-miR-335, hsa-miR-133b, hsa-miR-325,
hsa-miR-345, hsa-miR-346, ebv-miR-BHRF1-1, ebv-miR-BHRF1-2*,
ebv-miR-BHRF1-2, ebv-miR-BHRF1-3, ebv-miR-BART1-5p, ebv-miR-BART2,
hsa-miR-384, hsa-miR-196b, hsa-miR-422a, hsa-miR-423, hsa-miR-424,
hsa-miR-425-3p, hsa-miR-18b, hsa-miR-20b, hsa-miR-448, hsa-miR-429,
hsa-miR-449, hsa-miR-450, hcmv-miR-UL22A, hcmv-miR-UL22A*,
hcmv-miR-UL36, hcmv-miR-UL112, hcmv-miR-UL148D, hcmv-miR-US5-1,
hcmv-miR-US5-2, hcmv-miR-US25-1, hcmv-miR-US25-2-5p,
hcmv-miR-US25-2-3p, hcmv-miR-US33, hsa-miR-191*, hsa-miR-200a*,
hsa-miR-369-5p, hsa-miR-431, hsa-miR-433, hsa-miR-329, hsa-miR-453,
hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-409-5p,
hsa-miR-409-3p, hsa-miR-412, hsa-miR-410, hsa-miR-376b,
hsa-miR-483, hsa-miR-484, hsa-miR-485-5p, hsa-miR-485-3p,
hsa-miR-486, hsa-miR-487a, kshv-miR-K12-10a, kshv-miR-K12-10b,
kshv-miR-K12-11, kshv-miR-K12-1, kshv-miR-K12-2, kshv-miR-K12-9*,
kshv-miR-K12-9, kshv-miR-K12-8, kshv-miR-K12-7, kshv-miR-K12-6-5p,
kshv-miR-K12-6-3p, kshv-miR-K12-5, kshv-miR-K12-4-5p,
kshv-miR-K12-4-3p, kshv-miR-K12-3, kshv-miR-K12-3*, hsa-miR-488,
hsa-miR-489, hsa-miR-490, hsa-miR-491, hsa-miR-511, hsa-miR-146b,
hsa-miR-202*, hsa-miR-202, hsa-miR-492, hsa-miR-493-5p,
hsa-miR-432, hsa-miR-432*, hsa-miR-494, hsa-miR-495, hsa-miR-496,
hsa-miR-193b, hsa-miR-497, hsa-miR-181d, hsa-miR-512-5p,
hsa-miR-512-3p, hsa-miR-498, hsa-miR-520e, hsa-miR-515-5p,
hsa-miR-515-3p, hsa-miR-519e*, hsa-miR-519e, hsa-miR-520f,
hsa-miR-526c, hsa-miR-519c, hsa-miR-520a*, hsa-miR-520a,
hsa-miR-526b, hsa-miR-526b*, hsa-miR-519b, hsa-miR-525,
hsa-miR-525*, hsa-miR-523, hsa-miR-518f*, hsa-miR-518f,
hsa-miR-520b, hsa-miR-518b, hsa-miR-526a, hsa-miR-520c,
hsa-miR-518c*, hsa-miR-518c, hsa-miR-524*, hsa-miR-524,
hsa-miR-517*, hsa-miR-517a, hsa-miR-519d, hsa-miR-521,
hsa-miR-520d*, hsa-miR-520d, hsa-miR-517b, hsa-miR-520g,
hsa-miR-516-5p, hsa-miR-516-3p, hsa-miR-518e, hsa-miR-527,
hsa-miR-518a, hsa-miR-518d, hsa-miR-517c, hsa-miR-520h,
hsa-miR-522, hsa-miR-519a, hsa-miR-499, hsa-miR-500, hsa-miR-501,
hsa-miR-502, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa-miR-513,
hsa-miR-506, hsa-miR-507, hsa-miR-508, hsa-miR-509, hsa-miR-510,
hsa-miR-514, hsa-miR-532, hsa-miR-299-5p, hsa-miR-18a*,
hsa-miR-455, hsa-miR-493-3p, hsa-miR-539, hsa-miR-544, hsa-miR-545,
hsa-miR-487b, hsa-miR-551a, hsa-miR-552, hsa-miR-553, hsa-miR-554,
hsa-miR-92b, hsa-miR-555, hsa-miR-556, hsa-miR-557, hsa-miR-558,
hsa-miR-559, hsa-miR-560, hsa-miR-561, hsa-miR-562, hsa-miR-563,
hsa-miR-564, hsa-miR-565, hsa-miR-566, hsa-miR-567, hsa-miR-568,
hsa-miR-551b, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR-572,
hsa-miR-573, hsa-miR-574, hsa-miR-575, hsa-miR-576, hsa-miR-577,
hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582,
hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-548a, hsa-miR-586,
hsa-miR-587, hsa-miR-548b, hsa-miR-588, hsa-miR-589, hsa-miR-550,
hsa-miR-590, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-595,
hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,
hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,
hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,
hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615,
hsa-miR-616, hsa-miR-548c, hsa-miR-617, hsa-miR-618, hsa-miR-619,
hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624,
hsa-miR-625, hsa-miR-626, hsa-miR-627, hsa-miR-628, hsa-miR-629,
hsa-miR-630, hsa-miR-631, hsa-miR-33b, hsa-miR-632, hsa-miR-633,
hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638,
hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643,
hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648,
hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-548d,
hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-449b, hsa-miR-653,
hsa-miR-411, hsa-miR-654, hsa-miR-655, hsa-miR-656, hsa-miR-549,
hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-421,
hsa-miR-542-5p, hcmv-miR-US4, hcmv-miR-UL70-5p, hcmv-miR-UL70-3p,
hsa-miR-363*, hsa-miR-376a*, hsa-miR-542-3p, ebv-miR-BART1-3p,
hsa-miR-425-5p, ebv-miR-BART3-5p, ebv-miR-BART3-3p, ebv-miR-BART4,
ebv-miR-BART5, ebv-miR-BART6-5p, ebv-miR-BART6-3p, ebv-miR-BART7,
ebv-miR-BART8-5p, ebv-miR-BART8-3p, ebv-miR-BART9, ebv-miR-BART10,
ebv-miR-BART11-5p, ebv-miR-BART11-3p, ebv-miR-BART12,
ebv-miR-BART13, ebv-miR-BART14-5p, ebv-miR-BART14-3p,
kshv-miR-K12-12, ebv-miR-BART15, ebv-miR-BART16, ebv-miR-BART17-5p,
ebv-miR-BART17-3p, ebv-miR-BART18, ebv-miR-BART19,
ebv-miR-BART20-5p, ebv-miR-BART20-3p, hsv1-miR-H1, hsa-miR-758,
hsa-miR-671, hsa-miR-668, hsa-miR-767-5p, hsa-miR-767-3p,
hsa-miR-454-5p, hsa-miR-454-3p, hsa-miR-769-5p, hsa-miR-769-3p,
hsa-miR-766, hsa-miR-765, hsa-miR-768-5p, hsa-miR-768-3p,
hsa-miR-770-5p, hsa-miR-802, hsa-miR-801, hsa-miR-675
[0108] Preferred single stranded oligonucleotide according to the
invention are complementary to a microRNA sequence selected from
the group consisting of has-miR19b, hsa-miR21, hsa-miR 122, hsa-miR
142 a7b, hsa-miR 155, hsa-miR 375.
[0109] Preferred single stranded oligonucleotide according to the
invention are complementary to a microRNA sequence selected from
the group consisting of hsa-miR196b and has-181a.
[0110] In one embodiment, the oligonucleotide according to the
invention does not comprise a nucleobase at the 3' end that
corresponds to the first 5' end nucleotide of the target
microRNA.
[0111] In one embodiment, the first nucleobase of the single
stranded oligonucleotide according to the invention, counting from
the 3' end, is a nucleotide analogue, such as an LNA unit.
[0112] In one embodiment, the second nucleobase of the single
stranded oligonucleotide according to the invention, counting from
the 3' end, is a nucleotide analogue, such as an LNA unit.
[0113] In one embodiment, the ninth and/or the tenth nucleotide of
the single stranded oligonucleotide according to the invention,
counting from the 3' end, is a nucleotide analogue, such as an LNA
unit.
[0114] In one embodiment, the ninth nucleobase of the single
stranded oligonucleotide according to the invention, counting from
the 3' end is a nucleotide analogue, such as an LNA unit.
[0115] In one embodiment, the tenth nucleobase of the single
stranded oligonucleotide according to the invention, counting from
the 3' end is a nucleotide analogue, such as an LNA unit.
[0116] In one embodiment, both the ninth and the tenth nucleobase
of the single stranded oligonucleotide according to the invention,
calculated from the 3' end is a nucleotide analogue, such as an LNA
unit.
[0117] In one embodiment, the single stranded oligonucleotide
according to the invention does not comprise a region of more than
5 consecutive DNA nucleotide units. In one embodiment, the single
stranded oligonucleotide according to the invention does not
comprise a region of more than 6 consecutive DNA nucleotide units.
In one embodiment, the single stranded oligonucleotide according to
the invention does not comprise a region of more than 7 consecutive
DNA nucleotide units. In one embodiment, the single stranded
oligonucleotide according to the invention does not comprise a
region of more than 8 consecutive DNA nucleotide units. In one
embodiment, the single stranded oligonucleotide according to the
invention does not comprise a region of more than 3 consecutive DNA
nucleotide units. In one embodiment, the single stranded
oligonucleotide according to the invention does not comprise a
region of more than 2 consecutive DNA nucleotide units.
[0118] In one embodiment, the single stranded oligonucleotide
comprises at least region consisting of at least two consecutive
nucleotide analogue units, such as at least two consecutive LNA
units.
[0119] In one embodiment, the single stranded oligonucleotide
comprises at least region consisting of at least three consecutive
nucleotide analogue units, such as at least three consecutive LNA
units.
[0120] In one embodiment, the single stranded oligonucleotide of
the invention does not comprise a region of more than 7 consecutive
nucleotide analogue units, such as LNA units. In one embodiment,
the single stranded oligonucleotide of the invention does not
comprise a region of more than 6consecutive nucleotide analogue
units, such as LNA units. In one embodiment, the single stranded
oligonucleotide of the invention does not comprise a region of more
than 5 consecutive nucleotide analogue units, such as LNA units. In
one embodiment, the single stranded oligonucleotide of the
invention does not comprise a region of more than 4 consecutive
nucleotide analogue units, such as LNA units. In one embodiment,
the single stranded oligonucleotide of the invention does not
comprise a region of more than 3 consecutive nucleotide analogue
units, such as LNA units. In one embodiment, the single stranded
oligonucleotide of the invention does not comprise a region of more
than 2 consecutive nucleotide analogue units, such as LNA
units.
[0121] In one embodiment, the first or second 3' nucleobase of the
single stranded oligonucleotide corresponds to the second 5'
nucleotide of the microRNA sequence.
[0122] In one embodiment, nucleobase units 1 to 6 (inclusive) of
the single stranded oligonucleotide as measured from the 3' end the
region of the single stranded oligonucleotide are complementary to
the microRNA seed region sequence.
[0123] In one embodiment, nucleobase units 1 to 7 (inclusive) of
the single stranded oligonucleotide as measured from the 3' end the
region of the single stranded oligonucleotide are complementary to
the microRNA seed region sequence.
[0124] In one embodiment, nucleobase units 2 to 7 (inclusive) of
the single stranded oligonucleotide as measured from the 3' end the
region of the single stranded oligonucleotide are complementary to
the microRNA seed region sequence.
[0125] In one embodiment, the single stranded oligonucleotide
comprises at least one nucleotide analogue unit, such as at least
one LNA unit, in a position which is within the region
complementary to the miRNA seed region. The single stranded
oligonucleotide may, in one embodiment comprise at between one and
6 or between 1 and 7 nucleotide analogue units, such as between 1
and 6 and 1 and 7 LNA units, in a position which is within the
region complementary to the miRNA seed region.
[0126] In one embodiment, the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
(X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X) xxxXxx, (X)xxxxXx and
(X)xxxxxX, as read in a 3'-5' direction, wherein "X" denotes a
nucleotide analogue, (X) denotes an optional nucleotide analogue,
such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit.
[0127] In one embodiment, the single stranded oligonucleotide
comprises at least two nucleotide analogue units, such as at least
two LNA units, in positions which are complementary to the miRNA
seed region.
[0128] In one embodiment, the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
(X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx,
(X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX,
(X)xxxXXx, (X)xxxXxX and (X)xxxxXX, wherein "X" denotes a
nucleotide analogue, such as an LNA unit, (X) denotes an optional
nucleotide analogue, such as an LNA unit, and "x" denotes a DNA or
RNA nucleotide unit.
[0129] In one embodiment, the single stranded oligonucleotide
comprises at least three nucleotide analogue units, such as at
least three LNA units, in positions which are complementary to the
miRNA seed region.
[0130] In one embodiment, the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
(X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,
(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx,
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and
(X)XxXxXx, wherein "X" denotes a nucleotide analogue, such as an
LNA unit, (X) denotes an optional nucleotide analogue, such as an
LNA unit, and "x" denotes a DNA or RNA nucleotide unit.
[0131] In one embodiment, the single stranded oligonucleotide
comprises at least four nucleotide analogue units, such as at least
four LNA units, in positions which are complementary to the miRNA
seed region.
[0132] In one embodiment the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
(X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X) xXXXxX, (X)xXXXXx, (X)XxxXXXX,
(X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx,
(X)XXXxxX, (X)XXXxXx, and (X)XXXXxx, wherein "X" denotes a
nucleotide analogue, such as an LNA unit, (X) denotes an optional
nucleotide analogue, such as an LNA unit, and "x" denotes a DNA or
RNA nucleotide unit.
[0133] In one embodiment, the single stranded oligonucleotide
comprises at least five nucleotide analogue units, such as at least
five LNA units, in positions which are complementary to the miRNA
seed region.
[0134] In one embodiment, the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
(X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and
(X)XXXXXx, wherein "X" denotes a nucleotide analogue, such as an
LNA unit, (X) denotes an optional nucleotide analogue, such as an
LNA unit, and "x" denotes a DNA or RNA nucleotide unit.
[0135] In one embodiment, the single stranded oligonucleotide
comprises six or seven nucleotide analogue units, such as six or
seven LNA units, in positions which are complementary to the miRNA
seed region.
[0136] In one embodiment, the nucleobase sequence of the single
stranded oligonucleotide which is complementary to the sequence of
the microRNA seed region, is selected from the group consisting of
XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx,
wherein "X" denotes a nucleotide analogue, such as an LNA unit,
such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit.
[0137] In one embodiment, the two nucleobase motif at position 7 to
8, counting from the 3' end of the single stranded oligonucleotide
is selected from the group consisting of xx, XX, xX and Xx, wherein
"X" denotes a nucleotide analogue, such as an LNA unit, such as an
LNA unit, and "x" denotes a DNA or RNA nucleotide unit.
[0138] In one embodiment, the two nucleobase motif at position 7 to
8, counting from the 3' end of the single stranded oligonucleotide
is selected from the group consisting of XX, xX and Xx, wherein "X"
denotes a nucleotide analogue, such as an LNA unit, such as an LNA
unit, and "x" denotes a DNA or RNA nucleotide unit.
[0139] In one embodiment, the single stranded oligonucleotide
comprises at least 12 nucleobases and wherein the two nucleobase
motif at position 11 to 12, counting from the 3' end of the single
stranded oligonucleotide is selected from the group consisting of
xx, XX, xX and Xx, wherein "X" denotes a nucleotide analogue, such
as an LNA unit, such as an LNA unit, and "x" denotes a DNA or RNA
nucleotide unit.
[0140] In one embodiment, the single stranded oligonucleotide
comprises at least 12 nucleobases and wherein the two nucleobase
motif at position 11 to 12, counting from the 3' end of the single
stranded oligonucleotide is selected from the group consisting of
XX, xX and Xx, wherein "X" denotes a nucleotide analogue, such as
an LNA unit, such as an LNA unit, and "x" denotes a DNA or RNA
nucleotide unit.
[0141] In one embodiment, the single stranded oligonucleotide
comprises at least 13 nucleobases and wherein the three nucleobase
motif at position 11 to 13, counting from the 3' end, is selected
from the group consisting of xxx, Xxx, xXx, xxX, XXx, XxX, xXX and
XXX, wherein "X" denotes a nucleotide analogue, such as an LNA
unit, such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit.
[0142] In one embodiment, the three nucleobase motif at position 11
to 13, counting from the 3' end of the single stranded
oligonucleotide, is selected from the group consisting of Xxx, xXx,
xxX, XXx, XxX, xXX and XXX, wherein "X" denotes a nucleotide
analogue, such as an LNA unit, such as an LNA unit, and "x" denotes
a DNA or RNA nucleotide unit.
[0143] In one embodiment, the single stranded oligonucleotide
comprises at least 14 nucleobases and wherein the four nucleobase
motif at positions 11 to 14, counting from the 3' end, is selected
from the group consisting of xxxx, Xxxx, xXxx, xxXx, xxxX, XXxx,
XxXx, XxxX, xXXx, xXxX, xxXX, XXXx, XxXX, xXXX, XXxX and XXXX
wherein "X" denotes a nucleotide analogue, such as an LNA unit,
such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit.
[0144] In one embodiment, the four nucleobase motif at position 11
to 14 of the single stranded oligonucleotide, counting from the 3'
end, is selected from the group consisting of Xxxx, xXxx, xxXx,
xxxX, XXxx, XxXx, XxxX, xXXx, xXxX, xxXX, XXXx, XxXX, xXXX, XXxX
and XXXX, wherein "X" denotes a nucleotide analogue, such as an LNA
unit, and "x" denotes a DNA or RNA nucleotide unit.
[0145] In one embodiment, the single stranded oligonucleotide
comprises 15 nucleobases and the five nucleobase motif at position
11 to 15, counting from the 3' end, is selected from the group
consisting of Xxxxx, xXxxx, xxXxx, xxxXx, xxxxX, XXxxx, XxXxx,
XxxXx, XxxxX, xXXxx, xXxXx, xXxxX, xxXXx, xxXxX, xxxXX, XXXXX,
XXXXX, XXXXX, XXXXX, XXXXX, XXxXX, XxXxX, XXXXx, XXXxX, XXxXX,
XxXXXX, xXXXX, and XXXXX wherein "X" denotes a nucleotide analogue,
such as an LNA unit, such as an LNA unit, and "x" denotes a DNA or
RNA nucleotide unit.
[0146] In one embodiment, the single stranded oligonucleotide
comprises 16 nucleobases and the six nucleobase motif at positions
11 to 16, counting from the 3' end, is selected from the group
consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx, xxxxxX,
XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx,
xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX, xxxxXX, XXXxxx,
XXxXxx, XXxxXx, XXxxxX, XxXXxx, XxXxXx, XxXxxX, XxxXXx, XxxXxX,
XxxxXX, xXXXxx, xXXxXx, xXXxxX, xXxXXx, xXxXxX, xXxxXX, xxXXXx,
xxXXxX, xxXxXX, xxxXXX, XXXXxx, XXXxxX, XXxxXX, XxxXXX, xxXXXX,
xXxXXX, XxXxXX, XXxXxX, XXXxXx, xXXxXX, XxXXxX, XXxXXx, xXXXxX,
XxXXXx, xXXXXx, xXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX, XXXXXx, and
XXXXXX wherein "X" denotes a nucleotide analogue, such as an LNA
unit, such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit.
[0147] In one embodiment, the six nucleobase motif at positions 11
to 16 of the single stranded oligonucleotide, counting from the 3'
end, is xxXxxX, wherein "X" denotes a nucleotide analogue, such as
an LNA unit, such as an LNA unit, and "x" denotes a DNA or RNA
nucleotide unit.
[0148] In one embodiment, the three 5' most nucleobases, is
selected from the group consisting of Xxx, xXx, xxX, XXx, XxX, xXX
and XXX, wherein "X" denotes a nucleotide analogue, such as an LNA
unit, such as an LNA unit, and "x" denotes a DNA or RNA nucleotide
unit. In one embodiment, x'' denotes a DNA unit.
[0149] In one embodiment, the single stranded oligonucleotide
comprises a nucleotide analogue unit, such as an LNA unit, at the
5' end.
[0150] In one embodiment, the nucleotide analogue units, such as X,
are independently selected form the group consisting of:
2'-O-alkyl-RNA unit, 2'-OMe-RNA unit, 2'-amino-DNA unit,
2'-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit.
[0151] In one embodiment, all the nucleobases of the single
stranded oligonucleotide of the invention are nucleotide analogue
units.
[0152] In one embodiment, the nucleotide analogue units, such as X,
are independently selected form the group consisting of: 2'-OMe-RNA
units, 2'-fluoro-DNA units, and LNA units,
[0153] In one embodiment, the single stranded oligonucleotide
comprises said at least one LNA analogue unit and at least one
further nucleotide analogue unit other than LNA.
[0154] In one embodiment, the non-LNA nucleotide analogue unit or
units are independently selected from 2'-OMe RNA units and
2'-fluoro DNA units.
[0155] In one embodiment, the single stranded oligonucleotide
consists of at least one sequence XYX or YXY, wherein X is LNA and
Y is either a 2'-OMe RNA unit and 2'-fluoro DNA unit.
[0156] In one embodiment, the sequence of nucleobases of the single
stranded oligonucleotide consists of alternative X and Y units.
[0157] In one embodiment, the single stranded oligonucleotide
comprises alternating LNA and DNA units (Xx) or (xX).
[0158] In one embodiment, the single stranded oligonucleotide
comprises a motif of alternating LNA followed by 2 DNA units (Xxx),
xXx or xxX.
[0159] In one embodiment, at least one of the DNA or non-LNA
nucleotide analogue units are replaced with a LNA nucleobase in a
position selected from the positions identified as LNA nucleobase
units in any one of the embodiments referred to above.
[0160] In one embodiment, "X" donates an LNA unit.
[0161] In one embodiment, the single stranded oligonucleotide
comprises at least 2 nucleotide analogue units, such as at least 3
nucleotide analogue units, such as at least 4 nucleotide analogue
units, such as at least 5 nucleotide analogue units, such as at
least 6 nucleotide analogue units, such as at least 7 nucleotide
analogue units, such as at least 8 nucleotide analogue units, such
as at least 9 nucleotide analogue units, such as at least 10
nucleotide analogue units.
[0162] In one embodiment, the single stranded oligonucleotide
comprises at least 2 LNA units, such as at least 3 LNA units, such
as at least 4 LNA units, such as at least 5 LNA units, such as at
least 6 LNA units, such as at least 7 LNA units, such as at least 8
LNA units, such as at least 9 LNA units, such as at least 10 LNA
units.
[0163] In one embodiment wherein at least one of the nucleotide
analogues, such as LNA units, is either cytosine or guanine, such
as between 1-10 of the of the nucleotide analogues, such as LNA
units, is either cytosine or guanine, such as 2, 3, 4, 5, 6, 7, 8,
or 9 of the of the nucleotide analogues, such as LNA units, is
either cytosine or guanine.
[0164] In one embodiment at least two of the nucleotide analogues
such as LNA units is either cytosine or guanine. In one embodiment
at least three of the nucleotide analogues such as LNA units is
either cytosine or guanine. In one embodiment at least four of the
nucleotide analogues such as LNA units is either cytosine or
guanine. In one embodiment at least five of the nucleotide
analogues such as LNA units is either cytosine or guanine. In one
embodiment at least six of the nucleotide analogues such as LNA
units is either cytosine or guanine. In one embodiment at least
seven of the nucleotide analogues such as LNA units is either
cytosine or guanine. In one embodiment at least eight of the
nucleotide analogues such as LNA units is either cytosine or
guanine.
[0165] In a preferred embodiment the nucleotide analogues have a
higher thermal duplex stability a complementary RNA nucleotide than
the binding affinity of an equivalent DNA nucleotide to said
complementary RNA nucleotide.
[0166] In one embodiment, the nucleotide analogues confer enhanced
serum stability to the single stranded oligonucleotide.
[0167] In one embodiment, the single stranded oligonucleotide forms
an A-helix conformation with a complementary single stranded RNA
molecule.
[0168] A duplex between two RNA molecules typically exists in an
A-form conformation, where as a duplex between two DNA molecules
typically exits in a B-form conformation. A duplex between a DNA
and RNA molecule typically exists in a intermediate conformation
(A/B form). The use of nucleotide analogues, such as beta-D-oxy LNA
can be used to promote a more A form like conformation. Standard
circular dichromisms (CD) or NMR analysis is used to determine the
form of duplexes between the oligonucleotides of the invention and
complementary RNA molecules.
[0169] As recruitment by the RISC complex is thought to be
dependant upon the specific structural conformation of the
miRNA/mRNA target, the oligonucleotides according to the present
invention may, in one embodiment form a A/B-form duplex with a
complementary RNA molecule.
[0170] However, we have also determined that the use of nucleotide
analogues which promote the A-form structure can also be effective,
such as the alpha-L isomer of LNA.
[0171] In one embodiment, the single stranded oligonucleotide forms
an A/B-form conformation with a complementary single stranded RNA
molecule.
[0172] In one embodiment, the single stranded oligonucleotide forms
an A-from conformation with a complementary single stranded RNA
molecule.
[0173] In one embodiment, the single stranded oligonucleotide
according to the invention does not mediate RNAseH based cleavage
of a complementary single stranded RNA molecule. Typically a
stretch of at least 5 (typically not effective ofr RNAse H
recruitment), more preferably at least 6, more preferably at least
7 or 8 consecutive DNA nucleobases (or alternative nucleobases
which can recruit RNAseH, such as alpha L-amino LNA) are required
in order for an oligonucleotide to be effective in recruitment of
RNAseH.
[0174] EP 1 222 309 provides in vitro methods for determining
RNaseH activity, which may be used to determine the ability to
recruit RNaseH. A compound is deemed capable of recruiting RNase H
if, when provided with the complementary RNA target, it has an
initial rate, as measured in pmol/l/min, of at least 1%, such as at
least 5%, such as at least 10% or less than 20% of the equivalent
DNA only oligonucleotide, with no 2' substitutions, with
phosphorothiote linkage groups between all nucleotides in the
oligonucleotide, using the methodology provided by Example 91-95 of
EP 1 222 309.
[0175] A compound is deemed essentially incapable of recruiting
RNaseH if, when provided with the complementary RNA target, and
RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less
than 1%, such as less than 5%,such as less than 10% or less than
20% of the initial rate determined using the equivalent DNA only
oligonucleotide, with no 2' substitutions, with phosphiothiote
linkage groups between all nucleotides in the oligonucleotide,
using the methodology provided by Example 91-95 of EP 1 222
309.
[0176] In a highly preferred embodiment, the single stranded
oligonucleotide of the invention is capable of forming a duplex
with a complementary single stranded RNA nucleic acid molecule
(typically of about the same length of said single stranded
oligonucleotide) with phosphodiester internucleoside linkages,
wherein the duplex has a T.sub.m of at least about 60.degree. C.,
indeed it is preferred that the single stranded oligonucleotide is
capable of forming a duplex with a complementary single stranded
RNA nucleic acid molecule with phosphodiester internucleoside
linkages, wherein the duplex has a T.sub.m of between about
70.degree. C. to about 95.degree. C., such as a T.sub.m of between
about 70.degree. C. to about 90.degree. C., such as between about
70.degree. C. and about 85.degree. C.
[0177] In one embodiment, the single stranded oligonucleotide is
capable of forming a duplex with a complementary single stranded
DNA nucleic acid molecule with phosphodiester internucleoside
linkages, wherein the duplex has a T.sub.m of between about
50.degree. C. to about 95.degree. C., such as between about
50.degree. C. to about 90.degree. C., such as at least about
55.degree. C., such as at least about 60.degree. C., or no more
than about 95.degree. C.
[0178] The single stranded oligonucleotide may, in one embodiment
have a length of between 14-16 nucleobases, including 15
nucleobases.
[0179] In one embodiment, the LNA unit or units are independently
selected from the group consisting of oxy-LNA, thio-LNA, and
amino-LNA, in either of the D-.beta. and L-.alpha. configurations
or combinations thereof.
[0180] In one specific embodiment the LNA units may be an ENA
nucleobase.
[0181] In one the embodiment the LNA units are beta D oxy-LNA.
[0182] In one embodiment the LNA units are in alpha-L amino
LNA.
[0183] In a preferable embodiment, the single stranded
oligonucleotide comprises between 3 and 17 LNA units.
[0184] In one embodiment, the single stranded oligonucleotide
comprises at least one internucleoside linkage group which differs
from phosphate.
[0185] In one embodiment, the single stranded oligonucleotide
comprises at least one phosphorothioate internucleoside
linkage.
[0186] In one embodiment, the single stranded oligonucleotide
comprises phosphodiester and phosphorothioate linkages.
[0187] In one embodiment, the all the internucleoside linkages are
phosphorothioate linkages.
[0188] In one embodiment, the single stranded oligonucleotide
comprises at least one phosphodiester internucleoside linkage.
[0189] In one embodiment, all the internucleoside linkages of the
single stranded oligonucleotide of the invention are phosphodiester
linkages.
[0190] In one embodiment, pharmaceutical composition according to
the invention comprises a carrier such as saline or buffered
saline.
[0191] In one embodiment, the method for the synthesis of a single
stranded oligonucleotide targeted against a human microRNA, is
performed in the 3' to 5' direction a-f.
[0192] The method for the synthesis of the single stranded
oligonucleotide according to the invention may be performed using
standard solid phase oligonucleotide synthesis.
DEFINITIONS
[0193] The term `nucleobase` refers to nucleotides, such as DNA and
RNA, and nucleotide analogues.
[0194] The term "oligonucleotide" (or simply "oligo") refers, in
the context of the present invention, to a molecule formed by
covalent linkage of two or more nucleobases. When used in the
context of the oligonucleotide of the invention (also referred to
the single stranded oligonucleotide), the term "oligonucleotide"
may have, in one embodiment, for example between 8-26 nucleobases,
such as between 10 to 26 nucleobases such between 12 to 26
nucleobases. In a preferable embodiment, as detailed herein, the
oligonucleotide of the invention has a length of between 8-17
nucleobases, such as between 20-27 nucleobases such as between 8-16
nucleobases, such as between 12-nucleobases,
[0195] In such an embodiment, the oligonucleotide of the invention
may have a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17
nucleobases.
[0196] It will be recognised that for shorter oligonucleotides it
may be necessary to increase the proportion of (high affinity)
nucleotide analogues, such as LNA. Therefore in one embodiment at
least about 30% of the nucleobases are nucleotide analogues, such
as at least about 33%, such as at least about 40%, or at least
about 50% or at least about 60%, such as at least about 66%, such
as at least about 70%, such as at least about 80%, or at least
about 90%. It will also be apparent that the oligonucleotide may
comprise of a nucleobase sequence which consists of only nucleotide
analogue sequences.
[0197] Herein, the term "nitrogenous base" is intended to cover
purines and pyrimidines, such as the DNA nucleobases A, C, T and G,
the RNA nucleobases A, C, U and G, as well as non-DNA/RNA
nucleobases, such as 5-methylcytosine (.sup.MeC), isocytosine,
pseudoisocytosine, 5-bromouracil, 5-propynyluracil,
5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine,
2-aminopurine, inosine, 2,6-diaminopurine,
7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and
2-chloro-6-aminopurine, in particular .sup.MeC. It will be
understood that the actual selection of the non-DNA/RNA nucleobase
will depend on the corresponding (or matching) nucleotide present
in the microRNA strand which the oligonucleotide is intended to
target. For example, in case the corresponding nucleotide is G it
will normally be necessary to select a non-DNA/RNA nucleobase which
is capable of establishing hydrogen bonds to G. In this specific
case, where the corresponding nucleotide is G, a typical example of
a preferred non-DNA/RNA nucleobase is .sup.MeC.
[0198] The term "internucleoside linkage group" is intended to mean
a group capable of covalently coupling together two nucleobases,
such as between DNA units, between DNA units and nucleotide
analogues, between two non-LNA units, between a non-LNA unit and an
LNA unit, and between two LNA units, etc. Preferred examples
include phosphate, phosphodiester groups and phosphorothioate
groups.
[0199] The internucleoside linkage may be selected form the group
consisting of: --O--P(O).sub.2--O--, --O--P(O,S)--O--,
--O--P(S).sub.2--O--, --S--P(O).sub.2--O--, --S--P(O,S)--O--,
--S--P(S).sub.2--O--, --O--P(O).sub.2--S--, --O--P(O,S)--S--,
--S--P(O).sub.2--S--, --O--PO(R.sup.H)--O--, O--PO(OCH.sub.3)--O--,
--O--PO(NR.sup.H)--O--, --O--PO(OCH.sub.2CH.sub.2S--R)--O--,
--O--PO(BH.sub.3)--O--, --O--PO(NHR.sup.H)--O--,
--O--P(O).sub.2--NR.sup.H--, --NR.sup.H--P(O).sub.2--O--,
--NR.sup.H--CO--O--, --NR.sup.H--CO--NR.sup.H--, and/or the
internucleoside linkage may be selected form the group consisting
of: --O--CO--O--, --O--CO--NR.sup.H--, --NR.sup.H--CO--CH.sub.2--,
--O--CH.sub.2--CO--NR.sup.H--, --O--CH.sub.2--CH.sub.2--NR.sup.H--,
--CO--NR.sup.H--CH.sub.2--, --CH.sub.2--NR.sup.H--CO--,
--O--CH.sub.2--CH.sub.2--S--, --S--CH.sub.2--CH.sub.2--O--,
--S--CH.sub.2--CH.sub.2--S--, --CH.sub.2--SO.sub.2--CH.sub.2--,
--CH.sub.2--CO--NR.sup.H--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--CO--,
--CH.sub.2--NCH.sub.3--O--CH.sub.2--, where R.sup.H is selected
from hydrogen and C.sub.1-4-alkyl. Suitably, in some embodiments,
sulphur (S) containing internucleoside linkages as provided above
may be preferred
[0200] The terms "corresponding to" and "corresponds to" as used in
the context of oligonucleotides refers to the comparison between
either a nucleobase sequence of the compound of the invention, and
the reverse complement thereof, or in one embodiment between a
nucleobase sequence and an equivalent (identical) nucleobase
sequence which may for example comprise other nucleobases but
retains the same base sequence, or complement thereof. Nucleotide
analogues are compared directly to their equivalent or
corresponding natural nucleotides. Sequences which form the reverse
complement of a sequence are referred to as the complement sequence
of the sequence.
[0201] When referring to the length of a nucleotide molecule as
referred to herein, the length corresponds to the number of monomer
units, i.e. nucleobases, irrespective as to whether those monomer
units are nucleotides or nucleotide analogues. With respect to
nucleobases, the terms monomer and unit are used interchangeably
herein.
[0202] It should be understood that when the term "about" is used
in the context of specific values or ranges of values, the
disclosure should be read as to include the specific value or range
referred to.
[0203] Preferred DNA analogues includes DNA analogues where the
2'-H group is substituted with a substitution other than --OH(RNA)
e.g. by substitution with --O--CH.sub.3,
--O--CH.sub.2--CH.sub.2--O--CH.sub.3,
--O--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2,
--O--CH.sub.2--CH.sub.2--CH.sub.2--OH or --F.
[0204] Preferred RNA analogues includes RNA analogues which have
been modified in its 2'-OH group, e.g. by substitution with a group
other than --H (DNA), for example --O--CH.sub.3,
--O--CH.sub.2--CH.sub.2--O--CH.sub.3,
--O--CH.sub.2--CH.sub.2--CH.sub.2--NH.sub.2,
--O--CH.sub.2--CH.sub.2--CH.sub.2--OH or --F.
[0205] In one embodiment the nucleotide analogue is "ENA".
[0206] When used in the present context, the terms "LNA unit", "LNA
monomer", "LNA residue", "locked nucleic acid unit", "locked
nucleic acid monomer" or "locked nucleic acid residue", refer to a
bicyclic nucleoside analogue. LNA units are described in inter alia
WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO
03/006475 and WO 03/095467. The LNA unit may also be defined with
respect to its chemical formula. Thus, an "LNA unit", as used
herein, has the chemical structure shown in Scheme 1 below:
##STR00001##
wherein [0207] X is selected from the group consisting of O, S and
NR.sup.H, where R.sup.H is H or C.sub.1-4-alkyl; [0208] Y is
(--CH.sub.2).sub.r, where r is an integer of 1-4; and [0209] B is a
nitrogenous base.
[0210] When referring to substituting a DNA unit by its
corresponding LNA unit in the context of the present invention, the
term "corresponding LNA unit" is intended to mean that the DNA unit
has been replaced by an LNA unit containing the same nitrogenous
base as the DNA unit that it has replaced, e.g. the corresponding
LNA unit of a DNA unit containing the nitrogenous base A also
contains the nitrogenous base A. The exception is that when a DNA
unit contains the base C, the corresponding LNA unit may contain
the base C or the base .sup.MeC, preferably .sup.MeC.
[0211] Herein, the term "non-LNA unit" refers to a nucleoside
different from an LNA-unit, i.e. the term "non-LNA unit" includes a
DNA unit as well as an RNA unit. A preferred non-LNA unit is a DNA
unit.
[0212] The terms "unit", "residue" and "monomer" are used
interchangeably herein.
[0213] The term "at least one" encompasses an integer larger than
or equal to 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 and so forth.
[0214] The terms "a" and "an" as used about a nucleotide, an agent,
an LNA unit, etc., is intended to mean one or more. In particular,
the expression "a component (such as a nucleotide, an agent, an LNA
unit, or the like) selected from the group consisting of . . . " is
intended to mean that one or more of the cited components may be
selected. Thus, expressions like "a component selected from the
group consisting of A, B and C" is intended to include all
combinations of A, B and C, i.e. A, B, C, A+B, A+C, B+C and
A+B+C.
[0215] The term "thio-LNA unit" refers to an LNA unit in which X in
Scheme 1 is S. A thio-LNA unit can be in both the beta-D form and
in the alpha-L form. Generally, the beta-D form of the thio-LNA
unit is preferred. The beta-D-form and alpha-L-form of a thio-LNA
unit are shown in Scheme 3 as compounds 3A and 3B,
respectively.
[0216] The term "amino-LNA unit" refers to an LNA unit in which X
in Scheme 1 is NH or NR.sup.H, where R.sup.H is hydrogen or
C.sub.1-4-alkyl. An amino-LNA unit can be in both the beta-D form
and in the alpha-L form. Generally, the beta-D form of the
amino-LNA unit is preferred. The beta-D-form and alpha-L-form of an
amino-LNA unit are shown in Scheme 4 as compounds 4A and 4B,
respectively.
[0217] The term "oxy-LNA unit" refers to an LNA unit in which X in
Scheme 1 is O. An Oxy-LNA unit can be in both the beta-D form and
in the alpha-L form. Generally, the beta-D form of the oxy-LNA unit
is preferred. The beta-D form and the alpha-L form of an oxy-LNA
unit are shown in Scheme 5 as compounds 5A and 5B,
respectively.
[0218] In the present context, the term "C.sub.1-6-alkyl" is
intended to mean a linear or branched saturated hydrocarbon chain
wherein the longest chains has from one to six carbon atoms, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl. A
branched hydrocarbon chain is intended to mean a C.sub.1-6-alkyl
substituted at any carbon with a hydrocarbon chain.
[0219] In the present context, the term "C.sub.1-4-alkyl" is
intended to mean a linear or branched saturated hydrocarbon chain
wherein the longest chains has from one to four carbon atoms, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl
and tert-butyl. A branched hydrocarbon chain is intended to mean a
C.sub.1-4-alkyl substituted at any carbon with a hydrocarbon
chain.
[0220] When used herein the term "C.sub.1-6-alkoxy" is intended to
mean C.sub.1-6-alkyl-oxy, such as methoxy, ethoxy, n-propoxy,
isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy,
isopentoxy, neopentoxy and hexoxy.
[0221] In the present context, the term "C.sub.2-6-alkenyl" is
intended to mean a linear or branched hydrocarbon group having from
two to six carbon atoms and containing one or more double bonds.
Illustrative examples of C.sub.2-6-alkenyl groups include allyl,
homo-allyl, vinyl, crotyl, butenyl, butadienyl, pentenyl,
pentadienyl, hexenyl and hexadienyl. The position of the
unsaturation (the double bond) may be at any position along the
carbon chain.
[0222] In the present context the term "C.sub.2-6-alkynyl" is
intended to mean linear or branched hydrocarbon groups containing
from two to six carbon atoms and containing one or more triple
bonds. Illustrative examples of C.sub.2-6-alkynyl groups include
acetylene, propynyl, butynyl, pentynyl and hexynyl. The position of
unsaturation (the triple bond) may be at any position along the
carbon chain. More than one bond may be unsaturated such that the
"C.sub.2-6-alkynyl" is a di-yne or enedi-yne as is known to the
person skilled in the art.
[0223] As used herein, "hybridisation" means hydrogen bonding,
which may be Watson-Crick, Hoogsteen, reversed Hoogsteen hydrogen
bonding, etc., between complementary nucleoside or nucleotide
bases. The four nucleobases commonly found in DNA are G, A, T and C
of which G pairs with C, and A pairs with T. In RNA T is replaced
with uracil (U), which then pairs with A. The chemical groups in
the nucleobases that participate in standard duplex formation
constitute the Watson-Crick face. Hoogsteen showed a couple of
years later that the purine nucleobases (G and A) in addition to
their Watson-Crick face have a Hoogsteen face that can be
recognised from the outside of a duplex, and used to bind
pyrimidine oligonucleotides via hydrogen bonding, thereby forming a
triple helix structure.
[0224] In the context of the present invention "complementary"
refers to the capacity for precise pairing between two nucleotides
sequences with one another. For example, if a nucleotide at a
certain position of an oligonucleotide is capable of hydrogen
bonding with a nucleotide at the corresponding position of a DNA or
RNA molecule, then the oligonucleotide and the DNA or RNA are
considered to be complementary to each other at that position. The
DNA or RNA strand are considered complementary to each other when a
sufficient number of nucleotides in the oligonucleotide can form
hydrogen bonds with corresponding nucleotides in the target DNA or
RNA to enable the formation of a stable complex. To be stable in
vitro or in vivo the sequence of an oligonucleotide need not be
100% complementary to its target microRNA. The terms
"complementary" and "specifically hybridisable" thus imply that the
oligonucleotide binds sufficiently strong and specific to the
target molecule to provide the desired interference with the normal
function of the target whilst leaving the function of non-target
RNAs unaffected.
[0225] In a preferred example the oligonucleotide of the invention
is 100% complementary to a human microRNA sequence, such as one of
the microRNA sequences referred to herein.
[0226] In a preferred example, the oligonucleotide of the invention
comprises a contiguous sequence which is 100% complementary to the
seed region of the human microRNA sequence.
[0227] MicroRNAs are short, non-coding RNAs derived from endogenous
genes that act as post-transcriptional regulators of gene
expression. They are processed from longer (ca 70-80 nt)
hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme
Dicer. MicroRNAs assemble in ribonucleoprotein complexes termed
miRNPs and recognize their target sites by antisense
complementarity thereby mediating down-regulation of their target
genes. Near-perfect or perfect complementarity between the miRNA
and its target site results in target mRNA cleavage, whereas
limited complementarity between the microRNA and the target site
results in translational inhibition of the target gene.
[0228] The term "microRNA" or "miRNA", in the context of the
present invention, means an RNA oligonucleotide consisting of
between 18 to 25 nucleotides in length. In functional terms miRNAs
are typically regulatory endogenous RNA molecules.
[0229] The terms "target microRNA" or "target miRNA" refer to a
microRNA with a biological role in human disease, e.g. an
upregulated, oncogenic miRNA or a tumor suppressor miRNA in cancer,
thereby being a target for therapeutic intervention of the disease
in question.
[0230] The terms "target gene" or "target mRNA" refer to regulatory
mRNA targets of microRNAs, in which said "target gene" or "target
mRNA" is regulated post-transcriptionally by the microRNA based on
near-perfect or perfect complementarity between the miRNA and its
target site resulting in target mRNA cleavage; or limited
complementarity, often conferred to complementarity between the
so-called seed sequence (nucleotides 2-7 of the miRNA) and the
target site resulting in translational inhibition of the target
mRNA.
[0231] In the context of the present invention the oligonucleotide
is single stranded, this refers to the situation where the
oligonucleotide is in the absence of a complementary
oligonucleotide--i.e. it is not a double stranded oligonucleotide
complex, such as an siRNA. In one embodiment, the composition
according of the invention does not comprise a further
oligonucleotide which has a region of complementarity with the
single stranded oligonucleotide of five or more consecutive
nucleobases, such as eight or more, or 12 or more of more
consecutive nucleobases. It is considered that the further
oligonucleotide is not covalently linked to the single stranded
oligonucleotide.
Modification of Nucleotides in Positions 3 to 8, Counting from the
3' End
[0232] In the following embodiments which refer to the modification
of nucleotides in positions 3 to 8, counting from the 3' end, the
LNA units may be replaced with other nucleotide analogues, such as
those referred to herein. "X" may, therefore be selected from the
group consisting of 2'-O-alkyl-RNA unit, 2'-OMe-RNA unit,
2'-amino-DNA unit, 2'-fluoro-DNA unit, LNA unit, PNA unit, HNA
unit, INA unit. "x" is preferably DNA or RNA, most preferably
DNA.
[0233] In an interesting embodiment of the invention, the
oligonucleotides of the invention are modified in positions 3 to 8,
counting from the 3' end. The design of this sequence may be
defined by the number of non-LNA units present or by the number of
LNA units present. In a preferred embodiment of the former, at
least one, such as one, of the nucleotides in positions three to
eight, counting from the 3' end, is a non-LNA unit. In another
embodiment, at least two, such as two, of the nucleotides in
positions three to eight, counting from the 3' end, are non-LNA
units. In yet another embodiment, at least three, such as three, of
the nucleotides in positions three to eight, counting from the 3'
end, are non-LNA units. In still another embodiment, at least four,
such as four, of the nucleotides in positions three to eight,
counting from the 3' end, are non-LNA units. In a further
embodiment, at least five, such as five, of the nucleotides in
positions three to eight, counting from the 3' end, are non-LNA
units. In yet a further embodiment, all six nucleotides in
positions three to eight, counting from the 3' end, are non-LNA
units. In a preferred embodiment, said non-LNA unit is a DNA
unit.
[0234] Alternatively defined, in a preferred embodiment, the
oligonucleotide according to the invention comprises at least one
LNA unit in positions three to eight, counting from the 3' end. In
an embodiment thereof, the oligonucleotide according to the present
invention comprises one LNA unit in positions three to eight,
counting from the 3' end. The substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
may be selected from the group consisting of Xxxxxx, xXxxxx,
xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein "X" denotes an LNA unit
and "x" denotes a non-LNA unit.
[0235] In another embodiment, the oligonucleotide according to the
present invention comprises at least two LNA units in positions
three to eight, counting from the 3' end. In an embodiment thereof,
the oligonucleotide according to the present invention comprises
two LNA units in positions three to eight, counting from the 3'
end. The substitution pattern for the nucleotides in positions
three to eight, counting from the 3' end, may be selected from the
group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx,
xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and
xxxxXX, wherein "X" denotes an LNA unit and "x" denotes a non-LNA
unit. In a preferred embodiment, the substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
is selected from the group consisting of XxXxxx, XxxXxx, XxxxXx,
XxxxxX, xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit. In a more
preferred embodiment, the substitution pattern for the nucleotides
in positions three to eight, counting from the 3' end, is selected
from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX
and xxxXxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit. In an even more preferred embodiment, the
substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of xXxXxx, xXxxXx and xxXxXx, wherein "X" denotes an LNA
unit and "x" denotes a non-LNA unit. In a most preferred
embodiment, the substitution pattern for the nucleotides in
positions three to eight, counting from the 3' end, is xXxXxx,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0236] In yet another embodiment, the oligonucleotide according to
the present invention comprises at least three LNA units in
positions three to eight, counting from the 3' end. In an
embodiment thereof, the oligonucleotide according to the present
invention comprises three LNA units in positions three to eight,
counting from the 3' end. The substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
may be selected from the group consisting of XXXxxx, xXXXxx,
xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX,
XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit. In
a preferred embodiment, the substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX,
xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX,
xxXxXX, xXxXxX and XxXxXx, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit. In a more preferred embodiment, the
substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and
xXxXxX, wherein "X" denotes an LNA unit and "x" denotes a non-LNA
unit. In an even more preferred embodiment, the substitution
pattern for the nucleotides in positions three to eight, counting
from the 3' end, is xXxXxX or XxXxXx, wherein "X" denotes an LNA
unit and "x" denotes a non-LNA unit. In a most preferred
embodiment, the substitution pattern for the nucleotides in
positions three to eight, counting from the 3' end, is xXxXxX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0237] In a further embodiment, the oligonucleotide according to
the present invention comprises at least four LNA units in
positions three to eight, counting from the 3' end. In an
embodiment thereof, the oligonucleotide according to the present
invention comprises four LNA units in positions three to eight,
counting from the 3' end. The substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
may be selected from the group consisting of xxXXXX, xXxXXX,
xXXxXX, xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX,
XXxXxX, XXxXXx, XXXxxX, XXXxXx and XXXXxx, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit.
[0238] In yet a further embodiment, the oligonucleotide according
to the present invention comprises at least five LNA units in
positions three to eight, counting from the 3' end. In an
embodiment thereof, the oligonucleotide according to the present
invention comprises five LNA units in positions three to eight,
counting from the 3' end. The substitution pattern for the
nucleotides in positions three to eight, counting from the 3' end,
may be selected from the group consisting of xXXXXX, XxXXXX,
XXxXXX, XXXxXX, XXXXxX and XXXXXx, wherein "X" denotes an LNA unit
and "x" denotes a non-LNA unit.
[0239] Preferably, the oligonucleotide according to the present
invention comprises one or two LNA units in positions three to
eight, counting from the 3' end. This is considered advantageous
for the stability of the A-helix formed by the oligo:microRNA
duplex, a duplex resembling an RNA:RNA duplex in structure.
[0240] In a preferred embodiment, said non-LNA unit is a DNA
unit.
Variation of the Length of the Oligonucleotides
[0241] The length of the oligonucleotides of the invention need not
match the length of the target microRNAs exactly. Accordingly, the
length of the oligonucleotides of the invention may vary. Indeed it
is considered advantageous to have short oligonucleotides, such as
between 10-17 or 10-16 nucleobases.
[0242] In one embodiment, the oligonucleotide according to the
present has a length of from 8 to 24 nucleotides, such as 10 to 24,
between 12 to 24 nucleotides, such as a length of 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides,
preferably a length of from 10-22, such as between 12 to 22
nucleotides, such as a length of 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21 or 22 nucleotides, more preferably a length of from
10-20, such as between 12 to 20 nucleotides, such as a length of
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, even more
preferably a length of from 10 to 19, such as between 12 to 19
nucleotides, such as a length of 10, 11, 12, 13, 14, 15, 16, 17, 18
or 19 nucleotides, e.g. a length of from 10 to 18, such as between
12 to 18 nucleotides, such as a length of 10, 11, 12, 13, 14, 15,
16, 17 or 18 nucleotides, more preferably a length of from 10-17,
such as from 12 to 17 nucleotides, such as a length of 10, 11, 12,
13, 14, 15, 16 or 17 nucleotides, most preferably a length of from
10 to 16, such as between 12 to 16 nucleotides, such as a length of
10, 11, 12, 13, 14, 15 or 16 nucleotides.
Modification of Nucleotides from Position 11, Counting from the 3'
End, to the 5' End
[0243] The substitution pattern for the nucleotides from position
11, counting from the 3' end, to the 5' end may include nucleotide
analogue units (such as LNA) or it may not. In a preferred
embodiment, the oligonucleotide according to the present invention
comprises at least one nucleotide analogue unit (such as LNA), such
as one nucleotide analogue unit, from position 11, counting from
the 3' end, to the 5' end. In another preferred embodiment, the
oligonucleotide according to the present invention comprises at
least two nucleotide analogue units, such as LNA units, such as two
nucleotide analogue units, from position 11, counting from the 3'
end, to the 5' end.
[0244] In the following embodiments which refer to the modification
of nucleotides in the nucleobases from position 11 to the 5' end of
the oligonucleotide, the LNA units may be replaced with other
nucleotide analogues, such as those referred to herein. "X" may,
therefore be selected from the group consisting of 2'-O-alkyl-RNA
unit, 2'-OMe-RNA unit, 2'-amino-DNA unit, 2'-fluoro-DNA unit, LNA
unit, PNA unit, HNA unit, INA unit. "x" is preferably DNA or RNA,
most preferably DNA.
[0245] In one embodiment, the oligonucleotide according to the
present invention has the following substitution pattern, which is
repeated from nucleotide eleven, counting from the 3' end, to the
5' end: xXxX or XxXx, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit. In another embodiment, the oligonucleotide
according to the present invention has the following substitution
pattern, which is repeated from nucleotide eleven, counting from
the 3' end, to the 5' end: XxxXxx, xXxxXx or xxXxxX, wherein "X"
denotes an LNA unit and "x" denotes a non-LNA unit. In yet another
embodiment, the oligonucleotide according to the present invention
has the following substitution pattern, which is repeated from
nucleotide eleven, counting from the 3' end, to the 5' end:
XxxxXxxx, xXxxxXxx, xxXxxxXx or xxxXxxxX, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit.
[0246] The specific substitution pattern for the nucleotides from
position 11, counting from the 3' end, to the 5' end depends on the
number of nucleotides in the oligonucleotides according to the
present invention. In a preferred embodiment, the oligonucleotide
according to the present invention contains 12 nucleotides and the
substitution pattern for positions 11 to 12, counting from the 3'
end, is selected from the group consisting of xX and Xx, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit. In a more
preferred embodiment thereof, the substitution pattern for
positions 11 to 12, counting from the 3' end, is xX, wherein "X"
denotes an LNA unit and "x" denotes a non-LNA unit. Alternatively,
no LNA units are present in positions 11 to 12, counting from the
3' end, i.e. the substitution pattern is xx.
[0247] In another preferred embodiment, the oligonucleotide
according to the present invention contains 13 nucleotides and the
substitution pattern for positions 11 to 13, counting from the 3'
end, is selected from the group consisting of Xxx, xXx, xxX, XXx,
XxX, xXX and XXX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit. In a more preferred embodiment thereof, the
substitution pattern for positions 11 to 13, counting from the 3'
end, is selected from the group consisting of xXx, xxX and xXX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit. In
a most preferred embodiment thereof, the substitution pattern for
positions 11 to 13, counting from the 3' end, is xxX, wherein "X"
denotes an LNA unit and "x" denotes a non-LNA unit. Alternatively,
no LNA units are present in positions 11 to 13, counting from the
3' end, i.e. the substitution pattern is xxx.
[0248] In yet another preferred embodiment, the oligonucleotide
according to the present invention contains 14 nucleotides and the
substitution pattern for positions 11 to 14, counting from the 3'
end, is selected from the group consisting of Xxxx, xXxx, xxXx,
xxxX, XXxx, XxXx, XxxX, xXXx, xXxX and xxXX, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit. In a preferred embodiment
thereof, the substitution pattern for positions 11 to 14, counting
from the 3' end, is selected from the group consisting of xXxx,
xxXx, xxxX, xXxX and xxXX, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit. In a more preferred embodiment thereof, the
substitution pattern for positions 11 to 14, counting from the 3'
end, is xXxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit. Alternatively, no LNA units are present in positions
11 to 14, counting from the 3' end, i.e. the substitution pattern
is xxxx
[0249] In a further preferred embodiment, the oligonucleotide
according to the present invention contains 15 nucleotides and the
substitution pattern for positions 11 to 15, counting from the 3'
end, is selected from the group consisting of Xxxxx, xXxxx, xxXxx,
xxxXx, xxxxX, XXxxx, XxXxx, XxxXx, XxxxX, xXXxx, xXxXx, xXxxX,
xxXXx, xxXxX, xxxXX and XxXxX, wherein "X" denotes an LNA unit and
"x" denotes a non-LNA unit. In a preferred embodiment thereof, the
substitution pattern for positions 11 to 15, counting from the 3'
end, is selected from the group consisting of xxXxx, XxXxx, XxxXx,
xXxXx, xXxxX and xxXxX, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit. In a more preferred embodiment thereof, the
substitution pattern for positions 11 to 15, counting from the 3'
end, is selected from the group consisting of xxXxx, xXxXx, xXxxX
and xxXxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit. In an even more preferred embodiment thereof, the
substitution pattern for positions 11 to 15, counting from the 3'
end, is selected from the group consisting of xXxxX and xxXxX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit. In
a most preferred embodiment, the substitution pattern for positions
11 to 15, counting from the 3' end, is xxXxX, wherein "X" denotes
an LNA unit and "x" denotes a non-LNA unit. Alternatively, no LNA
units are present in positions 11 to 15, counting from the 3' end,
i.e. the substitution pattern is xxxxx
[0250] In yet a further preferred embodiment, the oligonucleotide
according to the present invention contains 16 nucleotides and the
substitution pattern for positions 11 to 16, counting from the 3'
end, is selected from the group consisting of Xxxxxx, xXxxxx,
xxXxxx, xxxXxx, xxxxXx, xxxxxX, XXxxxx, XxXxxx, XxxXxx, XxxxXx,
XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX,
xxxXXx, xxxXxX, xxxxXX, XXXxxx, XXxXxx, XXxxXx, XXxxxX, XxXXxx,
XxXxXx, XxXxxX, XxxXXx, XxxXxX, XxxxXX, xXXXxx, xXXxXx, xXXxxX,
xXxXXx, xXxXxX, xXxxXX, xxXXXx, xxXXxX, xxXxXX and xxxXXX, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit. In a
preferred embodiment thereof, the substitution pattern for
positions 11 to 16, counting from the 3' end, is selected from the
group consisting of XxxXxx, xXxXxx, xXxxXx, xxXxXx, xxXxxX, XxXxXx,
XxXxxX, XxxXxX, xXxXxX, xXxxXX and xxXxXX, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit. In a more preferred
embodiment thereof, the substitution pattern for positions 11 to
16, counting from the 3' end, is selected from the group consisting
of xXxXxx, xXxxXx, xxXxXx, xxXxxX, xXxXxX, xXxxXX and xxXxXX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit. In
an even more preferred embodiment thereof, the substitution pattern
for positions 11 to 16, counting from the 3' end, is selected from
the group consisting of xxXxxX, xXxXxX, xXxxXX and xxXxXX, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit. In a still
more preferred embodiment thereof, the substitution pattern for
positions 11 to 16, counting from the 3' end, is selected from the
group consisting of xxXxxX and xXxXxX, wherein "X" denotes an LNA
unit and "x" denotes a non-LNA unit. In a most preferred embodiment
thereof, the substitution pattern for positions 11 to 16, counting
from the 3' end, is xxXxxX, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit. Alternatively, no LNA units are present in
positions 11 to 16, counting from the 3' end, i.e. the substitution
pattern is xxxxxx
[0251] In a preferred embodiment of the invention, the
oligonucleotide according to the present invention contains an LNA
unit at the 5' end. In another preferred embodiment, the
oligonucleotide according to the present invention contains an LNA
unit at the first two positions, counting from the 5' end.
[0252] In a particularly preferred embodiment, the oligonucleotide
according to the present invention contains 13 nucleotides and the
substitution pattern, starting from the 3' end, is XXxXxXxxXXxxX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit. The
preferred sequence for this embodiment, starting from the 3' end,
is CCtCaCacTGttA, wherein a capital letter denotes a nitrogenous
base in an LNA-unit and a small letter denotes a nitrogenous base
in a non-LNA unit.
[0253] In another particularly preferred embodiment, the
oligonucleotide according to the present invention contains 15
nucleotides and the substitution pattern, starting from the 3' end,
is XXxXxXxxXXxxXxX, wherein "X" denotes an LNA unit and "x" denotes
a non-LNA unit. The preferred sequence for this embodiment,
starting from the 3' end, is CCtCaCacTGttAcC, wherein a capital
letter denotes a nitrogenous base in an LNA-unit and a small letter
denotes a nitrogenous base in a non-LNA unit.
Modification of the Internucleoside Linkage Group
[0254] Typical internucleoside linkage groups in oligonucleotides
are phosphate groups, but these may be replaced by internucleoside
linkage groups differing from phosphate. In a further interesting
embodiment of the invention, the oligonucleotide of the invention
is modified in its internucleoside linkage group structure, i.e.
the modified oligonucleotide comprises an internucleoside linkage
group which differs from phosphate. Accordingly, in a preferred
embodiment, the oligonucleotide according to the present invention
comprises at least one internucleoside linkage group which differs
from phosphate.
[0255] Specific examples of internucleoside linkage groups which
differ from phosphate (--O--P(O).sub.2--O--) include
--O--P(O,S)--O--, --O--P(S).sub.2--O--, --O--P(O).sub.2--S--,
--O--P(O,S)--S--, --S--P(O).sub.2--S--, --O--PO(R.sup.H)--O--,
O--PO(OCH.sub.3)--O--, --O--PO(NR.sup.H)--O--,
--O--PO(OCH.sub.2CH.sub.2S--R)--O--, --O--PO(BH.sub.3)--O--,
--O--PO(NHR.sup.H)--O--, --O--P(O).sub.2--NR.sup.H--,
--NR.sup.H--P(O).sub.2--O--, --NR.sup.H--CO--O--,
--NR.sup.H--CO--NR.sup.H--, --O--CO--O--, --O--CO--NR.sup.H--,
--NR.sup.H--CO--CH.sub.2--, --O--CH.sub.2--CO--NR.sup.H--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--, --CO--NR.sup.H--CH.sub.2--,
--CH.sub.2--NR.sup.H--CO--, --O--CH.sub.2--CH.sub.2--S--,
--S--CH.sub.2--CH.sub.2--O--, --S--CH.sub.2--CH.sub.2--S--,
--CH.sub.2--SO.sub.2--CH.sub.2--, --CH.sub.2--CO--NR.sup.H--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--CO--CH.sub.2--NCH.sub.3--O--CH.sub.2---
, where R.sup.H is hydrogen or C.sub.1-4-alkyl.
[0256] When the internucleoside linkage group is modified, the
internucleoside linkage group is preferably a phosphorothioate
group (--O--P(O,S)--O--). In a preferred embodiment, all
internucleoside linkage groups of the oligonucleotides according to
the present invention are phosphorothioate.
The LNA Unit
[0257] In a preferred embodiment, the LNA unit has the general
chemical structure shown in Scheme 1 below:
##STR00002##
wherein [0258] X is selected from the group consisting of O, S and
NR.sup.H, where R.sup.H is H or C.sub.1-4-alkyl; [0259] Y is
(--CH.sub.2).sub.r, where r is an integer of 1-4; and [0260] B is a
nitrogenous base.
[0261] In a preferred embodiment of the invention, r is 1 or 2, in
particular 1, i.e. a preferred LNA unit has the chemical structure
shown in Scheme 2 below:
##STR00003##
wherein X and B are as defined above.
[0262] In an interesting embodiment, the LNA units incorporated in
the oligonucleotides of the invention are independently selected
from the group consisting of thio-LNA units, amino-LNA units and
oxy-LNA units.
[0263] Thus, the thio-LNA unit may have the chemical structure
shown in Scheme 3 below:
##STR00004##
wherein B is as defined above.
[0264] Preferably, the thio-LNA unit is in its beta-D-form, i.e.
having the structure shown in 3A above.
[0265] likewise, the amino-LNA unit may have the chemical structure
shown in Scheme 4 below:
##STR00005##
wherein B and R.sup.H are as defined above.
[0266] Preferably, the amino-LNA unit is in its beta-D-form, i.e.
having the structure shown in 4A above.
[0267] The oxy-LNA unit may have the chemical structure shown in
Scheme 5 below:
##STR00006##
wherein B is as defined above.
[0268] Preferably, the oxy-LNA unit is in its beta-D-form, i.e.
having the structure shown in 5A above.
[0269] As indicated above, B is a nitrogenous base which may be of
natural or non-natural origin. Specific examples of nitrogenous
bases include adenine (A), cytosine (C), 5-methylcytosine
(.sup.MeC), isocytosine, pseudoisocytosine, guanine (G), thymine
(T), uracil (U), 5-bromouracil, 5-propynyluracil,
5-propyny-6,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine,
inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine,
7-propyne-7-deazaguanine and 2-chloro-6-aminopurine.
Terminal Groups
[0270] Specific examples of terminal groups include terminal groups
selected from the group consisting of hydrogen, azido, halogen,
cyano, nitro, hydroxy, Prot-O--, mercapto, Prot-S--,
C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate including protected
monophosphate, monothiophosphate including protected
monothiophosphate, diphosphate including protected diphosphate,
dithiophosphate including protected dithiophosphate, triphosphate
including protected triphosphate, trithiophosphate including
protected trithiophosphate, where Prot is a protection group for
--OH, --SH and --NH(R.sup.H), and R.sup.H is hydrogen or
C.sub.1-6-alkyl.
[0271] Examples of phosphate protection groups include
S-acetylthioethyl (SATE) and S-pivaloylthioethyl
(t-butyl-SATE).
[0272] Still further examples of terminal groups include DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, ligands, carboxy,
sulphono, hydroxymethyl, Prot-O--CH.sub.2--, Act-O--CH.sub.2--,
aminomethyl, Prot-N(R.sup.H)--CH.sub.2--,
Act-N(R.sup.H)--CH.sub.2--, carboxymethyl, sulphonomethyl, where
Prot is a protection group for --OH, --SH and --NH(R.sup.H), and
Act is an activation group for --OH, --SH, and --NH(R.sup.H), and
R.sup.H is hydrogen or C.sub.1-6-alkyl.
[0273] Examples of protection groups for --OH and --SH groups
include substituted trityl, such as 4,4'-dimethoxytrityloxy (DMT),
4-monomethoxytrityloxy (MMT); trityloxy, optionally substituted
9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted
methoxytetrahydro-pyranyloxy (mthp); silyloxy, such as
trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),
tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy,
phenyldimethylsilyloxy; tert-butylethers; acetals (including two
hydroxy groups); acyloxy, such as acetyl or halogen-substituted
acetyls, e.g. chloroacetyloxy or fluoroacetyloxy, isobutyryloxy,
pivaloyloxy, benzoyloxy and substituted benzoyls, methoxymethyloxy
(MOM), benzyl ethers or substituted benzyl ethers such as
2,6-dichlorobenzyloxy (2,6-Cl.sub.2Bzl). Moreover, when Z or Z* is
hydroxyl they may be protected by attachment to a solid support,
optionally through a linker.
[0274] Examples of amine protection groups include
fluorenylmethoxycarbonylamino (Fmoc), tert-butyloxycarbonylamino
(BOC), trifluoroacetylamino, allyloxycarbonylamino (alloc, AOC),
Z-benzyloxycarbonylamino (Cbz), substituted benzyloxycarbonylamino,
such as 2-chloro benzyloxycarbonylamino (2-ClZ),
monomethoxytritylamino (MMT), di methoxytritylamino (DMT),
phthaloylamino, and 9-(9-phenyl)xanthenylamino (pixyl).
[0275] In the present context, the term "phosphoramidite" means a
group of the formula --P(OR.sup.x)--N(R.sup.y).sub.2, wherein
R.sup.x designates an optionally substituted alkyl group, e.g.
methyl, 2-cyanoethyl, or benzyl, and each of R.sup.y designates
optionally substituted alkyl groups, e.g. ethyl or isopropyl, or
the group --N(R.sup.y).sub.2 forms a morpholino group
(--N(CH.sub.2CH.sub.2).sub.2O). R.sup.x preferably designates
2-cyanoethyl and the two R.sup.y are preferably identical and
designates isopropyl. Accordingly, a particularly preferred
phosphoramidite is
N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.
[0276] The most preferred terminal groups are hydroxy, mercapto and
amino, in particular hydroxy.
Designs for Specific microRNAs
[0277] The following table provides examples of oligonucleotide
according to the present invention, such as those used in
pharmaceutical compositions, as compared to prior art type of
molecules.
TABLE-US-00001 target: hsa-miR-122a MIMAT0000421 SEQ ID
uggagugugacaaugguguuugu SEQ ID NO 535 screened in HUH-7 cell line
expressing miR-122 Oligo #, target microRNA, oligo sequence Design
3962: miR-122 5'-ACAAacaccattgtcacacTCCA-3' Full complement, gap
SEQ ID NO 536 3965: miR-122 5'-acaaacACCATTGTcacactcca-3' Full
complement, block SEQ ID NO 537 3972: miR-122
5'-acAaaCacCatTgtCacActCca-3' Full complement, LNA_3 SEQ ID NO 538
3549 (3649): miR-122 5'-CcAttGTcaCaCtCC-3' New design SEQ ID NO 539
3975: miR-122 5'-CcAtTGTcaCACtCC-3' Enhanced new design SEQ ID NO
540 3975': miR-122 5'-ATTGTcACACtCC-3' ED-13 mer SEQ ID NO 541
3975'': miR-122 5'-TGTcACACtCC-3' ED-11 mer SEQ ID NO 542 3549'
(3649): miR-122 5' New design-2'MOE SEQ ID NO 543
CC.sup.MAT.sup.MT.sup.MGTC.sup.MA.sup.MCA.sup.MCT.sup.MCC-3' 3549''
(3649): miR-122 5' New design-2'Fluoro SEQ ID NO 544
CC.sup.FAT.sup.FT.sup.FGTC.sup.FA.sup.FCA.sup.FCT.sup.FCC-3'
target: hsa-miR-19b MIMAT0000074 ugugcaaauccaugcaaaacuga SEQ ID NO
545 screened HeLa cell line expressing miR-19b Oligo #, target
microRNA, oligo sequence Design 3963: miR-19b 5'-TCAG
gcatggatttgCACA-3' Full complement, gap SEQ ID NO 546 3967: miR-19b
5'-tcagttTTGCATGGatttgcaca-3' Full complement, block SEQ ID NO 547
3973: miR-19b 5'-tcAgtTttGcaTggAttTgcAca-3' Full complement, LNA_3
SEQ ID NO 548 3560: miR-19b 5'-TgCatGGatTtGcAC-3' New design SEQ ID
NO 549 3976: miR-19b 5'-TgCaTGGatTTGcAC-3' Enhanced new design SEQ
ID NO 550 3976': miR-19b 5'-CaTGGaTTTGcAC-3' ED-13 mer SEQ ID NO
551 3976'': miR-19b 5'TGGaTTTGcAC-3' ED-11 mer SEQ ID NO 552 3560':
miR-19b 5'
TG.sup.MCA.sup.MT.sup.MGGA.sup.MT.sup.MTT.sup.MGC.sup.MAC-3' New
design-2'MOE SEQ ID NO 553 3560'': miR-19b
5'-TG.sup.FCA.sup.FT.sup.FGGA.sup.FT.sup.FTT.sup.FGC.sup.FAC-3' New
design-2'MOE SEQ ID NO 554 target: hsa-miR-155 MIMAT0000646
uuaaugcuaaucgugauagggg SEQ ID NO 555 screen in 518A2 cell line
expressing miR-155 Oligo #, target microRNA, oligo sequence Design
3964: miR-155 5'-CCCCtatcacgattagcaTTAA-3' Full complement, gap SEQ
ID NO 556 3968: miR-155 5'-cccctaTCACGATTagcattaa-3' Full
complement, block SEQ ID NO 557 3974: miR-155
5'-cCccTatCacGatTagCatTaa-3' Full complement, LNA_3 SEQ ID NO 558
3758: miR-155 5'-TcAcgATtaGcAtTA-3' New design SEQ ID NO 559 3818:
miR-155 5'-TcAcGATtaGCAtTA-3' Enhanced new design SEQ ID NO 560
3818': miR-155 5'-ACGATtAGCAtTA-3' ED-13 mer SEQ ID NO 561 3818'':
miR-155 5'-GATtAGCaTTA-3' ED-11 mer SEQ ID NO 562 3758': miR-155
5'-TC.sup.MAC.sup.MG.sup.MATTA.sup.MGC.sup.MA.sup.MTA-3' New
design-2'MOE SEQ ID NO 563 3758'': miR-155
5'-TC.sup.FAC.sup.FG.sup.FATT.sup.FA.sup.FGC.sup.FAT.sup.FTA-3' New
design-2'Fluoro SEQ ID NO 564 target: hsa-miR-21 MIMAT0000076
uagcuuaucagacugauguuga SEQ ID NO 565 miR-21
5'-TCAAcatcagtctgataaGCTA-3' Full complement, gap SEQ ID NO 566
miR-21 5'-tcaacaTCAGTCTGataagcta-3' Full complement, block SEQ ID
NO 567 miR-21 5'-tcAtcAtcAgtCtgAtaAGcTta-3' Full complement, LNA_3
SEQ ID NO 568 miR-21 5'- TcAgtCTgaTaAgCT-3' New design SEQ ID NO
569 miR-21 5'- TcAgTCTgaTAAgCT-3'- Enhanced new design SEQ ID NO
570 miR-21 5'- AGTCTgATAAgCT-3'- ED-13 mer SEQ ID NO 571 miR-21 5'-
TCTgAtAAGCT-3'- ED-11 mer SEQ ID NO 572 miR-21
5'-TC.sup.MAG.sup.MT.sup.MCTG.sup.MA.sup.MTA.sup.MAG.sup.MCT-3' New
design-2'MOE SEQ ID NO 573 miR-21
5'-TC.sup.FAG.sup.FT.sup.FCTG.sup.FA.sup.FTA.sup.FAG.sup.FCT-3' New
design-2'Fluoro SEQ ID NO 574 target: hsa-miR-375 MIMAT0000728
uuuguucguucggcucgcguga SEQ ID NO 575 miR-375
5'-TCTCgcgtgccgttcgttCTTT-3' Full complement, gap SEQ ID NO 576
miR-375 5'-tctcgcGTGCCGTTcgttcttt-3' Full complement, block SEQ ID
NO 577 miR-375 5'-tcTcgCgtGccGttCgtTctTt-3' Full complement, LNA_3
SEQ ID NO 578 miR-375 5'-GtGccGTtcGtTcTT 3' New design SEQ ID NO
579 miR-375 5'-GtGcCGTtcGTTcTT 3' Enhanced new design SEQ ID NO 580
miR-375 5'-GCCGTtCgTTCTT 3' ED-13 mer SEQ ID NO 581 miR-375
5'-CGTTcGTTCTT 3' ED-11 mer SEQ ID NO 582 miR-375
5'-GT.sup.MGC.sup.MC.sup.MGTT.sup.MC.sup.MGT.sup.MTC.sup.MTT 3' New
design-2'MOE SEQ ID NO 583 miR-375
5'-GT.sup.FGC.sup.FC.sup.FGTT.sup.FC.sup.FGT.sup.FTC.sup.FTT 3' New
design-2'Fluoro SEQ ID NO 584
[0278] Capital Letters without a superscript M or F, refer to LNA
units. Lower case=DNA, except for lower case in bold=RNA. The LNA
cytosines may optionally be methylated). Capital letters followed
by a superscript M refer to 2'OME RNA units, Capital letters
followed by a superscript F refer to 2' fluoro DNA units, lowercase
letter refer to DNA. The above oligos may in one embodiment be
entirely phosphorothioate, but other nucleobase linkages as herein
described bay be used. In one embodiment the nucleobase linkages
are all phosphodiester. It is considered that for use within the
brain/spinal cord it is preferable to use phosphodiester linkages,
for example for the use of antimiRs targeting miR21.
[0279] Table 2 below provides non-limiting examples of
oligonucleotide designs against known human microRNA sequences in
miRBase microRNA database version 8.1.
[0280] The oligonucleotides according to the invention, such as
those disclosed in table 2 may, in one embodiment, have a sequence
of nucleobases 5'-3' selected form the group consisting of:
LdLddLLddLdLdLL (New design) LdLdLLLddLLLdLL (Enhanced new design)
LMLMMLLMMLMLMLL (New design--2'MOE) LMLMLLLMMLLLMLL (Enhanced new
design--2'MOE) LFLFFLLFFLFLFLL (New design--2' Fluoro)
LFLFLLLFFLLLFLL (Enhanced new design--2' Fluoro)
LddLddLddL(d)(d)(L)(d)(d)(L)(d) `Every third`
dLddLddLdd(L)(d)(d)(L)(d)(d)(L) `Every third`
ddLddLddLd(d)(L)(d)(d)(L)(d)(d) `Every third`
LMMLMMLMML(M)(M)(L)(M)(M)(L)(M) `Every third`
MLMMLMMLMM(L)(M)(M)(L)(M)(M)(L) `Every third`
MMLMMLMMLM(M)(L)(M)(M)(L)(M)(M) `Every third`
LFFLFFLFFL(F)(F)(L)(F)(F)(L)(F) `Every third`
FLFFLFFLFF(L)(F)(F)(L)(F)(F)(L) `Every third`
FFLFFLFFLF(F)(L)(F)(F)(L)(F)(F) `Every third`
dLdLdLdLdL(d)(L)(d)(L)(d)(L)(d) `Every second`
LdLdLdLdL(d)(L)(d)(L)(d)(L)(d)(L) `Every second`
MLMLMLMLML(M)(L)(M)(L)(M)(L)(M) `Every second`
LMLMLMLML(M)(L)(M)(L)(M)(L)(M)(L) `Every second`
FLFLFLFLFL(F)(L)(F)(L)(F)(L)(F) `Every second`
LFLFLFLFL(F)(L)(F)(L)(F)(L)(F)(L) `Every second`
[0281] Wherein L=LNA unit, d=DNA units, M=2'MOE RNA, F=2'Fluoro and
residues in brackets are optional
Conjugates
[0282] The invention also provides for conjugates comprising the
oligonucleotide according of the invention.
[0283] In one embodiment of the invention the oligomeric compound
is linked to ligands/conjugates, which may be used, e.g. to
increase the cellular uptake of antisense oligonucleotides. This
conjugation can take place at the terminal positions 5'/3'-OH but
the ligands may also take place at the sugars and/or the bases. In
particular, the growth factor to which the antisense
oligonucleotide may be conjugated, may comprise transferrin or
folate. Transferrin-polylysine-oligonucleotide complexes or
folate-polylysine-oligonucleotide complexes may be prepared for
uptake by cells expressing high levels of transferrin or folate
receptor. Other examples of conjugates/ligands are cholesterol
moieties, duplex intercalators such as acridine, poly-L-lysine,
"end-capping" with one or more nuclease-resistant linkage groups
such as phosphoromonothioate, and the like. The invention also
provides for a conjugate comprising the compound according to the
invention as herein described, and at least one non-nucleotide or
non-polynucleotide moiety covalently attached to said compound.
Therefore, in one embodiment where the compound of the invention
consists of s specified nucleic acid, as herein disclosed, the
compound may also comprise at least one non-nucleotide or
non-polynucleotide moiety (e.g. not comprising one or more
nucleotides or nucleotide analogues) covalently attached to said
compound. The non-nucleobase moiety may for instance be or comprise
a sterol such as cholesterol.
[0284] Therefore, it will be recognised that the oligonucleotide of
the invention, such as the oligonucleotide used in pharmaceutical
(therapeutic) formulations may comprise further non-nucleobase
components, such as the conjugates herein defined.
Therapy and Pharmaceutical Compositions
[0285] As explained initially, the oligonucleotides of the
invention will constitute suitable drugs with improved properties.
The design of a potent and safe drug requires the fine-tuning of
various parameters such as affinity/specificity, stability in
biological fluids, cellular uptake, mode of action, pharmacokinetic
properties and toxicity.
[0286] Accordingly, in a further aspect the present invention
relates to a pharmaceutical composition comprising an
oligonucleotide according to the invention and a pharmaceutically
acceptable diluent, carrier or adjuvant. Preferably said carrier is
saline of buffered saline.
[0287] In a still further aspect the present invention relates to
an oligonucleotide according to the present invention for use as a
medicament.
[0288] As will be understood, dosing is dependent on severity and
responsiveness of the disease state to be treated, and the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Optimum dosages may vary depending on the relative potency of
individual oligonucleotides. Generally it can be estimated based on
EC50s found to be effective in in vitro and in vivo animal models.
In general, dosage is from 0.01 .mu.g to 1g per kg of body weight,
and may be given once or more daily, weekly, monthly or yearly, or
even once every 2 to 10 years or by continuous infusion for hours
up to several months. The repetition rates for dosing can be
estimated based on measured residence times and concentrations of
the drug in bodily fluids or tissues. Following successful
treatment, it may be desirable to have the patient undergo
maintenance therapy to prevent the recurrence of the disease
state.
Pharmaceutical Compositions
[0289] As indicated above, the invention also relates to a
pharmaceutical composition, which comprises at least one
oligonucleotide of the invention as an active ingredient. It should
be understood that the pharmaceutical composition according to the
invention optionally comprises a pharmaceutical carrier, and that
the pharmaceutical composition optionally comprises further
compounds, such as chemotherapeutic compounds, anti-inflammatory
compounds, antiviral compounds and/or immuno-modulating
compounds.
[0290] The oligonucleotides of the invention can be used "as is" or
in form of a variety of pharmaceutically acceptable salts. As used
herein, the term "pharmaceutically acceptable salts" refers to
salts that retain the desired biological activity of the
herein-identified oligonucleotides and exhibit minimal undesired
toxicological effects. Non-limiting examples of such salts can be
formed with organic amino acid and base addition salts formed with
metal cations such as zinc, calcium, bismuth, barium, magnesium,
aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and
the like, or with a cation formed from ammonia,
N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or
ethylenediamine.
[0291] In one embodiment of the invention, the oligonucleotide may
be in the form of a pro-drug. Oligonucleotides are by virtue
negatively charged ions. Due to the lipophilic nature of cell
membranes the cellular uptake of oligonucleotides are reduced
compared to neutral or lipophilic equivalents. This polarity
"hindrance" can be avoided by using the pro-drug approach (see e.g.
Crooke, R. M. (1998) in Crooke, S. T. Antisense research and
Application. Springer-Verlag, Berlin, Germany, vol. 131, pp.
103-140). Pharmaceutically acceptable binding agents and adjuvants
may comprise part of the formulated drug.
[0292] Examples of delivery methods for delivery of the therapeutic
agents described herein, as well as details of pharmaceutical
formulations, salts, may are well described elsewhere for example
in U.S. provisional application 60/838,710 and 60/788,995, which
are hereby incorporated by reference, and Danish applications, PA
2006 00615 which is also hereby incorporated by reference.
[0293] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids. Delivery of drug to tumour tissue may
be enhanced by carrier-mediated delivery including, but not limited
to, cationic liposomes, cyclodextrins, porphyrin derivatives,
branched chain dendrimers, polyethylenimine polymers, nanoparticles
and microspheres (Dass C R. J Pharm Phammacol 2002; 54(1):3-27).
The pharmaceutical formulations of the present invention, which may
conveniently be presented in unit dosage form, may be prepared
according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product. The compositions of the present invention may be
formulated into any of many possible dosage forms such as, but not
limited to, tablets, capsules, gel capsules, liquid syrups, soft
gels and suppositories. The compositions of the present invention
may also be formulated as suspensions in aqueous, non-aqueous or
mixed media. Aqueous suspensions may further contain substances
which increase the viscosity of the suspension including, for
example, sodium carboxymethyl-cellulose, sorbitol and/or dextran.
The suspension may also contain stabilizers. The compounds of the
invention may also be conjugated to active drug substances, for
example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic. In another embodiment, compositions
of the invention may contain one or more oligonucleotide compounds,
targeted to a first microRNA and one or more additional
oligonucleotide compounds targeted to a second microRNA target. Two
or more combined compounds may be used together or
sequentially.
[0294] The compounds disclosed herein are useful for a number of
therapeutic applications as indicated above. In general,
therapeutic methods of the invention include administration of a
therapeutically effective amount of an oligonucleotide to a mammal,
particularly a human. In a certain embodiment, the present
invention provides pharmaceutical compositions containing (a) one
or more compounds of the invention, and (b) one or more
chemotherapeutic agents. When used with the compounds of the
invention, such chemotherapeutic agents may be used individually,
sequentially, or in combination with one or more other such
chemotherapeutic agents or in combination with radiotherapy. All
chemotherapeutic agents known to a person skilled in the art are
here incorporated as combination treatments with compound according
to the invention. Other active agents, such as anti-inflammatory
drugs, including but not limited to nonsteroidal anti-inflammatory
drugs and corticosteroids, antiviral drugs, and immuno-modulating
drugs may also be combined in compositions of the invention. Two or
more combined compounds may be used together or sequentially.
[0295] Examples of therapeutic indications which may be treated by
the pharmaceutical compositions of the invention:
TABLE-US-00002 microRNA Possible medical indications miR-21
Glioblastoma, breast cancer miR-122 hypercholesterolemia, hepatitis
C, hemochromatosis miR-19b lymphoma and other tumour types miR-155
lymphoma, breast and lung cancer miR-375 diabetes, metabolic
disorders miR-181 myoblast differentiation, auto immune
disorders
[0296] Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been
indicated as a target of miR-21. Myotrophin (mtpn) mRNA has been
indicated as a target of miR 375.
[0297] In an even further aspect, the present invention relates to
the use of an oligonucleotide according to the invention for the
manufacture of a medicament for the treatment of a disease selected
from the group consisting of: atherosclerosis, hypercholesterolemia
and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma,
lung cancer; diabetes, metabolic disorders; myoblast
differentiation; immune disorders.
[0298] The invention further refers to an oligonucleotides
according to the invention for the use in the treatment of from a
disease selected from the group consisting of: atherosclerosis,
hypercholesterolemia and hyperlipidemia; cancer, glioblastoma,
breast cancer, lymphoma, lung cancer; diabetes, metabolic
disorders; myoblast differentiation; immune disorders.
[0299] The invention provides for a method of treating a subject
suffering from a disease or condition selected from the group
consisting of: atherosclerosis, hypercholesterolemia and
hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung
cancer; diabetes, metabolic disorders; myoblast differentiation;
immune disorders, the method comprising the step of administering
an oligonucleotide or pharmaceutical composition of the invention
to the subject in need thereof.
Cancer
[0300] In an even further aspect, the present invention relates to
the use of an oligonucleotide according to the invention for the
manufacture of a medicament for the treatment of cancer. In another
aspect, the present invention concerns a method for treatment of,
or prophylaxis against, cancer, said method comprising
administering an oligonucleotide of the invention or a
pharmaceutical composition of the invention to a patient in need
thereof.
[0301] Such cancers may include lymphoreticular neoplasia,
lymphoblastic leukemia, brain tumors, gastric tumors,
plasmacytomas, multiple myeloma, leukemia, connective tissue
tumors, lymphomas, and solid tumors.
[0302] In the use of a compound of the invention for the
manufacture of a medicament for the treatment of cancer, said
cancer may suitably be in the form of a solid tumor. Analogously,
in the method for treating cancer disclosed herein said cancer may
suitably be in the form of a solid tumor.
[0303] Furthermore, said cancer is also suitably a carcinoma. The
carcinoma is typically selected from the group consisting of
malignant melanoma, basal cell carcinoma, ovarian carcinoma, breast
carcinoma, non-small cell lung cancer, renal cell carcinoma,
bladder carcinoma, recurrent superficial bladder cancer, stomach
carcinoma, prostatic carcinoma, pancreatic carcinoma, lung
carcinoma, cervical carcinoma, cervical dysplasia, laryngeal
papillomatosis, colon carcinoma, colorectal carcinoma and carcinoid
tumors. More typically, said carcinoma is selected from the group
consisting of malignant melanoma, non-small cell lung cancer,
breast carcinoma, colon carcinoma and renal cell carcinoma. The
malignant melanoma is typically selected from the group consisting
of superficial spreading melanoma, nodular melanoma, lentigo
maligna melanoma, acral melagnoma, amelanotic melanoma and
desmoplastic melanoma.
[0304] Alternatively, the cancer may suitably be a sarcoma. The
sarcoma is typically in the form selected from the group consisting
of osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous
histiocytoma, fibrosarcoma and Kaposi's sarcoma.
[0305] Alternatively, the cancer may suitably be a glioma.
[0306] A further embodiment is directed to the use of an
oligonucleotide according to the invention for the manufacture of a
medicament for the treatment of cancer, wherein said medicament
further comprises a chemotherapeutic agent selected from the group
consisting of adrenocorticosteroids, such as prednisone,
dexamethasone or decadron; altretamine (hexylen, hexamethylmelamine
(HMM)); amifostine (ethyol); aminoglutethimide (cytadren);
amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as
testosterone; asparaginase (elspar); bacillus calmette-gurin;
bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran);
carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil
(leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin);
cisplatin (platinol); cytosine arabinoside (cytarabine);
dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen);
daunorubicin (cerubidine); docetaxel (taxotere); doxorubicin
(adriomycin); epirubicin; estramustine (emcyt); estrogens, such as
diethylstilbestrol (DES); etopside (VP-16, VePesid, etopophos);
fludarabine (fludara); flutamide (eulexin); 5-FUDR (floxuridine);
5-fluorouracil (5-FU); gemcitabine (gemzar); goserelin (zodalex);
herceptin (trastuzumab); hydroxyurea (hydrea); idarubicin
(idamycin); ifosfamide; IL-2 (proleukin, aldesleukin); interferon
alpha (intron A, roferon A); irinotecan (camptosar); leuprolide
(lupron); levamisole (ergamisole); lomustine (CCNU);
mechlorathamine (mustargen, nitrogen mustard); melphalan (alkeran);
mercaptopurine (purinethol, 6-MP); methotrexate (mexate);
mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide
(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin
(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;
tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon,
VM-26); thiotepa; topotecan (hycamtin); tretinoin (vesanoid,
all-trans retinoic acid); vinblastine (valban); vincristine
(oncovin) and vinorelbine (navelbine). Suitably, the further
chemotherapeutic agent is selected from taxanes such as Taxol,
Paclitaxel or Docetaxel.
[0307] Similarly, the invention is further directed to the use of
an oligonucleotide according to the invention for the manufacture
of a medicament for the treatment of cancer, wherein said treatment
further comprises the administration of a further chemotherapeutic
agent selected from the group consisting of adrenocorticosteroids,
such as prednisone, dexamethasone or decadron; altretamine
(hexylen, hexamethylmelamine (HMM)); amifostine (ethyol);
aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole
(arimidex); androgens, such as testosterone; asparaginase (elspar);
bacillus calmette-gurin; bicalutamide (casodex); bleomycin
(blenoxane); busulfan (myleran); carboplatin (paraplatin);
carmustine (BCNU, BiCNU); chlorambucil (leukeran);
chlorodeoxyadenosine (2-CDA, cladribine, leustatin); cisplatin
(platinol); cytosine arabinoside (cytarabine); dacarbazine (DTIC);
dactinomycin (actinomycin-D, cosmegen); daunorubicin (cerubidine);
docetaxel (taxotere); doxorubicin (adriomycin); epirubicin;
estramustine (emcyt); estrogens, such as diethylstilbestrol (DES);
etopside (VP-16, VePesid, etopophos); fludarabine (fludara);
flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);
gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);
hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2
(proleukin, aldesleukin); interferon alpha (intron A, roferon A);
irinotecan (camptosar); leuprolide (lupron); levamisole
(ergamisole); lomustine (CCNU); mechlorathamine (mustargen,
nitrogen mustard); melphalan (alkeran); mercaptopurine (purinethol,
6-MP); methotrexate (mexate); mitomycin-C (mutamucin); mitoxantrone
(novantrone); octreotide (sandostatin); pentostatin
(2-deoxycoformycin, nipent); plicamycin (mithramycin, mithracin);
prorocarbazine (matulane); streptozocin; tamoxifin (nolvadex);
taxol (paclitaxel); teniposide (vumon, VM-26); thiotepa; topotecan
(hycamtin); tretinoin (vesanoid, all-trans retinoic acid);
vinblastine (valban); vincristine (oncovin) and vinorelbine
(navelbine). Suitably, said treatment further comprises the
administration of a further chemotherapeutic agent selected from
taxanes, such as Taxol, Paclitaxel or Docetaxel.
[0308] Alternatively stated, the invention is furthermore directed
to a method for treating cancer, said method comprising
administering an oligonucleotide of the invention or a
pharmaceutical composition according to the invention to a patient
in need thereof and further comprising the administration of a
further chemotherapeutic agent. Said further administration may be
such that the further chemotherapeutic agent is conjugated to the
compound of the invention, is present in the pharmaceutical
composition, or is administered in a separate formulation.
Infectious Diseases
[0309] It is contemplated that the compounds of the invention may
be broadly applicable to a broad range of infectious diseases, such
as diphtheria, tetanus, pertussis, polio, hepatitis B, hepatitis C,
hemophilus influenza, measles, mumps, and rubella.
[0310] Hsa-miR122 is indicated in hepatitis C infection and as such
oligonucleotides according to the invention which target miR-122
may be used to treat Hepatitis C infection.
[0311] Accordingly, in yet another aspect the present invention
relates the use of an oligonucleotide according to the invention
for the manufacture of a medicament for the treatment of an
infectious disease, as well as to a method for treating an
infectious disease, said method comprising administering an
oligonucleotide according to the invention or a pharmaceutical
composition according to the invention to a patient in need
thereof.
Inflammatory Diseases
[0312] The inflammatory response is an essential mechanism of
defense of the organism against the attack of infectious agents,
and it is also implicated in the pathogenesis of many acute and
chronic diseases, including autoimmune disorders. In spite of being
needed to fight pathogens, the effects of an inflammatory burst can
be devastating. It is therefore often necessary to restrict the
symptomatology of inflammation with the use of anti-inflammatory
drugs. Inflammation is a complex process normally triggered by
tissue injury that includes activation of a large array of enzymes,
the increase in vascular permeability and extravasation of blood
fluids, cell migration and release of chemical mediators, all aimed
to both destroy and repair the injured tissue.
[0313] In yet another aspect, the present invention relates to the
use of an oligonucleotide according to the invention for the
manufacture of a medicament for the treatment of an inflammatory
disease, as well as to a method for treating an inflammatory
disease, said method comprising administering an oligonucleotide
according to the invention or a pharmaceutical composition
according to the invention to a patient in need thereof.
[0314] In one preferred embodiment of the invention, the
inflammatory disease is a rheumatic disease and/or a connective
tissue diseases, such as rheumatoid arthritis, systemic lupus
erythematous (SLE) or Lupus, scleroderma, polymyositis,
inflammatory bowel disease, dermatomyositis, ulcerative colitis,
Crohn's disease, vasculitis, psoriatic arthritis, exfoliative
psoriatic dermatitis, pemphigus vulgaris and Sjorgren's syndrome,
in particular inflammatory bowel disease and Crohn's disease.
[0315] Alternatively, the inflammatory disease may be a
non-rheumatic inflammation, like bursitis, synovitis, capsulitis,
tendinitis and/or other inflammatory lesions of traumatic and/or
sportive origin.
Metabolic Diseases
[0316] A metabolic disease is a disorder caused by the accumulation
of chemicals produced naturally in the body. These diseases are
usually serious, some even life threatening. Others may slow
physical development or cause mental retardation. Most infants with
these disorders, at first, show no obvious signs of disease. Proper
screening at birth can often discover these problems. With early
diagnosis and treatment, metabolic diseases can often be managed
effectively.
[0317] In yet another aspect, the present invention relates to the
use of an oligonucleotide according to the invention or a conjugate
thereof for the manufacture of a medicament for the treatment of a
metabolic disease, as well as to a method for treating a metabolic
disease, said method comprising administering an oligonucleotide
according to the invention or a conjugate thereof, or a
pharmaceutical composition according to the invention to a patient
in need thereof.
[0318] In one preferred embodiment of the invention, the metabolic
disease is selected from the group consisting of Amyloidosis,
Biotimidase, OMIM (Online Mendelian Inheritance in Man), Crigler
Najjar Syndrome, Diabetes, Fabry Support & Information Group,
Fatty acid Oxidation Disorders, Galactosemia, Glucose-6-Phosphate
Dehydrogenase (G6PD) deficiency, Glutaric aciduria, International
Organization of Glutaric Acidemia, Glutaric Acidemia Type I,
Glutaric Acidemia, Type II, Glutaric Acidemia Type I, Glutaric
Acidemia Type-II, F-HYPDRR-Familial Hypophosphatemia, Vitamin D
Resistant Rickets, Krabbe Disease, Long chain 3 hydroxyacyl CoA
dehydrogenase deficiency (LCHAD), Mannosidosis Group, Maple Syrup
Urine Disease, Mitochondrial disorders, Mucopolysaccharidosis
Syndromes: Niemann Pick, Organic acidemias, PKU, Pompe disease,
Porphyria, Metabolic Syndrome, Hyperlipidemia and inherited lipid
disorders, Trimethylaminuria: the fish malodor syndrome, and Urea
cycle disorders.
Liver Disorders
[0319] In yet another aspect, the present invention relates to the
use of an oligonucleotide according to the invention or a conjugate
thereof for the manufacture of a medicament for the treatment of a
liver disorder, as well as to a method for treating a liver
disorder, said method comprising administering an oligonucleotide
according to the invention or a conjugate thereof, or a
pharmaceutical composition according to the invention to a patient
in need thereof.
[0320] In one preferred embodiment of the invention, the liver
disorder is selected from the group consisting of Biliary Atresia,
Alagille Syndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal
Hepatitis, and Wilson Disease.
Other Uses
[0321] The oligonucleotides of the present invention can be
utilized for as research reagents for diagnostics, therapeutics and
prophylaxis. In research, the oligonucleotide may be used to
specifically inhibit the synthesis of target genes in cells and
experimental animals thereby facilitating functional analysis of
the target or an appraisal of its usefulness as a target for
therapeutic intervention. In diagnostics the oligonucleotides may
be used to detect and quantitate target expression in cell and
tissues by Northern blotting, in-situ hybridisation or similar
techniques. For therapeutics, an animal or a human, suspected of
having a disease or disorder, which can be treated by modulating
the expression of target is treated by administering the
oligonucleotide compounds in accordance with this invention.
Further provided are methods of treating an animal particular mouse
and rat and treating a human, suspected of having or being prone to
a disease or condition, associated with expression of target by
administering a therapeutically or prophylactically effective
amount of one or more of the oligonucleotide compounds or
compositions of the invention.
Therapeutic Use of Oligonucleotides Targeting MiR-122a
[0322] In the examples section, it is demonstrated that a
LNA-antimiR.TM., such as SPC3372, targeting miR-122a reduces plasma
cholesterol levels. Therefore, another aspect of the invention is
use of the above described oligonucleotides targeting miR-122a as
medicine. Still another aspect of the invention is use of the above
described oligonucleotides targeting miR-122a for the preparation
of a medicament for treatment of increased plasma cholesterol
levels. The skilled man will appreciate that increased plasma
cholesterol levels is undesirable as it increases the risk of
various conditions, e.g. atherosclerosis.
[0323] Still another aspect of the invention is use of the above
described oligonucleotides targeting miR-122a for upregulating the
mRNA levels of Nrdg3, Aldo A, Bckdk or CD320.
Further Embodiments
[0324] The following embodiments may be combined with the other
embodiments as described herein:
[0325] 1. An oligonucleotide having a length of from 12 to 26
nucleotides, wherein [0326] i) the first nucleotide, counting from
the 3' end, is a locked nucleic acid (LNA) unit; [0327] ii) the
second nucleotide, counting from the 3' end, is an LNA unit; and
[0328] iii) the ninth and/or the tenth nucleotide, counting from
the 3' end, is an LNA unit.
[0329] 2. The oligonucleotide according to claim 1, wherein the
ninth nucleotide, counting from the 3' end, is an LNA unit.
[0330] 3. The oligonucleotide according to embodiment 1, wherein
the tenth nucleotide, counting from the 3' end, is an LNA unit.
[0331] 4. The oligonucleotide according to embodiment 1, wherein
both the ninth and the tenth nucleotide, calculated from the 3'
end, are LNA units.
[0332] 5. The oligonucleotide according to any of embodiments 1-4,
wherein said oligonucleotide comprises at least one LNA unit in
positions three to eight, counting from the 3' end.
[0333] 6. The oligonucleotide according to embodiment 5, wherein
said oligonucleotide comprises one LNA unit in positions three to
eight, counting from the 3' end.
[0334] 7. The oligonucleotide according to embodiment 6, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0335] 8. The oligonucleotide according to embodiment 5, wherein
said oligonucleotide comprises at least two LNA units in positions
three to eight, counting from the 3' end.
[0336] 9. The oligonucleotide according to embodiment 8, wherein
said oligonucleotide comprises two LNA units in positions three to
eight, counting from the 3' end.
[0337] 10. The oligonucleotide according to embodiment 9, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx,
xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and
xxxxXX, wherein "X" denotes an LNA unit and "x" denotes a non-LNA
unit.
[0338] 11. The oligonucleotide according to embodiment 10, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxXxx, xXxxXx,
xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein "X" denotes an LNA unit
and "x" denotes a non-LNA unit.
[0339] 12. The oligonucleotide according to embodiment 11, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0340] 13. The oligonucleotide according to embodiment 12, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of xXxXxx, xXxxXx and xxXxXx, wherein "X" denotes an LNA
unit and "x" denotes a non-LNA unit.
[0341] 14. The oligonucleotide according to embodiment 13, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is xXxXxx, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit.
[0342] 15. The oligonucleotide according to embodiment 5, wherein
said oligonucleotide comprises at least three LNA units in
positions three to eight, counting from the 3' end.
[0343] 16. The oligonucleotide according to embodiment 15, wherein
said oligonucleotide comprises three LNA units in positions three
to eight, counting from the 3' end.
[0344] 17. The oligonucleotide according to embodiment 16, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx,
XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx,
xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein "X" denotes an LNA unit
and "x" denotes a non-LNA unit.
[0345] 18. The oligonucleotide according to embodiment 17, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX,
XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx,
wherein "X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0346] 19. The oligonucleotide according to embodiment 18, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is selected from the group
consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and
xXxXxX, wherein "X" denotes an LNA unit and "x" denotes a non-LNA
unit.
[0347] 20. The oligonucleotide according to embodiment 18, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is xXxXxX or XxXxXx, wherein "X"
denotes an LNA unit and "x" denotes a non-LNA unit.
[0348] 21. The oligonucleotide according to embodiment 20, wherein
the substitution pattern for the nucleotides in positions three to
eight, counting from the 3' end, is xXxXxX, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit.
[0349] 22. The oligonucleotide according to any of embodiment 7-21,
wherein said non-LNA unit is a DNA unit.
[0350] 23. The oligonucleotide according to any of the preceding
embodiments , wherein said nucleotide has a length of from 12 to 24
nucleotides, such as a length of from 12 to 22 nucleotides,
preferably a length of from 12 to 20 nucleotides, such as a length
of from 12 to 19 nucleotides, more preferably a length of from 12
to 18 nucleotides, such as a length of from 12 to 17 nucleotides,
even more preferably a length of from 12 to 16 nucleotides.
[0351] 24. The oligonucleotide according to any of the preceding
embodiments, wherein said oligonucleotide comprises at least one
LNA unit, such as one LNA unit, from position 11, counting from the
3' end, to the 5' end.
[0352] 25. The oligonucleotide according to any of the preceding
embodiments, wherein said oligonucleotide comprises at least two
LNA units, such as two LNA units, from position 11, counting from
the 3' end, to the 5' end.
[0353] 26. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises 12 nucleotides and the
substitution pattern for positions 11 to 12, counting from the 3'
end, is selected from the group consisting of xX and Xx, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0354] 27. The oligonucleotide according to embodiment 26, wherein
the substitution pattern for positions 11 to 12, counting from the
3' end, is xX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit.
[0355] 28. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises 13 nucleotides and the
substitution pattern for positions 11 to 13, counting from the 3'
end, is selected from the group consisting of Xxx, xXx, xxX, XXx,
XxX, xXX and XXX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit.
[0356] 29. The oligonucleotide according to embodiment 28, wherein
the substitution pattern for positions 11 to 13, counting from the
3' end, is xxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit.
[0357] 30. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises 14 nucleotides and the
substitution pattern for positions 11 to 14, counting from the 3'
end, is selected from the group consisting of Xxxx, xXxx, xxXx,
xxxX, XXxx, XxXx, XxxX, xXXx, xXxX and xxXX, wherein "X" denotes an
LNA unit and "x" denotes a non-LNA unit.
[0358] 31. The oligonucleotide according to embodiment 30, wherein
the substitution pattern for positions 11 to 14, counting from the
3' end, is xXxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit.
[0359] 32. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises 15 nucleotides and the
substitution pattern for positions 11 to 15, counting from the 3'
end, is selected from the group consisting of Xxxxx, xXxxx, xxXxx,
xxxXx, xxxxX, XXxxx, XxXxx, XxxXx, XxxxX, xXXxx, xXxXx, xXxxX,
xxXXx, xxXxX and xxxXX, wherein "X" denotes an LNA unit and "x"
denotes a non-LNA unit.
[0360] 33. The oligonucleotide according to embodiment 32, wherein
the substitution pattern for positions 11 to 15, counting from the
3' end, is xxXxX, wherein "X" denotes an LNA unit and "x" denotes a
non-LNA unit.
[0361] 34. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises 16 nucleotides and the
substitution pattern for positions 11 to 16, counting from the 3'
end, is selected from the group consisting of Xxxxxx, xXxxxx,
xxXxxx, xxxXxx, xxxxXx, xxxxxX, XXxxxx, XxXxxx, XxxXxx, XxxxXx,
XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX,
xxxXXx, xxxXxX, xxxxXX, XXXxxx, XXxXxx, XXxxXx, XXxxxX, XxXXxx,
XxXxXx, XxXxxX, XxxXXx, XxxXxX, XxxxXX, xXXXxx, xXXxXx, xXXxxX,
xXxXXx, xXxXxX, xXxxXX, xxXXXx, xxXXxX, xxXxXX and xxxXXX, wherein
"X" denotes an LNA unit and "x" denotes a non-LNA unit.
[0362] 35. The oligonucleotide according to embodiment 34, wherein
the substitution pattern for positions 11 to 16, counting from the
3' end, is xxXxxX, wherein "X" denotes an LNA unit and "x" denotes
a non-LNA unit.
[0363] 36. The oligonucleotide according to embodiment 24 or 25,
wherein said oligonucleotide comprises an LNA unit at the 5'
end.
[0364] 37. The oligonucleotide according to embodiment 36
containing an LNA unit at the first two positions, counting from
the 5' end.
[0365] 38. The oligonucleotide according to any of the preceding
embodiments, wherein the oligonucleotide comprises at least one
internucleoside linkage group which differs from phosphate.
[0366] 39. The oligonucleotide according to embodiment 38, wherein
said internucleoside linkage group, which differs from phosphate,
is phosphorothioate.
[0367] 40. The oligonucleotide according to embodiment 39, wherein
all internucleoside linkage groups are phosphorothioate.
[0368] 41. The oligonucleotide according to any of the preceding
embodiments, wherein said LNA units are independently selected from
the group consisting of thio-LNA units, amino-LNA units and oxy-LNA
units.
[0369] 42. The oligonucleotide according to embodiment 41, wherein
said LNA units are in the beta-D-form.
[0370] 43. The oligonucleotide according to embodiment 41, wherein
said LNA units are oxy-LNA units in the beta-D-form.
[0371] 44. The oligonucleotide according to any of the preceding
embodiments for use as a medicament.
[0372] 45. A pharmaceutical composition comprising an
oligonucleotide according to any of embodiments 1-43 and a
pharmaceutically acceptable carrier.
[0373] 46. The composition according to embodiment 45, wherein said
carrier is saline or buffered saline.
[0374] 47. Use of an oligonucleotide according to any of
embodiments 1-43 for the manufacture of a medicament for the
treatment of cancer.
[0375] 48. A method for the treatment of cancer, comprising the
step of administering an oligonucleotide according to any of
embodiments 1-43 or a composition according to embodiment 45.
REFERENCES
[0376] Abelson, J. F. et al. 2005. Science 310: 317-20. [0377]
Bartel, D. P. 2004. Cell 116: 281-297. [0378] Boehm, M., Slack, F.
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USA 99: 15524-15529. [0381] Calin, G. A. et al. 2004. Proc. Natl.
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EXPERIMENTAL
Example 1
Monomer Synthesis
[0412] The LNA monomer building blocks and derivatives thereof were
prepared following published procedures and references cited
therein, see, e.g. WO 03/095467 A1 and D. S. Pedersen, C.
Rosenbohm, T. Koch (2002) Preparation of LNA Phosphoramidites,
Synthesis 6, 802-808.
Example 2
Oligonucleotide Synthesis
[0413] Oligonucleotides were synthesized using the phosphoramidite
approach on an Expedite 8900/MOSS synthesizer (Multiple
Oligonucleotide Synthesis System) at 1 .mu.mol or 15 .mu.mol scale.
For larger scale synthesis an Akta Oligo Pilot (GE Healthcare) was
used. At the end of the synthesis (DMT-on), the oligonucleotides
were cleaved from the solid support using aqueous ammonia for 1-2
hours at room temperature, and further deprotected for 4 hours at
65.degree. C. The oligonucleotides were purified by reverse phase
HPLC (RP-HPLC). After the removal of the DMT-group, the
oligonucleotides were characterized by AE-HPLC, RP-HPLC, and CGE
and the molecular mass was further confirmed by ESI-MS. See below
for more details.
Preparation of the LNA-Solid Support:
Preparation of the LNA Succinyl Hemiester
[0414] 5'-O-Dmt-3'-hydroxy-LNA monomer (500 mg), succinic anhydride
(1.2 eq.) and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The
reaction was stirred at room temperature overnight. After
extractions with NaH.sub.2PO.sub.4 0.1 M pH 5.5 (2.times.) and
brine (1.times.), the organic layer was further dried with
anhydrous Na.sub.2SO.sub.4 filtered and evaporated. The hemiester
derivative was obtained in 95% yield and was used without any
further purification.
Preparation of the LNA-Support
[0415] The above prepared hemiester derivative (90 .mu.mol) was
dissolved in a minimum amount of DMF, DIEA and pyBOP (90 .mu.mol)
were added and mixed together for 1 min. This pre-activated mixture
was combined with LCAA-CPG (500 .ANG., 80-120 mesh size, 300 mg) in
a manual synthesizer and stirred. After 1.5 hours at room
temperature, the support was filtered off and washed with DMF, DCM
and MeOH. After drying, the loading was determined to be 57
.mu.mol/g (see Tom Brown, Dorcas J. S. Brown. Modern machine-aided
methods of oligodeoxyribonucleotide synthesis. In: F. Eckstein,
editor. Oligonucleotides and Analogues A Practical Approach.
Oxford: IRL Press, 1991: 13-14).
Elongation of the Oligonucleotide
[0416] The coupling of phosphoramidites (A(bz), G(lbu),
5-methyl-C(bz)) or T-.beta.-cyanoethyl-phosphoramidite) is
performed by using a solution of 0.1 M of the 5'-O-DMT-protected
amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in
acetonitrile (0.25 M) as activator. The thiolation is carried out
by using xanthane chloride (0.01 M in acetonitrile:pyridine 10%).
The rest of the reagents are the ones typically used for
oligonucleotide synthesis.
Purification by RP-HPLC:
Column: Xterra RP.sub.18
[0417] Flow rate: 3 mL/min Buffers: 0.1 M ammonium acetate pH 8 and
acetonitrile
Abbreviations:
DMT: Dimethoxytrityl
DCI: 4,5-Dicyanoimidazole
DMAP: 4-Dimethylaminopyridine
DCM: Dichloromethane
DMF: Dimethylformamide
THF: Tetrahydrofurane
DIEA: N,N-diisopropylethylamine
[0418] PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate
Bz: Benzoyl
Ibu: Isobutyryl
Example 3
Design of the LNA Anti-MiR Oligonucleotides and Melting
Temperatures
Target MicroRNA:
TABLE-US-00003 [0419] miR-122a: SEQ ID NO : 535
5'-uggagugugacaaugguguuugu-3' miR-122a 3' to 5': (SEQ ID NO : 535
reverse orientation) 3'-uguuugugguaacagugugaggu-5'
TABLE-US-00004 TABLE 1 LNA anti-miR oligonucleotide sequences and
T.sub.m: SEQ ID Tm NO: Oligo ID SED ID Sequence: (.degree. C.) 2
SPC3370 XxxX SEQ ID 585 5'-cCatTgtCacActCca- PS 75 design 3'
backbone 3 SPC3372 XxxX SEQ ID 586 5'-ccAttGtcAcaCtcCa- PS 69
design 3' backbone 4 SPC3375 Gapmer SEQ ID 587 5'- PS 69
CCAttgtcacacTCCa-3' backbone 5 SPC3549 15-mer SEQ ID 588
5'-CcAttGTcaCaCtCC- PS 78 3' backbone 6 SPC3550 mismatch SEQ ID 589
5'-CcAttCTgaCcCtAC- PS 32 control 3' backbone 7 SPC3373 mismatch
SEQ ID 590 5'-ccAttGtcTcaAtcCa- PS 46 control 3' backbone 8 SPC3548
13-mer SEQ ID 591 5'-AttGTcaCaCtCC-3' PS backbone lower case: DNA,
uppercase: LNA (all LNA C were methylated), underlined:
mismatch
[0420] The melting temperatures were assessed towards the mature
miR-122a sequence, using a synthetic miR-122a RNA oligonucleotide
with phosphorothioate linkaged.
[0421] The LNA anti-miR/miR-122a oligo duplex was diluted to 3
.mu.M in 500 .mu.l RNase free H.sub.20, which was then mixed with
500 .mu.l 2.times. dimerization buffer (final oligo/duplex conc.
1.5 .mu.M, 2.times.Tm buffer: 200 mM NaCl, 0.2 mM EDTA, 20 mM NaP,
pH 7,0, DEPC treated to remove RNases). The mix was first heated to
95 degrees for 3 minutes, then allowed to cool at room temperature
(RT) for 30 minutes.
[0422] Following RT incubation T.sub.m was measured on Lambda 40
UV/VIS Spectrophotometer with peltier temperature programmer PTP6
using PE Termplab software (Perkin Elmer). The Temperature was
ramped up from 20.degree. C. to 95.degree. C. and then down again
to 20.degree. C., continuously recording absorption at 260 nm.
First derivative and local maximums of both the melting and
annealing was used to assess melting/annealing point (T.sub.m),
both should give similar/same T.sub.m values. For the first
derivative 91 points was used to calculate the slope.
[0423] By substituting the antimir oligonucleotide and the
complementary RNA molecule, the above assay can be used to
determine the T.sub.m of other oligonucleotides such as the
oligonucleotides according to the invention.
[0424] However, in one embodiment the T.sub.m may be made with a
complementary DNA (phosphorothioate linkages) molecule. Typically
the T.sub.m measured against a DNA complementary molecule is about
10.degree. C. lower than the T.sub.m with an equivalent RNA
complement. The T.sub.m measured using the DNA complement may
therefore be used in cases where the duplex has a very high
T.sub.m.
Melting Temperature (T.sub.m) Measurements:
TABLE-US-00005 [0425] oligo to miR-122 RNA complement T.sub.m
SPC3372 + miR-122a, RNA 69.degree. C. SPC3648 + miR-122a, RNA
74.degree. C. SPC3649 + miR-122a, RNA 79.degree. C.
TABLE-US-00006 oligo to DNA complement T.sub.m SPC3372 + 122R, DNA
57.degree. C. SPC3649 + 122R, DNA 66.degree. C.
[0426] It is recognised that for oligonucleotides with very high
T.sub.m, the above T.sub.m assays may be insufficient to determine
the T.sub.m. In such an instance the use of a phosphorothioated DNA
complementary molecule may further lower the T.sub.m.
[0427] The use of formamide is routine in the analysis of
oligonucleotide hybridisation (see Hutton 1977, NAR 4 (10)
3537-3555). In the above assay the inclusion of 15% formamide
typically lowers the T.sub.m by about 9.degree. C., and the
inclusion of 50% formamide typically lowers the T.sub.m by about
30.degree. C. Using these ratios, it is therefore possible to
determine the comparative T.sub.m of an oligonucleotide against its
complementary RNA (phosphodiester) molecule, even when the T.sub.m
of the duplex is, for example higher than 95.degree. C. (in the
absence of formamide).
[0428] For oligonucleotides with a very high T.sub.m, an
alternative method of determining the T.sub.m, is to make
titrations and run it out on a gel to see single strand versus
duplex and by those concentrations and ratios determine Kd (the
dissociation constant) which is related to deltaG and also
T.sub.m.
Example 4
Stability of LNA Oligonucleotides in Human or Rat Plasma
[0429] LNA oligonucleotide stability was tested in plasma from
human or rats (it could also be mouse, monkey or dog plasma). In 45
.mu.l plasma, 5 .mu.l LNA oligonucleotide is added (at a final
concentration of 20 .mu.M). The LNA oligonucleotides are incubated
in plasma for times ranging from 0 to 96 hours at 37.degree. C.
(the plasma is tested for nuclease activity up to 96 hours and
shows no difference in nuclease cleavage-pattern).
[0430] At the indicated time the sample were snap frozen in liquid
nitrogen. 2 .mu.L (equals 40 .mu.mol) LNA oligonucleotide in plasma
was diluted by adding 15 .mu.L of water and 3 .mu.L 6.times.
loading dye (Invitrogen). As marker a 10 by ladder (Invitrogen, USA
10821-015) is used. To 1 .mu.l ladder, 1 .mu.l 6.times. loading and
4 .mu.l water is added. The samples are mixed, heated to 65.degree.
C. for 10 min and loaded to a pre-run gel (16% acrylamide, 7 M
UREA, 1.times.TBE, pre-run at 50 Watt for 1 h) and run at 50-60
Watt for 21/2 hours. Subsequently, the gel is stained with
1.times.SyBR gold (molecular probes) in 1.times.TBE for 15 min. The
bands were visualised using a phosphoimager from BioRad.
Example 5
In Vitro Model: Cell Culture
[0431] The effect of LNA oligonucleotides on target nucleic acid
expression (amount) can be tested in any of a variety of cell types
provided that the target nucleic acid is present at measurable
levels. Target can be expressed endogenously or by transient or
stable transfection of a nucleic acid encoding said nucleic
acid.
[0432] The expression level of target nucleic acid can be routinely
determined using, for example, Northern blot analysis (including
microRNA northern), Quantitative PCR (including microRNA qPCR),
Ribonuclease protection assays. The following cell types are
provided for illustrative purposes, but other cell types can be
routinely used, provided that the target is expressed in the cell
type chosen.
[0433] Cells were cultured in the appropriate medium as described
below and maintained at 37.degree. C. at 95-98% humidity and 5%
CO.sub.2. Cells were routinely passaged 2-3 times weekly.
[0434] 15PC3: The human prostate cancer cell line 15PC3 was kindly
donated by Dr. F. Baas, Neurozintuigen Laboratory, AMC, The
Netherlands and was cultured in DMEM (Sigma)+10% fetal bovine serum
(FBS)+Glutamax I+gentamicin.
[0435] PC3: The human prostate cancer cell line PC3 was purchased
from ATCC and was cultured in F12 Coon's with glutamine (Gibco)+10%
FBS+gentamicin.
[0436] 518A2: The human melanoma cancer cell line 518A2 was kindly
donated by Dr. B. Jansen, Section of experimental Oncology,
Molecular Pharmacology, Department of Clinical Pharmacology,
University of Vienna and was cultured in DMEM (Sigma)+10% fetal
bovine serum (FBS)+Glutamax I+gentamicin.
[0437] HeLa: The cervical carcinoma cell line HeLa was cultured in
MEM (Sigma) containing 10% fetal bovine serum gentamicin at
37.degree. C., 95% humidity and 5% CO.sub.2.
[0438] MPC-11: The murine multiple myeloma cell line MPC-11 was
purchased from ATCC and maintained in DMEM with 4 mM Glutamax+10%
Horse Serum.
[0439] DU-145: The human prostate cancer cell line DU-145 was
purchased from ATCC and maintained in RPMI with Glutamax+10%
FBS.
[0440] RCC-4+/-VHL: The human renal cancer cell line RCC4 stably
transfected with plasmid expressing VHL or empty plasmid was
purchased from ECACC and maintained according to manufacturers
instructions.
[0441] 786-0: The human renal cell carcinoma cell line 786-0 was
purchased from ATCC and maintained according to manufacturers
instructions
[0442] HUVEC: The human umbilical vein endothelial cell line HUVEC
was purchased from Camcrex and maintained in EGM-2 medium.
[0443] K562: The human chronic myelogenous leukaemia cell line K562
was purchased from ECACC and maintained in RPMI with Glutamax+10%
FBS. U87MG: The human glioblastoma cell line U87MG was purchased
from ATCC and maintained according to the manufacturers
instructions.
[0444] B16: The murine melanoma cell line B16 was purchased from
ATCC and maintained according to the manufacturers
instructions.
[0445] LNCap: The human prostate cancer cell line LNCap was
purchased from ATCC and maintained in RPMI with Glutamax+10%
FBS
[0446] Huh-7: Human liver, epithelial like cultivated in Eagles MEM
with 10% FBS, 2 mM Glutamax I, 1.times. non-essential amino acids,
Gentamicin 25 .mu.g/ml
[0447] L428: (Deutsche Sammlung fur Mikroorganismen (DSM,
Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI
1640 supplemented with 10% FCS, L-glutamine and antibiotics.
[0448] L1236: (Deutsche Sammlung fur Mikroorganismen (DSM,
Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI
1640 supplemented with 10% FCS, L-glutamine and antibiotics.
Example 6
In Vitro Model: Treatment with LNA Anti-MiR Antisense
Oligonucleotide
[0449] The miR-122a expressing cell line Huh-7 was transfected with
LNA anti-miRs at 1 and 100 nM concentrations according to optimized
lipofectamine 2000 (LF2000, Invitrogen) protocol (as follows).
[0450] Huh-7 cells were cultivated in Eagles MEM with 10% FBS, 2 mM
Glutamax I, 1.times. non-essential amino acids, Gentamicin 25
.mu.g/ml. The cells were seeded in 6-well plates (300000 cells per
well), in a total vol. of 2.5 ml the day before transfection. At
the day of transfection a solution containing LF2000 diluted in
Optimem (Invitrogen) was prepared (1.2 ml optimem+3.75 .mu.l LF2000
per well, final 2.5 .mu.g LF2000/ml, final tot vol 1.5 ml).
[0451] LNA Oligonucleotides (LNA anti-miRs) were also diluted in
optimem. 285 .mu.l optimem+15 .mu.l LNA oligonucleotide (10 .mu.M
oligonucleotide stock for final concentration 100 nM and 0.1 .mu.M
for final concentration 1 nM) Cells were washed once in optimem
then the 1.2 ml optimem/LF2000 mix were added to each well. Cells
were incubated 7 min at room temperature in the LF2000 mix where
after the 300 .mu.l oligonucleotide optimem solution was added.
[0452] Cell were further incubated for four hours with
oligonucleotide and lipofectamine2000 (in regular cell incubator at
37.degree. C., 5% CO2). After these four hours the medium/mix was
removed and regular complete medium was added. Cells were allowed
to grow for another 20 hours. Cells were harvested in Trizol
(Invitrogen) 24 hours after transfection. RNA was extracted
according to a standard Trizol protocol according to the
manufacturer's instructions (Invitrogen), especially to retain the
microRNA in the total RNA extraction.
Example 7
In Vitro and In Vivo Model: Analysis of Oligonucleotide Inhibition
of MiR Expression by MicroRNA Specific Quantitative PCR
[0453] miR-122a levels in the RNA samples were assessed on an ABI
7500 Fast real-time PCR instrument (Applied Biosystems, USA) using
a miR-122a specific qRT-PCR kit, mirVana (Ambion, USA) and miR-122a
primers (Ambion, USA). The procedure was conducted according to the
manufacturers protocol.
Results:
[0454] The miR-122a-specific new LNA anti-miR oligonucleotide
design (ie SPC3349 (also referred to as SPC 3549)), was more
efficient in inhibiting miR-122a at 1 nM compared to previous
design models, including "every-third" and "gap-mer" (SPC3370,
SPC3372, SPC3375) motifs were at 100 nM. The mismatch control was
not found to inhibit miR-122a (SPC3350). Results are shown in FIG.
1.
Example 8
Assessment of LNA Antago-Mir Knock-Down Specificity Using miRNA
Microarray Expression Profiling
[0455] A) RNA Labeling for miRNA Microarray Profiling
[0456] Total RNA was extracted using Trizol reagent (Invitrogen)
and 3' end labeled using T4 RNA ligase and Cy3- or Cy5-labeled RNA
linker (5'-PO4-rUrUrU-Cy3/dT-3' or 5'-PO4-rUrUrU-Cy5/dT-3'). The
labeling reactions contained 2-5 .mu.g total RNA, 15 .mu.M RNA
linker, 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 1 mM ATP,
16% polyethylene glycol and 5 unit T4 RNA ligase (Ambion, USA) and
were incubated at 30.degree. C. for 2 hours followed by heat
inactivation of the T4 RNA ligase at 80.degree. C. for 5
minutes.
B) Microarray Hybridization and Post-Hybridization Washes
[0457] LNA-modified oligonucleotide capture probes comprising
probes for all annotated miRNAs annotated from mouse (Mus musculus)
and human (Homo sapiens) in the miRBase MicroRNA database Release
7.1 including a set of positive and negative control probes were
purchased from Exiqon (Exiqon, Denmark) and used to print the
microarrays for miRNA profiling. The capture probes contain a
5'-terminal C6-amino modified linker and were designed to have a Tm
of 72.degree. C. against complementary target miRNAs by adjustment
of the LNA content and length of the capture probes. The capture
probes were diluted to a final concentration of 10 .mu.M in 150 mM
sodium phosphate buffer (pH 8.5) and spotted in quadruplicate onto
Codelink slides (Amersham Biosciences) using the MicroGrid II
arrayer from BioRobotics at 45% humidity and at room temperature.
Spotted slides were post-processed as recommended by the
manufacturer.
[0458] Labeled RNA was hybridized to the LNA microarrays overnight
at 65.degree. C. in a hybridization mixture containing 4.times.SSC,
0.1% SDS, 1 .mu.g/.mu.l Herring Sperm DNA and 38% formamide. The
hybridized slides were washed three times in 2.times.SSC, 0.025%
SDS at 65.degree. C., followed by three times in 0.08.times.SSC and
finally three times in 0.4.times.SSC at room temperature.
C) Array Scanning, Image Analysis and Data Processing
[0459] The microarrays were scanned using the ArrayWorx scanner
(Applied Precision, USA) according to the manufacturer's
recommendations. The scanned images were imported into TIGR
Spotfinder version 3.1 (Saeed et al., 2003) for the extraction of
mean spot intensities and median local background intensities,
excluding spots with intensities below median local
background+4.times. standard deviations. Background-correlated
intensities were normalized using variance stabilizing
normalization package version 1.8.0 (Huber et al., 2002) for R
(www.r-project.org). Intensities of replicate spots were averaged
using Microsoft Excel. Probes displaying a coefficient of variance
>100% were excluded from further data analysis.
Example 9
Detection of MicroRNAs by In Situ Hybridization Detection of
MicroRNAs in Formalin-Fixed Paraffin-Embedded Tissue Sections by In
Situ Hybridization
A) Preparation of the Formalin-Fixed, Paraffin-Embedded Sections
for In Situ Hybridization
[0460] Archival paraffin-embedded samples are retrieved and
sectioned at 5 to 10 mm sections and mounted in positively-charged
slides using floatation technique. Slides are stored at 4.degree.
C. until the in situ experiments are conducted.
B) In Situ Hybridization
[0461] Sections on slides are deparaffinized in xylene and then
rehydrated through an ethanol dilution series (from 100% to 25%).
Slides are submerged in DEPC-treated water and subject to HCl and
0.2% Glycine treatment, re-fixed in 4% paraformaldehyde and treated
with acetic anhydride/triethanolamine; slides are rinsed in several
washes of 1.times.PBS in-between treatments. Slides are
pre-hybridized in hyb solution (50% formamide, 5.times.SSC, 500
mg/mL yeast tRNA, 1.times.Denhardt) at 50.degree. C. for 30 min.
Then, 3 .mu.mol of a FITC-labeled LNA probe (Exiqon, Denmark)
complementary to each selected miRNA is added to the hyb. solution
and hybridized for one hour at a temperature 20-25.degree. C. below
the predicted Tm of the probe (typically between 45-55.degree. C.
depending on the miRNA sequence). After washes in 0.1.times. and
0.5.times.SCC at 65.degree. C., a tyramide signal amplification
reaction was carried out using the Genpoint Fluorescein (FITC) kit
(DakoCytomation, Denmark) following the vendor's recommendations.
Finally, slides are mounted with Prolong Gold solution.
Fluorescence reaction is allowed to develop for 16-24 hr before
documenting expression of the selected miRNA using an
epifluorescence microscope.
Detection of MicroRNAs by Whole-Mount In Situ Hybridization of
Zebrafish, Xenopus and Mouse Embryos.
[0462] All washing and incubation steps are performed in 2 ml
eppendorf tubes. Embryos are fixed overnight at 4.degree. C. in 4%
paraformaldehyde in PBS and subsequently transferred through a
graded series (25% MeOH in PBST (PBS containing 0.1% Tween-20), 50%
MeOH in PBST, 75% MeOH in PBST) to 100% methanol and stored at
-20.degree. C. up to several months. At the first day of the in
situ hybridization embryos are rehydrated by successive incubations
for 5 min in 75% MeOH in PBST, 50% MeOH in PBST, 25% MeOH in PBST
and 100% PBST (4.times.5 min).
[0463] Fish, mouse and Xenopus embryos are treated with proteinaseK
(10 .mu.g/ml in PBST) for 45 min at 37.degree. C., refixed for 20
min in 4% paraformaldehyde in PBS and washed 3.times.5 min with
PBST. After a short wash in water, endogenous alkaline phosphatase
activity is blocked by incubation of the embryos in 0.1 M
tri-ethanolamine and 2.5% acetic anhydride for 10 min, followed by
a short wash in water and 5.times.5 min washing in PBST. The
embryos are then transferred to hybridization buffer (50%
Formamide, 5.times.SSC, 0.1% Tween, 9.2 mM citric acid, 50 ug/ml
heparin, 500 ug/ml yeast RNA) for 2-3 hour at the hybridization
temperature. Hybridization is performed in fresh pre-heated
hybridization buffer containing 10 nM of 3' DIG-labeled LNA probe
(Roche Diagnostics) complementary to each selected miRNA.
Post-hybridization washes are done at the hybridization temperature
by successive incubations for 15 min in HM- (hybridization buffer
without heparin and yeast RNA), 75% HM-/25% 2.times.SSCT (SSC
containing 0.1% Tween-20), 50% HM-/50% 2.times.SSCT, 25% HM-/75%
2.times.SSCT, 100% 2.times.SSCT and 2.times.30 min in
0.2.times.SSCT.
[0464] Subsequently, embryos are transferred to PBST through
successive incubations for 10 min in 75% 0.2.times.SSCT/25% PBST,
50% 0.2.times.SSCT/50% PBST, 25% 0.2.times.SSCT/75% PBST and 100%
PBST. After blocking for 1 hour in blocking buffer (2% sheep
serum/2 mg:ml BSA in PBST), the embryos are incubated overnight at
4.degree. C. in blocking buffer containing anti-DIG-AP FAB
fragments (Roche, 1/2000). The next day, zebrafish embryos are
washed 6.times.15 min in PBST, mouse and X. tropicalis embryos are
washed 6.times.1 hour in TBST containing 2 mM levamisole and then
for 2 days at 4.degree. C. with regular refreshment of the wash
buffer.
[0465] After the post-antibody washes, the embryos are washed
3.times.5 min in staining buffer (100 mM tris HCl pH9.5, 50 mM
MgCl2, 100 mM NaCl, 0.1% tween 20). Staining was done in buffer
supplied with 4.5 .mu.l/ml NBT (Roche, 50 mg/ml stock) and 3.5
.mu.l/ml BCIP (Roche, 50 mg/ml stock). The reaction is stopped with
1 mM EDTA in PBST and the embryos are stored at 4.degree. C. The
embryos are mounted in Murray's solution (2:1
benzylbenzoate:benzylalcohol) via an increasing methanol series
(25% MeOH in PBST, 50% MeOH in PBST, 75% MeOH in PBST, 100% MeOH)
prior to imaging.
Example 10
In Vitro Model: Isolation and Analysis of mRNA Expression (Total
RNA Isolation and cDNA Synthesis for mRNA Analysis)
[0466] Total RNA was isolated either using RNeasy mini kit (Qiagen)
or using the Trizol reagent (Invitrogen). For total RNA isolation
using RNeasy mini kit (Qiagen), cells were washed with PBS, and
Cell Lysis Buffer (RTL, Qiagen) supplemented with 1%
mercaptoethanol was added directly to the wells. After a few
minutes, the samples were processed according to manufacturer's
instructions.
[0467] For in vivo analysis of mRNA expression tissue samples were
first homogenised using a Retsch 300 mM homogeniser and total RNA
was isolated using the Trizol reagent or the RNeasy mini kit as
described by the manufacturer.
[0468] First strand synthesis (cDNA from mRNA) was performed using
either OmniScript Reverse Transcriptase kit or M-MLV Reverse
transcriptase (essentially described by manufacturer (Ambion))
according to the manufacturer's instructions (Qiagen). When using
OmniScript Reverse Transcriptase 0.5 .mu.g total RNA each sample,
was adjusted to 12 .mu.l and mixed with 0.2 .mu.l
poly(dT).sub.12-18 (0.5 .mu.g/.mu.l) (Life Technologies), 2 .mu.l
dNTP mix (5 mM each), 2 .mu.l 10.times.RT buffer, 0.5 .mu.l
RNAguard.TM. RNase Inhibitor (33 units/ml, Amersham) and 1 .mu.l
OmniScript Reverse Transcriptase followed by incubation at
37.degree. C. for 60 min. and heat inactivation at 93.degree. C.
for 5 min.
[0469] When first strand synthesis was performed using random
decamers and M-MLV-Reverse Transcriptase (essentially as described
by manufacturer (Ambion)) 0.25 .mu.g total RNA of each sample was
adjusted to 10.8 .mu.l in H.sub.2O. 2 .mu.l decamers and 2 .mu.l
dNTP mix (2.5 mM each) was added. Samples were heated to 70.degree.
C. for 3 min. and cooled immediately in ice water and added 3.25
.mu.l of a mix containing (2 .mu.l 10.times.RT buffer; 1 .mu.l
M-MLV Reverse Transcriptase; 0.25 .mu.l RNAase inhibitor). cDNA is
synthesized at 42.degree. C. for 60 min followed by heating
inactivation step at 95.degree. C. for 10 min and finally cooled to
4.degree. C. The cDNA can further be used for mRNA quantification
by for example Real-time quantitative PCR.
[0470] mRNA expression can be assayed in a variety of ways known in
the art. For example, mRNA levels can be quantitated by, e.g.,
Northern blot analysis, competitive polymerase chain reaction
(PCR), Ribonuclease protection assay (RPA) or real-time PCR.
Real-time quantitative PCR is presently preferred. RNA analysis can
be performed on total cellular RNA or mRNA.
[0471] Methods of RNA isolation and RNA analysis such as Northern
blot analysis are routine in the art and is taught in, for example,
Current Protocols in Molecular Biology, John Wiley and Sons.
[0472] Real-time quantitative (PCR) can be conveniently
accomplished using the commercially available iQ Multi-Color Real
Time PCR Detection System available from BioRAD. Real-time
Quantitative PCR is a technique well-known in the art and is taught
in for example Heid et al. Real time quantitative PCR, Genome
Research (1996), 6: 986-994.
Example 11
LNA Oligonucleotide Uptake and Efficacy In Vivo
[0473] In vivo study: Six groups of animals (5 mice per group) were
treated in the following manner. Group 1 animals were injected with
0.2 ml saline by i.v. on 3 successive days, Group 2 received 2.5
mg/kg SPC3372, Group 3 received 6.25 mg/kg, Group 4 received 12.5
mg/kg and Group 5 received 25 mg/kg, while Group 6 received 25
mg/kg SPC 3373 (mismatch LNA-antimiR.TM. oligonucleotide), all in
the same manner. All doses were calculated from the Day 0 body
weights of each animal.
[0474] Before dosing (Day 0) and 24 hour after last dose (Day 3),
retro-orbital blood was collected in tubes containing EDTA and the
plasma fraction harvested and stored frozen -80.degree. C. for
cholesterol analysis. At sacrifice livers were dissected and one
portion was cut into 5 mm cubes and immersed in 5 volumes of
ice-cold RNAlater. A second portion was snap frozen in liquid
nitrogen and stored for cryo-sectioning.
[0475] Total RNA was extracted from liver samples as described
above and analysed for miR-122a levels by microRNA specific QPCR.
FIG. 5 demonstrates a clear dose-response obtained with SPC3372
with an IC50 at ca 3-5 mg/kg, whereas no miR-122a inhibition was
detected using the mismatch LNA antago-mir SPC 3373 for
miR-122a.
Example 12
LNA-antimiR-122a Dose-Response In Vivo in C57/BL/J Female Mice
[0476] In vivo study: Ten groups of animals (female C57/BL6; 3 mice
per group) were treated in the following manner. Group 1 animals
were injected with 0.2 ml saline by i.p. on day 0, day 2 and day 4.
Groups 2-10 were dosed by i.p. with three different conc. (25
mg/kg, 5 mg/kg and 1 mg/kg) of either LNA antimiR-122a/SPC3372
(group 2-4), LNA antimir-122a/SPC3548 (group 5-7) or LNA
antimir-122a/SPC3549 (group 8-10); the LNA antimir-122a sequences
are given in the Table 1. All three LNA antimiR-122a
oligonucleotides target the liver-specific miR-122a. The doses were
calculated from the Day 0 body weights of each animal.
[0477] The animals were sacrificed 48 hours after last dose (Day
6), retro-orbital blood was collected in tubes containing EDTA and
the plasma fraction harvested and stored frozen -80.degree. C. for
cholesterol analysis. At sacrifice livers were dissected and one
portion was cut into 5 mm cubes and immersed in 5 volumes of
ice-cold RNAlater. A second portion was snap frozen in liquid
nitrogen and stored for cryo-sectioning.
[0478] Total RNA was extracted from liver samples using Trizol
reagent according to the manufacturer's recommendations
(Invitrogen, USA) and analysed for miR-122a levels by
microRNA-specific QPCR according to the manufacturer's
recommendations (Ambion, USA). FIG. 2 demonstrates a clear
dose-response obtained with all three LNA antimir-122a molecules
(SPC3372, SPC3548, SPC3549). Both SPC3548 and SPC3549 show
significantly improved efficacy in vivo in miR-122a silencing (as
seen from the reduced miR-122a levels) compared to SPC3372, with
SPC3549 being most potent (IC.sub.50 ca mg/kg).
[0479] The above example was repeated using SPC3372 and SPC 3649
using 5 mice per group and the data combined (total of eight mice
per group) is shown in FIG. 2b.
Example 12a
Northern Blot
[0480] MicroRNA specific northern blot showing enhanced miR-122
blocking by SPC3649 compared to SPC3372 in LNA-antimiR treated
mouse livers.
Oligos Used in this Example:
TABLE-US-00007 SPC3649: 5'-CcAttGTcaCaCtCC-3' New design (SEQ ID
539) SPC3372 : 5'-CcAttGtcAcaCtcCa-3' Old design (SEQ ID 586)
[0481] We decided to assess the effect of SPC3649 on miR-122 miRNA
levels in the livers of SPC3649-treated mice. The LNA-antimiRs
SPC3649 and SPC3372 were administered into mice by three i.p.
injections on every second day over a six-day-period at indicated
doses followed by sacrificing the animals 48 hours after the last
dose. Total RNA was extracted from the livers. miR-122 levels were
assessed by microRNA specific northern blot (FIG. 6)
[0482] Treatment of normal mice with SPC3649 resulted in
dramatically improved, dose-dependent reduction of miR-122.
MicroRNA specific northern blot comparing SPC3649 with SPC3372 was
performed (FIG. 6). SPC3649 completely blocked miR-122 at both 5
and 25 mg/kg as seen by the absence of mature single stranded
miR-122 and only the presence of the duplex band between the
LNA-antimiR and miR-122. Comparing duplex versus mature band on the
northern blot SPC3649 seem equally efficient at 1 mg/kg as SPC3372
at 25 mg/kg.
Example 13
Assessment of Cholesterol Levels in Plasma in LNA anti-miR122
Treated Mice
[0483] Total cholesterol level was measured in plasma using a
colometric assay Cholesterol CP from ABX Pentra. Cholesterol was
measured following enzymatic hydrolysis and oxidation (2,3). 21.5
.mu.l water was added to 1.5 .mu.l plasma. 250 .mu.l reagent was
added and within 5 min the cholesterol content measured at a
wavelength of 540 nM. Measurements on each animal were made in
duplicate. The sensitivity and linearity was tested with 2-fold
diluted control compound (ABX Pentra N control). The cholesterol
level was determined by subtraction of the background and presented
relative to the cholesterol levels in plasma of saline treated
mice.
[0484] FIG. 3 demonstrates a markedly lowered level of plasma
cholesterol in the mice that received SPC3548 and SPC3549 compared
to the saline control at Day 6.
Example 14
Assessment of MiR-122a Target mRNA Levels in LNA AntimiR-122a
Treated Mice
[0485] The saline control and different LNA-antimiR-122a treated
animals were sacrificed 48 hours after last dose (Day 6), and total
RNA was extracted from liver samples as using Trizol reagent
according to the manufacturer's recommendations (Invitrogen, USA).
The mRNA levels were assessed by real-time quantitative RT-PCR for
two miR-122a target genes, Bckdk (branched chain ketoacid
dehydrogenase kinase, ENSMUSG00000030802) and aldolase A (aldoA,
ENSMUSG00000030695), respectively, as well as for GAPDH as control,
using Taqman assays according to the manufacturer's instructions
(Applied biosystems, USA). FIGS. 4a and 4b demonstrate a clear
dose-dependent upregulation of the two miR-122a target genes, Bckdk
and AldoA, respectively, as a response to treatment with all three
LNA antimiR-122a molecules (SPC3372, SPC3548, SPC3549). In
contrast, the qPCR assays for GAPDH control did not reveal any
differences in the GAPD mRNA levels in the LNA-antimiR-122a treated
mice compared to the saline control animals (FIG. 4c). The Bckdk
and AldoA mRNA levels were significantly higher in the SPC3548 and
SPC3549 treated mice compared to the SPC3372 treated mice (FIGS. 4a
and 4b), thereby demonstrating their improved in vivo efficacy.
Example 15
LNA Oligonucleotide Duration of Action In Vivo
[0486] In vivo study: Two groups of animals (21 mice per group)
were treated in the following manner. Group 1 animals were injected
with 0.2 ml saline by i.v. on 3 successive days, Group 2 received
25 mg/kg SPC3372 in the same manner. All doses were calculated from
the Day 0 body weights of each animal.
[0487] After last dose (Day 3), 7 animals from each group were
sacrificed on Day 9, Day 16 and Day 23, respectively. Prior to
this, on each day, retro-orbital blood was collected in tubes
containing EDTA and the plasma fraction harvested and stored frozen
-80.degree. C. for cholesterol analysis from each day. At sacrifice
livers were dissected and one portion was cut into 5 mm cubes and
immersed in 5 volumes of ice-cold RNAlater. A second portion was
snap frozen in liquid nitrogen and stored for cryo-sectioning.
[0488] Total RNA was extracted from liver samples as described
above and analysed for miR-122a levels by microRNA specific QPCR.
FIG. 7 (Sacrifice day 9, 16 or 23 correspond to sacrifice 1, 2 or 3
weeks after last dose) demonstrates a two-fold inhibition in the
mice that received SPC3372 compared to the saline control, and this
inhibition could still be detected at Day 16, while by Day 23 the
mi122a levels approached those of the saline group.
Example 16
LNA Oligonucleotide Duration of Action In Vivo
[0489] In vivo study: Two groups of animals (21 mice per group)
were treated in the following manner. Group 1 animals were injected
with 0.2 ml saline by i.v. on 3 successive days, Group 2 received
25 mg/kg SPC3372 in the same manner. All doses were calculated from
the Day 0 body weights of each animal.
[0490] After last dose (Day 3), 7 animals from each group were
sacrificed on Day 9, Day 16 and Day 23, respectively. Prior to
this, on each day, retro-orbital blood was collected in tubes
containing EDTA and the plasma fraction harvested and stored frozen
-80.degree. C. for cholesterol analysis from each day. At sacrifice
livers were dissected and one portion was cut into 5 mm cubes and
immersed in 5 volumes of ice-cold RNAlater. A second portion was
snap frozen in liquid nitrogen and stored for cryo-sectioning.
[0491] Total RNA was extracted from liver samples as described
above and analysed for miR-122a levels by microRNA specific QPCR.
FIG. 8 demonstrates a two-fold inhibition in the mice that received
SPC3372 compared to the saline control, and this inhibition could
still be detected at Day 16, while by Day 23 the miR-122a levels
approached those of the saline group.
As to Examples 17-22, the Following Procedures Apply:
[0492] NMRI mice were administered intravenously with SPC3372 using
daily doses ranging from 2.5 to 25 mg/kg for three consecutive
days. Animals were sacrificed 24 hours, 1, 2 or 3 weeks after last
dose. Livers were harvested divided into pieces and submerged in
RNAlater (Ambion) or snap-frozen. RNA was extracted with Trizol
reagent according to the manufacturer's instructions (Invitrogen)
from the RNAlater tissue, except that the precipitated RNA was
washed in 80% ethanol and not vortexed. The RNA was used for mRNA
TaqMan qPCR according to manufacturer (Applied biosystems) or
northern blot (see below).The snap-frozen pieces were
cryo-sectioned for in situ hybridizations.
[0493] Further, as to FIGS. 9-14, SPC3372 is designated LNA-antimiR
and SPC3373 (the mismatch control) is designated "mm" instead of
using the SPC number.
Example 17
Dose Dependent MiR-122a Target mRNA Induction by SPC3372 Inhibition
of miR-122a
[0494] Mice were treated with different SPC3372 doses for three
consecutive days, as described above and sacrificed 24 hours after
last dose. Total RNA extracted from liver was subjected to qPCR.
Genes with predicted miR-122 target site and observed to be
upregulated by microarray analysis were investigated for
dose-dependent induction by increasing SPC3372 doses using qPCR.
Total liver RNA from 2 to 3 mice per group sacrificed 24 hours
after last dose were subjected to qPCR for the indicated genes.
Shown in FIG. 9 is mRNA levels relative to Saline group, n=2-3
(2.5-12.5 mg/kg/day: n=2, no SD). Shown is also the mismatch
control (m, SPC3373).
[0495] Assayed genes: Nrdg3 Aldo A, Bckdk, CD320 with predicted
miR-122 target site. Aldo B and Gapdh do not have a predicted
miR-122a target site.
[0496] A clear dose-dependent induction was seen of the miR-122a
target genes after treatment with different doses of SPC3372.
Example 18
Transient Induction of MiR-122a Target mRNAs Following SPC3372
Treatment
[0497] NMRI female mice were treated with 25 mg/kg/day SPC3372
along with saline control for three consecutive days and sacrificed
1, 2 or 3 weeks after last dose, respectively. RNA was extracted
from livers and mRNA levels of predicted miR-122a target mRNAs,
selected by microarray data were investigated by qPCR. Three
animals from each group were analysed.
[0498] Assayed genes: Nrdg3 Aldo A, Bckdk, CD320 with predicted
miR-122 target site. Gapdh does not have a predicted miR-122a
target site.
[0499] A transient induction followed by a restoration of normal
expression levels in analogy with the restoration of normal
miR-122a levels was seen (FIG. 10).
[0500] mRNA levels are normalized to the individual GAPDH levels
and to the mean of the Saline treated group at each individual time
point. Included are also the values from the animals sacrificed 24
hours after last dose. Shown is mean and standard deviation, n=3
(24 h n=3)
Example 19
Induction of Vldlr in Liver by SPC3372 Treatment
[0501] The same liver RNA samples as in previous example were
investigated for Vldlr induction.
[0502] A transient up-regulation was seen after SPC3372 treatment,
as with the other predicted miR-122a target mRNAs (FIG. 11)
Example 20
Stability of miR-122a/SPC3372 Duplex in Mouse Plasma
[0503] Stability of SPC3372 and SPC3372/miR-122a duplex were tested
in mouse plasma at 37.degree. C. over 96 hours. Shown in FIG. 12 is
a SYBR-Gold stained PAGE.
[0504] SPC3372 was completely stable over 96 hours. The
SPC3372/miR-122a duplex was immediately truncated (degradation of
the single stranded miR-122a region not covered by SPC3372) but
thereafter almost completely stable over 96 hours.
[0505] The fact that a preformed SPC3372/miR-122 duplex showed
stability in serum over 96 hours together with the high thermal
duplex stability of SPC3372 molecule supported our notion that
inhibition of miR-122a by SPC3372 was due to stable duplex
formation between the two molecules, which has also been reported
in cell culture (Naguibneva et al. 2006).
Example 21
Sequestering of Mature MiR-122a by SPC3372 Leads to Duplex
Formation
[0506] The liver RNA was also subjected to microRNA Northern blot.
Shown in FIG. 13 is a membrane probed with a miR-122a specific
probe (upper panel) and re-probed with a Let-7 specific probe
(lower panel). With the miR-122 probe, two bands could be detected,
one corresponding to mature miR-122 and one corresponding to a
duplex between SPC3372 and miR-122.
[0507] To confirm silencing of miR-122, liver RNA samples were
subjected to small RNA northern blot analysis, which showed
significantly reduced levels of detectable mature miR-122, in
accordance with our real-time RT-PCR results. By comparison, the
levels of the let-7a control were not altered. Interestingly, we
observed dose-dependent accumulation of a shifted miR-122/SPC3372
heteroduplex band, suggesting that SPC3372 does not target miR-122
for degradation, but rather binds to the microRNA, thereby
sterically hindering its function.
Northern Blot Analysis was Performed as Follows:
[0508] Preparation of northern membranes was done as described in
Sempere et al. 2002, except for the following changes: Total RNA,
10 .mu.g per lane, in formamide loading buffer (47.5% formamide, 9
mM EDTA, 0.0125% Bromophenol Blue, 0.0125% Xylene Cyanol, 0.0125%
SDS) was loaded onto a 15% denaturing Novex TBE-Urea polyacrylamide
gel (Invitrogen) without preheating the RNA. The RNA was
electrophoretically transferred to a GeneScreen plus Hybridization
Transfer Membrane (PerkinElmer) at 200 mA for 35 min. Membranes
were probed with 32P-labelled LNA-modified oligonucleotides
complimentary to the mature microRNAs*. The LNA oligonucleotides
were labelled and hybridized to the membrane as described in
(Valoczi et al. 2004) except for the following changes: The
prehybridization and hybridization solutions contained 50%
formamide, 0.5% SDS, 5.times.SSC, 5.times.Denhardt's solution and
20 .mu.g/ml sheared denatured herring sperm DNA. Hybridizations
were performed at 45.degree. C. The blots were visualized by
scanning in a Storm 860 scanner. The signal of the background
membrane was subtracted from the radioactive signals originating
from the miRNA bands. The values of the miR-122 signals were
corrected for loading differences based on the let-7a signal. To
determine the size of the radioactive signals the Decade Marker
System (Ambion) was used according to the suppliers'
recommendations.
Example 22
MiR-122a Sequestering by SPC3372 Along with SPC3372 Distribution
Assessed by In Situ Hybridization of Liver Sections
[0509] Liver cryo-sections from treated animals were subjected to
in situ hybridizations for detection and localization of miR-122
and SPC3372 (FIG. 14). A probe complementary to miR-122 could
detect miR-122a. A second probe was complementary to SPC3372. Shown
in FIG. 14 is an overlay, in green is distribution and apparent
amounts of miR-122a and SPC3372 and blue is DAPI nuclear stain, at
10.times. magnification. 100.times. magnifications reveal the
intracellular distribution of miR-122a and SPC3372 inside the mouse
liver cells. The liver sections from saline control animals showed
a strong miR-122 staining pattern over the entire liver section,
whereas the sections from SPC3372 treated mice showed a
significantly reduced patchy staining pattern. In contrast, SPC3372
molecule was readily detected in SPC3372 treated liver, but not in
the untreated saline control liver. Higher magnification localized
miR-122a to the cytoplasm in the hepatocytes, where the miR-122 in
situ pattern was clearly compartmentalized, while SPC3372 molecule
was evenly distributed in the entire cytoplasm.
Example 23
Micro Array Analysis
[0510] We carried out genome-wide expression profiling of total RNA
samples from saline LNA-antimiR-122 treated and LNA mismatch
control treated mice livers 24 hours after the last dose using
Affymetrix Mouse Genome 430 2.0 arrays. Analysis of the array data
revealed 455 transcripts that were upregulated in the LNA-antimiR
treated mice livers compared to saline and LNA mismatch controls,
while 54 transcripts were downregulated (FIG. 15a). A total of 415
of the upregulated and 53 downregulated transcripts could be
identified in the Ensembl database. We subsequently examined the 3'
untranslated regions (UTRs) of the differentially expressed mRNAs
for the presence of the 6 nt sequence CACTCC, corresponding to the
reverse complement of the nucleotide 2-7 seed region in mature
miR-122. The number of transcripts having at least one miR-122
recognition sequence was 213 (51%) among the upregulated
transcripts, and 10 (19%) within the down-regulated transcripts,
while the frequency in a random sequence population was 25%,
implying that a significant pool of the upregulated mRNAs represent
direct miR-122 targets in the liver (FIG. 15b).
[0511] The LNA-antimiR treatment showed maximal reduction of
miR-122 levels at 24 hours, 50% reduction at one week and matched
saline controls at three weeks after last LNA dose (Example 12 "old
design"). This coincided with a markedly reduced number of
differentially expressed genes between the two mice groups at the
later time points. Compared to the 509 mRNAs 24 hours after the
last LNA dose we identified 251 differentially expressed genes
after one week, but only 18 genes after three weeks post treatment
(FIGS. 15c and 15d). In general genes upregulated 24 hours after
LNA-antimiR treatment then reverted towards control levels over the
next two weeks (FIG. 15d).
[0512] In conclusion, a large portion of up-regulated/de-repressed
genes after LNA-antimiR treatment are miR-122 targets, indicating a
very specific effect for blocking miR-122. Also genes
up-regulated/de-repressed approach normal levels 3 weeks after end
of treatment, suggest a relative long therapeutic effect, but
however not cause a permanent alteration, ie the effect is
reversible.
Methods:
Gene Expression Profiling of LNA-AntimiR Treated Mice.
[0513] Expression profiles of livers of saline and LNA-antimiR
treated mice were compared. NMRI female mice were treated with 25
mg/kg/day of LNA-antimiR along with saline control for three
consecutive days and sacrificed 24 h, 1, 2 or 3 weeks after last
dose. Additionally, expression profiles of livers of mice treated
with the mismatch LNA control oligonucleotide 24 h after last dose
were obtained. Three mice from each group were analyzed, yielding a
total of 21 expression profiles. RNA quality and concentration was
measured using an Agilent 2100 Bioanalyzer and Nanodrop ND-1000,
respectively. Total RNA was processed following the GeneChip
Expression 3'-Amplification Reagents One-cycle cDNA synthesis kit
instructions (Affymetrix Inc, Santa Clara, Calif., USA) to produce
double-stranded cDNA. This was used as a template to generate
biotin-labeled cRNA following manufacturer's specifications.
Fifteen micrograms of biotin-labeled cRNA was fragmented to strands
between 35 and 200 bases in length, of which 10 micrograms were
hybridised onto Affymetrix Mouse Genome 430 2.0 arrays overnight in
the GeneChip Hybridisation oven 6400 using standard procedures. The
arrays were washed and stained in a GeneChip Fluidics Station 450.
Scanning was carried out using the GeneChip Scanner 3000 and image
analysis was performed using GeneChip Operating Software.
Normalization and statistical analysis were done using the LIMMA
software package for the R programming environment 27. Probes
reported as absent by GCOS software in all hybridizations were
removed from the dataset. Additionally, an intensity filter was
applied to the dataset to remove probes displaying
background-corrected intensities below 16. Data were normalized
using quantile normalization 28. Differential expression was
assessed using a linear model method. P values were adjusted for
multiple testing using the Benjamini and Hochberg. Tests were
considered to be significant if the adjusted p values were
p<0.05. Clustering and visualization of Affymetrix array data
were done using the MultiExperiment Viewer software 29.
Target Site Prediction
[0514] Transcripts with annotated 3' UTRs were extracted from the
Ensembl database (Release 41) using the EnsMart data mining tool 30
and searched for the presence of the CACTCC sequence which is the
reverse complement of the nucleotide 2-7 seed in the mature miR-122
sequence. As a background control, a set of 1000 sequences with a
length of 1200 nt, corresponding to the mean 3' UTR length of the
up- and downregulated transcripts at 24 h after last LNA-antimiR
dose, were searched for the 6 nucleotide miR-122 seed matches. This
was carried out 500 times and the mean count was used for
comparison
Example 24
Dose-Dependent Inhibition of MiR-122 in Mouse Liver by LNA-AntimiR
is Enhanced as Compared to Antagomir Inhibition of miR-122
[0515] NMRI female mice were treated with indicated doses of
LNA-antimiR (SPC3372) along with a mismatch control (m, SPC3373),
saline and antagomir (SPC3595) for three consecutive days and
sacrificed 24 hours after last dose (as in example 11 "old design",
n=5). miR-122 levels were analyzed by qPCR and normalized to the
saline treated group. Genes with predicted miR-122 target site and
up regulated in the expression profiling (AldoA, Nrdg3, Bckdk and
CD320) showed dose-dependent de-repression by increasing
LNA-antimiR doses measured by qPCR.
[0516] The de-repression was consistently higher on all tested
miR-122 target mRNAs (AldoA, Bckdk, CD320 and Nrdg3 FIG. 17, 18,
19, 20) in LNA-antimiR treated mice compared to antagomir treated
mice. This was also indicated when analysing the inhibition of
miR-122 by miR-122 specific qPCR (FIG. 16). Hence LNA-antimiRs give
a more potent functional inhibition of miR-122 than corresponding
dose antagomir.
Example 25
Inhibition of MiR-122 by LNA-AntimiR in Hypercholesterolemic Mice
Along with Cholesterol Reduction and MiR-122 Target mRNA
De-Repression
[0517] C57BL/63 female mice were fed on high fat diet for 13 weeks
before the initiation of the SPC3649 treatment. This resulted in
increased weight to 30-35 g compared to the weight of normal mice,
which was just under 20 g, as weighed at the start of the
LNA-antimiR treatment. The high fat diet mice lead to significantly
increased total plasma cholesterol level of about 130 mg/dl, thus
rendering the mice hypercholesterolemic compared to the normal
level of about 70 mg/dl. Both hypercholesterolemic and normal mice
were treated i.p. twice weekly with 5 mg/kg SPC3649 and the
corresponding mismatch control SPC3744 for a study period of 51/2
weeks. Blood samples were collected weekly and total plasma
cholesterol was measured during the entire course of the study.
Upon sacrificing the mice, liver and blood samples were prepared
for total RNA extraction, miRNA and mRNA quantification, assessment
of the serum transaminase levels, and liver histology.
[0518] Treatment of hypercholesterolemic mice with SPC3649 resulted
in reduction of total plasma cholesterol of about 30% compared to
saline control mice already after 10 days and sustained at this
level during the entire study (FIG. 21). The effect was not as
pronounced in the normal diet mice. By contrast, the mismatch
control SPC3744 did not affect the plasma cholesterol levels in
neither hypercholesterolemic nor normal mice.
[0519] Quantification of miR-122 inhibition and miR-122 target gene
mRNA de-repression (AldoA and Bckdk) after the long-term treatment
with SPC3649 revealed a comparable profile in both
hypercholesterolemic and normal mice (FIG. 22, 23, 24), thereby
demonstrating the potency of SPC3649 in miR-122 antagonism in both
animal groups. The miR-122 qPCR assay indicated that also the
mismatch control SPC3744 had an effect on miR-122 levels in the
treated mice livers, albeit to a lesser extent compared to SPC3649.
This might be a reduction associated with the stem-loop qPCR.
Consistent with this notion, treatment of mice with the mismatch
control SPC3744 did not result in any functional de-repression of
the direct miR-122 target genes (FIGS. 23 and 24) nor reduction of
plasma cholesterol (FIG. 21), implying that SPC3649-mediated
antagonism of miR-122 is highly specific in vivo.
[0520] Liver enzymes in hypercholesterolemic and normal mice livers
were assessed after long term SPC3649 treatment. No changes in the
alanine and aspartate aminotransferase (ALT and AST) levels were
detected in the SPC3649 treated hypercholesterolemic mice compared
to saline control mice (FIGS. 25 and 26). A possibly elevated ALT
level was observed in the normal mice after long-term treatment
with SPC3649 (FIG. 26).
Example 26
Methods for Performing the LNA-AntimiR/Hypercholesterolemic
Experiment and Analysis
Mice and Dosing.
[0521] C57BL/63 female mice (Taconic M&B Laboratory Animals,
Ejby, Denmark) were used. All substances were formulated in
physiological saline (0.9% NaCl) to final concentration allowing
the mice to receive an intraperitoneal injection volume of 10
ml/kg.
[0522] In the diet induced obesity study, the mice received a high
fat (60EN %) diet (D12492, Research Diets) for 13 weeks to increase
their blood cholesterol level before the dosing started. The dose
regimen was stretched out to 51/2 weeks of 5 mg/kg LNA-antimiR.TM.
twice weekly. Blood plasma was collected once a week during the
entire dosing period. After completion of the experiment the mice
were sacrificed and RNA extracted from the livers for further
analysis. Serum was also collected for analysis of liver
enzymes.
Total RNA Extraction.
[0523] The dissected livers from sacrificed mice were immediately
stored in RNA later (Ambion). Total RNA was extracted with Trizol
reagent according to the manufacturer's instructions (Invitrogen),
except that the precipitated RNA pellet was washed in 80% ethanol
and not vortexed.
MicroRNA-Specific Quantitative RT-PCR.
[0524] The miR-122 and let-7a microRNA levels were quantified with
TaqMan microRNA Assay (Applied Biosystems) following the
manufacturer's instructions. The RT reaction was diluted ten times
in water and subsequently used for real time PCR amplification
according to the manufacturer's instructions. A two-fold cDNA
dilution series from liver total RNA of a saline-treated animal or
mock transfected cells cDNA reaction (using 2.5 times more total
RNA than in samples) served as standard to ensure a linear range
(Ct versus relative copy number) of the amplification. Applied
Biosystems 7500 or 7900 real-time PCR instrument was used for
amplification.
Quantitative RT-PCR
[0525] mRNA quantification of selected genes was done using
standard TaqMan assays (Applied Biosystems). The reverse
transcription reaction was carried out with random decamers, 0.5
.mu.g total RNA, and the M-MLV RT enzyme from Ambion according to a
standard protocol. First strand cDNA was subsequently diluted 10
times in nuclease-free water before addition to the RT-PCR reaction
mixture. A two-fold cDNA dilution series from liver total RNA of a
saline-treated animal or mock transfected cells cDNA reaction
(using 2.5 times more total RNA than in samples) served as standard
to ensure a linear range (Ct versus relative copy number) of the
amplification. Applied Biosystems 7500 or 7900 real-time PCR
instrument was used for amplification.
Metabolic Measurements.
[0526] Immediately before sacrifice retro-orbital sinus blood was
collected in EDTA-coated tubes followed by isolation of the plasma
fraction. Total plasma cholesterol was analysed using ABX Pentra
Cholesterol CP (Horiba Group, Horiba ABX Diagnostics) according to
the manufacturer's instructions.
Liver Enzymes (ALT and AST) Measurement
[0527] Serum from each individual mouse was prepared as follows:
Blood samples were stored at room temperature for 2 h before
centrifugation (10 min, 3000 rpm at room temperature). After
centrifugation, serum was harvested and frozen at -20.degree.
C.
[0528] ALT and AST measurement was performed in 96-well plates
using ALT and AST reagents from ABX Pentra according to the
manufacturer's instructions. In short, serum samples were diluted
2.5 fold with H.sub.2O and each sample was assayed in duplicate.
After addition of 50 .mu.l diluted sample or standard (multical
from ABX Pentra) to each well, 200 .mu.l of 37.degree. C. AST or
ALT reagent mix was added to each well. Kinetic measurements were
performed for 5 min with an interval of 30s at 340 nm and
37.degree. C. using a spectrophotometer.
Example 27
Modulation of Hepatitis C Replication by LNA-AntimiR (SPC3649)
[0529] Oligos used in this example (uppercase: LNA, lowercase DNA,
LNA Cs are methyl--.sup.mC, and LNAs are preferably B-D-oxy (o
subscript after LNA residue e.g. C.sub.s.sup.o):
TABLE-US-00008 SPC3649 (LNA-antimiR targeting miR-122, was in the
initial small scale synthesis desig- nated SPC3549) SEQ ID 558
5'-.sup.mC.sub.s.sup.oc.sub.sA.sub.s.sup.ot.sub.st.sub.sG.sub.s.sup.oT.sub-
.s.sup.oc.sub.sa.sub.s.sup.mC.sub.s.sup.oa.sub.s.sup.mC.sub.s.sup.ot.sub.s-
.sup.mC.sub.s.sup.omC.sup.o-3' SPC3648 (LNA-antimiR targeting
miR-122, was in the initial small scale synthesis desig- nated
SPC3548)
5'-A.sub.s.sup.ot.sub.st.sub.sG.sub.s.sup.oT.sub.s.sup.oc.sub.sa.sub.s.sup-
.mC.sub.s.sup.oa.sub.s.sup.mC.sub.s.sup.ot.sub.s.sup.mC.sub.s.sup.omC.sup.-
o-3' SPC3550 (4 nt mismatch control to SPC3649) SEQ ID 592
5'-.sup.mC.sub.s.sup.oc.sub.sA.sub.s.sup.ot.sub.st.sub.s.sup.mC.sub.s.sup.-
oT.sub.s.sup.og.sub.sa.sub.s.sup.mC.sub.s.sup.oc.sub.s.sup.mC.sub.s.sup.ot-
.sub.sA.sub.s.sup.omC.sup.o-3' 2'OMe anti-122: full length (23 nt)
2'OMe modified oligo complementary to miR-122 2'OMe Ctrl: scrambled
2'OMe modified control
[0530] Hepatitis C(HCV) replication has been shown to be
facilitated by miR-122 and consequently, antagonizing miR-122 has
been demonstrated to affect HCV replication in a hepatoma cell
model in vitro. We assess the efficacy of SPC3649 reducing HCV
replication in the Huh-7 based cell model. The different
LNA-antimiR molecules along with a 2' OMe antisense and scramble
oligonucleotide are transfected into Huh-7 cells, HCV is allowed to
replicate for 48 hours. Total RNA samples extracted from the Huh-7
cells are subjected to Northern blot analysis.
[0531] A significant reduction of HCV RNA was observed in cells
treated with SPC3649 as compared to the mock and SPC3550 mismatch
control. The inhibition was clearly dose-dependent with both
SPC3649 and SPC3648. Interestingly, using a 2'OMe oligonucleotide
fully complementary to miR-122 at 50 nM was much less efficient
than SPC3649 at the same final concentration. Notably, the 13 nt
SPC3648 LNA-antimiR showed comparable efficacy with SPC3649.
Example 28
Enhanced LNA-AntimiR.TM. Antisense Oligonucleotide Targeting
miR-21
[0532] Mature MiR-21 Sequence from Sanger Institute MiRBase:
TABLE-US-00009 >hsa-miR-21 MIMAT0000076 UAGCUUAUCAGACUGAUGUUGA
(SEQ ID NO 565) >mmu-miR-21 MIMAT0000530 UAGCUUAUCAGACUGAUGUUGA
SEQ ID NO 593)
Sequence of Compounds:
TABLE-US-00010 [0533] SPC3521 miR-21 5'-FAM TCAgtctgataaGCTa-3'
(gap-mer design) (SEQ ID NO 594) SPC3870 miR-21(mm) 5'-FAM
TCCgtcttagaaGATa-3' (SEQ ID NO 595) SPC3825 miR-21 5'-FAM
TcTgtCAgaTaCgAT-3' (new design) (SEQ ID NO 596) SPC3826 miR-21(mm)
5'-FAM TcAgtCTgaTaAgCT-3' (SEQ ID NO 597) SPC3827 miR-21 5'-FAM
TcAGtCTGaTaAgCT-3' (new, enhanced design) (SEQ ID NO 598)
[0534] All compounds preferably have a fully or almost fully
thiolated backbone (preferably fully) and have here also a FAM
label in the 5' end (optional).
[0535] miR-21 has been show to be up-regulated in both glioblastoma
(Chan et al. Cancer Research 2005, 65 (14), p 6029) and breast
cancer (Iorio et al. Cancer Research 2005, 65 (16), p 7065) and
hence has been considered a potential `oncogenic` microRNA. Chan et
al. also show induction of apoptosis in glioblastoma cells by
antagonising miR-21 with 2'OMe or LNA modified antisense
oligonucleotides. Hence, agents antagonising miR-21 have the
potential to become therapeutics for treatment of glioblastoma and
other solid tumours, such as breast cancer. We present an enhanced
LNA modified oligonucleotide targeting miR-21, an LNA-antimiR.TM.,
with surprisingly good properties to inhibit miR-21 suited for the
abovementioned therapeutic purposes.
[0536] Suitable therapeutic administration routes are, for example,
intracranial injections in glioblastomas, intratumoural injections
in glioblastoma and breast cancer, as well as systemic delivery in
breast cancer
Inhibition of miR-21 in 0373 Glioblastoma Cell Line and MCF-7
Breast Cancer Cell Line.
[0537] Efficacy of current LNA-antimiR.TM. is assessed by
transfection at different concentrations, along with control
oligonucleotides, into U373 and MCF-7 cell lines known to express
miR-21 (or others miR-21 expressing cell lines as well).
Transfection is performed using standard Lipofectamine2000 protocol
(Invitrogen). 24 hours post transfection, the cells are harvested
and total RNA extracted using the Trizol protocol (Invitrogen).
Assessment of miR-21 levels, depending on treatment and
concentration used is done by miR-21 specific, stem-loop real-time
RT-PCR (Applied Biosystems), or alternatively by miR-21 specific
non-radioactive northern blot analyses. The detected miR-21 levels
compared to vehicle control reflects the inhibitory potential of
the LNA-antimiR.TM..
Functional Inhibition of miR-21 by Assessment of miR-21 Target Gene
Up-Regulation.
[0538] The effect of miR-21 antagonism is investigated through
cloning of the perfect match miR-21 target sequence behind a
standard Renilla luciferase reporter system (between coding
sequence and 3' UTR, psiCHECK-2, Promega)--see Example 29. The
reporter construct and LNA-antimiR.TM. will be co-transfected into
miR-21 expressing cell lines (f. ex. U373, MCF-7). The cells are
harvested 24 hours post transfection in passive lysis buffer and
the luciferase activity is measured according to a standard
protocol (Promega, Dual Luciferase Reporter Assay System). The
induction of luciferase activity is used to demonstrate the
functional effect of LNA-antimiR.TM. antagonising miR-21.
Example 29
Luciferase Reporter Assay for Assessing Functional Inhibition of
MicroRNA by LNA-AntimiRs and Other MicroRNA Targeting Oligos:
Generalisation of New and Enhanced New Design as Preferred Design
for Blocking MicroRNA Function
[0539] Oligos used in this example (uppercase: LNA, lowercase: DNA)
to assess LNA-antimiR de-repressing effect on luciferase reporter
with microRNA target sequence cloned by blocking respective
microRNA:
TABLE-US-00011 target: hsa-miR-122a MIMAT0000421
uggagugugacaaugguguuugu screened in HUH-7 cell line expressing
miR-122 Oligo #, target microRNA, oligo sequence Design 3962:
miR-122 5'-ACAAacaccattgtcacacTCCA-3' Full complement, gap 3965:
miR-122 5'-acaaacACCATTGTcacactcca-3' Full complement, block 3972:
miR-122 5'-acAaaCacCatTgtCacActCca-3' Full complement, LNA_3 3549
(3649): miR-122 5'-CcAttGTcaCaCtCC-3' New design 3975: miR-122
5'-CcAtTGTcaCACtCC-3' Enhanced new design target: hsa-miR-19b
MIMAT0000074 ugugcaaauccaugcaaaacuga screened HeLa cell line
expressing miR-19b Oligo #, target microRNA, oligo sequence Design
3963: miR-19b 5'-TCAGttttgcatggatttgCACA-3' Full complement, gap
3967: miR-19b 5'-tcagttTTGCATGGatttgcaca-3' Full complement, block
3973: miR-19b 5'-tcAgtTttGcaTggAttTgcAca-3' Full complement, LNA_3
3560: miR-19b 5'-TgCatGGatTtGcAC-3' New design 3976: miR-19b
5'-TgCaTGGatTTGcAC-3' Enhanced new design target: hsa-miR-155
MIMAT0000646 uuaaugcuaaucgugauagggg screen in 518A2 cell line
expressing miR-155 Oligo #, target microRNA, oligo sequence Design
3964: miR-155 5'-CCCCtatcacgattagcaTTAA-3' Full complement, gap
3968: miR-155 5'-cccctaTCACGATTagcattaa-3' Full complement, block
3974: miR-155 5'-cCccTatCacGatTagCatTaa-3' Full complement, LNA_3
3758: miR-155 5'-TcAcgATtaGcAtTA-3' New design 3818: miR-155
5'-TcAcGATtaGCAtTA-3' Enhanced new design SEQ ID NOs as before.
[0540] A reporter plasmid (psiCheck-2 Promega) encoding both the
Renilla and the Firefly variants of luciferase was engineered so
that the 3'UTR of the Renilla luciferase includes a single copy of
a sequence fully complementary to the miRNA under
investigation.
[0541] Cells endogenously expressing the investigated miRNAs (HuH-7
for miR-122a, HeLa for miR-19b, 518A2 for miR-155) were
co-transfected with LNA-antimiRs or other miR binding
oligonucleotides (the full complementary ie full length) and the
corresponding microRNA target reporter plasmid using Lipofectamine
2000 (Invitrogen). The transfection and measurement of luciferase
activity were carried out according to the manufacturer's
instructions (Invitrogen Lipofectamine 2000/Promega Dual-luciferase
kit) using 150 000 to 300 000 cells per well in 6-well plates. To
compensate for varying cell densities and transfection efficiencies
the Renilla luciferase signal was normalized with the Firefly
luciferase signal. All experiments were done in triplicate.
[0542] Surprisingly, new design and new enhanced design were the
best functional inhibitors for all three microRNA targets, miR-155,
miR-19b and miR-122 (FIG. 27, 28, 29). The results are summarized
in following table 3.
Result Summary:
TABLE-US-00012 [0543] TABLE 3 Degree of de-repression of endogenous
miR-155, miR-19b and miR-122a function by various designs of
LNA-antimiR's. Design miR-155 miR-19b miR-122a New enhanced design
*** *** no data New design *** *** *** Full complement, LNA_3 **
*** ** Full complement, block ** ** ** Full complement, gap * not
signif. not signif.
Example 30
Design of a LNA AntimiR Library for all Human MicroRNA Sequences in
miRBase MicroRNA Database Version 8.1, Griffiths-Jones, S.,
Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. 2006.
MiRBase: MicroRNA Sequences, Targets and Gene Nomenclature. Nucleic
Acids Res. 34: D140-4
(http://microrna.sanger.ac.uk/sequences/index.shtml)
[0544] LNA nucleotides are shown in uppercase letters, DNA
nucleotides in lowercase letters, LNA
[0545] C nucleotides denote LNA methyl-C (mC). The LNA-antimiR
oligonucleotides can be conjugated with a variety of haptens or
fluorochromes for monitoring uptake into cells and tissues using
standard methods.
TABLE-US-00013 TABLE 2 Accession Example LNA antimiR 5'- microRNA
nr. SEQ ID NO 3' hsa-let-7a MIMAT0000062 SEQ ID NO 1
AcAacCTacTaCcTC hsa-let-7b MIMAT0000063 SEQ ID NO 2 AcAacCTacTaCcTC
hsa-let-7c MIMAT0000064 SEQ ID NO 3 AcAacCTacTaCcTC hsa-let-7d
MIMAT0000065 SEQ ID NO 4 GcAacCTacTaCcTC hsa-let-7e MIMAT0000066
SEQ ID NO 5 AcAacCTccTaCcTC hsa-let-7f MIMAT0000067 SEQ ID NO 6
AcAatCTacTaCcTC hsa-miR-15a MIMAT0000068 SEQ ID NO 7
CcAttATgtGcTgCT hsa-miR-16 MIMAT0000069 SEQ ID NO 8 TaTttACgtGcTgCT
hsa-miR-17-5p MIMAT0000070 SEQ ID NO 9 CaCtgTAagCaCtTT
hsa-miR-17-3p MIMAT0000071 SEQ ID NO 10 GtGccTTcaCtGcAG hsa-miR-18a
MIMAT0000072 SEQ ID NO 11 CaCtaGAtgCaCcTT hsa-miR-19a MIMAT0000073
SEQ ID NO 12 TgCatAGatTtGcAC hsa-miR-19b MIMAT0000074 SEQ ID NO 13
TgCatGGatTtGcAC hsa-miR-20a MIMAT0000075 SEQ ID NO 14
CaCtaTAagCaCtTT hsa-miR-21 MIMAT0000076 SEQ ID NO 15
TcAgtCTgaTaAgCT hsa-miR-22 MIMAT0000077 SEQ ID NO 16
CtTcaACtgGcAgCT hsa-miR-23a MIMAT0000078 SEQ ID NO 17
TcCctGGcaAtGtGA hsa-miR-189 MIMAT0000079 SEQ ID NO 18
TcAgcTCagTaGgCA hsa-miR-24 MIMAT0000080 SEQ ID NO 19
CtGctGAacTgAgCC hsa-miR-25 MIMAT0000081 SEQ ID NO 20
CgAgaCAagTgCaAT hsa-miR-26a MIMAT0000082 SEQ ID NO 21
TcCtgGAttAcTtGA hsa-miR-26b MIMAT0000083 SEQ ID NO 22
TcCtgAAttAcTtGA hsa-miR-27a MIMAT0000084 SEQ ID NO 23
AcTtaGCcaCtGtGA hsa-miR-28 MIMAT0000085 SEQ ID NO 24
AgActGTgaGcTcCT hsa-miR-29a MIMAT0000086 SEQ ID NO 25
AtTtcAGatGgTgCT hsa-miR-30a-5p MIMAT0000087 SEQ ID NO 26
GtCgaGGatGtTtAC hsa-miR-30a-3p MIMAT0000088 SEQ ID NO 27
AaCatCCgaCtGaAA hsa-miR-31 MIMAT0000089 SEQ ID NO 28
AtGccAGcaTcTtGC hsa-miR-32 MIMAT0000090 SEQ ID NO 29
TtAgtAAtgTgCaAT hsa-miR-33 MIMAT0000091 SEQ ID NO 30
TgCaaCTacAaTgCA hsa-miR-92 MIMAT0000092 SEQ ID NO 31
CgGgaCAagTgCaAT hsa-miR-93 MIMAT0000093 SEQ ID NO 32
GcAcgAAcaGcAcTT hsa-miR-95 MIMAT0000094 SEQ ID NO 33
AtAaaTAccCgTtGA hsa-miR-96 MIMAT0000095 SEQ ID NO 34
AtGtgCTagTgCcAA hsa-miR-98 MIMAT0000096 SEQ ID NO 35
AcAacTTacTaCcTC hsa-miR-99a MIMAT0000097 SEQ ID NO 36
AtCggATctAcGgGT hsa-miR-100 MIMAT0000098 SEQ ID NO 37
TtCggATctAcGgGT hsa-miR-101 MIMAT0000099 SEQ ID NO 38
TtAtcACagTaCtGT hsa-miR-29b MIMAT0000100 SEQ ID NO 39
AtTtcAGatGgTgCT hsa-miR-103 MIMAT0000101 SEQ ID NO 40
CcTgtACaaTgCtGC hsa-miR-105 MIMAT0000102 SEQ ID NO 41
GaGtcTGagCaTtTG hsa-miR-106a MIMAT0000103 SEQ ID NO 42
CaCtgTAagCaCtTT hsa-miR-107 MIMAT0000104 SEQ ID NO 43
CcTgtACaaTgCtGC hsa-miR-192 MIMAT0000222 SEQ ID NO 44
TcAatTCatAgGtCA hsa-miR-196a MIMAT0000226 SEQ ID NO 45
AaCatGAaaCtAcCT hsa-miR-197 MIMAT0000227 SEQ ID NO 46
TgGagAAggTgGtGA hsa-miR-198 MIMAT0000228 SEQ ID NO 47
AtCtcCCctCtGgAC hsa-miR-199a MIMAT0000231 SEQ ID NO 48
TaGtcTGaaCaCtGG hsa-miR-199a* MIMAT0000232 SEQ ID NO 49
TgTgcAGacTaCtGT hsa-miR-208 MIMAT0000241 SEQ ID NO 50
TtTttGCtcGtCtTA hsa-miR-129 MIMAT0000242 SEQ ID NO 51
CcCagACcgCaAaAA hsa-miR-148a MIMAT0000243 SEQ ID NO 52
TtCtgTAgtGcAcTG hsa-miR-30c MIMAT0000244 SEQ ID NO 53
GtGtaGGatGtTtAC hsa-miR-30d MIMAT0000245 SEQ ID NO 54
GtCggGGatGtTtAC hsa-miR-139 MIMAT0000250 SEQ ID NO 55
AcAcgTGcaCtGtAG hsa-miR-147 MIMAT0000251 SEQ ID NO 56
AaGcaTTtcCaCaCA hsa-miR-7 MIMAT0000252 SEQ ID NO 57 AaTcaCTagTcTtCC
hsa-miR-10a MIMAT0000253 SEQ ID NO 58 TcGgaTCtaCaGgGT hsa-miR-10b
MIMAT0000254 SEQ ID NO 59 TcGgtTCtaCaGgGT hsa-miR-34a MIMAT0000255
SEQ ID NO 60 AgCtaAGacAcTgCC hsa-miR-181a MIMAT0000256 SEQ ID NO 61
GaCagCGttGaAtGT hsa-miR-181b MIMAT0000257 SEQ ID NO 62
GaCagCAatGaAtGT hsa-miR-181c MIMAT0000258 SEQ ID NO 63
CgAcaGGttGaAtGT hsa-miR-182 MIMAT0000259 SEQ ID NO 64
TtCtaCCatTgCcAA hsa-miR-182* MIMAT0000260 SEQ ID NO 65
GgCaaGTctAgAaCC hsa-miR-183 MIMAT0000261 SEQ ID NO 66
TtCtaCCagTgCcAT hsa-miR-187 MIMAT0000262 SEQ ID NO 67
GcAacACaaGaCaCG hsa-miR-199b MIMAT0000263 SEQ ID NO 68
TaGtcTAaaCaCtGG hsa-miR-203 MIMAT0000264 SEQ ID NO 69
GtCctAAacAtTtCA hsa-miR-204 MIMAT0000265 SEQ ID NO 70
AgGatGAcaAaGgGA hsa-miR-205 MIMAT0000266 SEQ ID NO 71
CcGgtGGaaTgAaGG hsa-miR-210 MIMAT0000267 SEQ ID NO 72
GcTgtCAcaCgCaCA hsa-miR-211 MIMAT0000268 SEQ ID NO 73
AgGatGAcaAaGgGA hsa-miR-212 MIMAT0000269 SEQ ID NO 74
TgActGGagAcTgTT hsa-miR-181a* MIMAT0000270 SEQ ID NO 75
AtCaaCGgtCgAtGG hsa-miR-214 MIMAT0000271 SEQ ID NO 76
TgTctGTgcCtGcTG hsa-miR-215 MIMAT0000272 SEQ ID NO 77
TcAatTCatAgGtCA hsa-miR-216 MIMAT0000273 SEQ ID NO 78
TtGccAGctGaGaTT hsa-miR-217 MIMAT0000274 SEQ ID NO 79
AgTtcCTgaTgCaGT hsa-miR-218 MIMAT0000275 SEQ ID NO 80
GtTagATcaAgCaCA hsa-miR-219 MIMAT0000276 SEQ ID NO 81
TgCgtTTggAcAaTC hsa-miR-220 MIMAT0000277 SEQ ID NO 82
GtCagATacGgTgTG hsa-miR-221 MIMAT0000278 SEQ ID NO 83
AgCagACaaTgTaGC hsa-miR-222 MIMAT0000279 SEQ ID NO 84
GtAgcCAgaTgTaGC hsa-miR-223 MIMAT0000280 SEQ ID NO 85
AtTtgACaaAcTgAC hsa-miR-224 MIMAT0000281 SEQ ID NO 86
AaCcaCTagTgAcTT hsa-miR-200b MIMAT0000318 SEQ ID NO 87
TtAccAGgcAgTaTT hsa-let-7g MIMAT0000414 SEQ ID NO 88
AcAaaCTacTaCcTC hsa-let-7i MIMAT0000415 SEQ ID NO 89
AcAaaCTacTaCcTC hsa-miR-1 MIMAT0000416 SEQ ID NO 90 AcTtcTTtaCaTtCC
hsa-miR-15b MIMAT0000417 SEQ ID NO 91 CcAtgATgtGcTgCT hsa-miR-23b
MIMAT0000418 SEQ ID NO 92 TcCctGGcaAtGtGA hsa-miR-27b MIMAT0000419
SEQ ID NO 93 AcTtaGCcaCtGtGA hsa-miR-30b MIMAT0000420 SEQ ID NO 94
GtGtaGGatGtTtAC hsa-miR-122a MIMAT0000421 SEQ ID NO 95
CcAttGTcaCaCtCC hsa-miR-124a MIMAT0000422 SEQ ID NO 96
TcAccGCgtGcCtTA hsa-miR-125b MIMAT0000423 SEQ ID NO 97
GtTagGGtcTcAgGG hsa-miR-128a MIMAT0000424 SEQ ID NO 98
GaCcgGTtcAcTgTG hsa-miR-130a MIMAT0000425 SEQ ID NO 99
TtTtaACatTgCaCT hsa-miR-132 MIMAT0000426 SEQ ID NO 100
TgGctGTagAcTgTT hsa-miR-133a MIMAT0000427 SEQ ID NO 101
GgTtgAAggGgAcCA hsa-miR-135a MIMAT0000428 SEQ ID NO 102
GgAatAAaaAgCcAT hsa-miR-137 MIMAT0000429 SEQ ID NO 103
GtAttCTtaAgCaAT hsa-miR-138 MIMAT0000430 SEQ ID NO 104
AtTcaCAacAcCaGC hsa-miR-140 MIMAT0000431 SEQ ID NO 105
AtAggGTaaAaCcAC hsa-miR-141 MIMAT0000432 SEQ ID NO 106
TtAccAGacAgTgTT hsa-miR-142-5p MIMAT0000433 SEQ ID NO 107
TgCttTCtaCtTtAT hsa-miR-142-3p MIMAT0000434 SEQ ID NO 108
AgTagGAaaCaCtAC hsa-miR-143 MIMAT0000435 SEQ ID NO 109
AcAgtGCttCaTcTC hsa-miR-144 MIMAT0000436 SEQ ID NO 110
CaTcaTCtaTaCtGT hsa-miR-145 MIMAT0000437 SEQ ID NO 111
CcTggGAaaAcTgGA hsa-miR-152 MIMAT0000438 SEQ ID NO 112
TtCtgTCatGcAcTG hsa-miR-153 MIMAT0000439 SEQ ID NO 113
TtTtgTGacTaTgCA hsa-miR-191 MIMAT0000440 SEQ ID NO 114
TtTtgGGatTcCgTT hsa-miR-9 MIMAT0000441 SEQ ID NO 115
GcTagATaaCcAaAG hsa-miR-9* MIMAT0000442 SEQ ID NO 116
CgGttATctAgCtTT hsa-miR-125a MIMAT0000443 SEQ ID NO 117
TaAagGGtcTcAgGG hsa-miR-126* MIMAT0000444 SEQ ID NO 118
AcCaaAAgtAaTaAT hsa-miR-126 MIMAT0000445 SEQ ID NO 119
AtTacTCacGgTaCG hsa-miR-127 MIMAT0000446 SEQ ID NO 120
GcTcaGAcgGaTcCG hsa-miR-134 MIMAT0000447 SEQ ID NO 121
TgGtcAAccAgTcAC hsa-miR-136 MIMAT0000448 SEQ ID NO 122
TcAaaACaaAtGgAG hsa-miR-146a MIMAT0000449 SEQ ID NO 123
TgGaaTTcaGtTcTC
hsa-miR-149 MIMAT0000450 SEQ ID NO 124 AaGacACggAgCcAG hsa-miR-150
MIMAT0000451 SEQ ID NO 125 TaCaaGGgtTgGgAG hsa-miR-154 MIMAT0000452
SEQ ID NO 126 CaAcaCGgaTaAcCT hsa-miR-154* MIMAT0000453 SEQ ID NO
127 TcAacCGtgTaTgAT hsa-miR-184 MIMAT0000454 SEQ ID NO 128
AtCagTTctCcGtCC hsa-miR-185 MIMAT0000455 SEQ ID NO 129
AcTgcCTttCtCtCC hsa-miR-186 MIMAT0000456 SEQ ID NO 130
AaAggAGaaTtCtTT hsa-miR-188 MIMAT0000457 SEQ ID NO 131
CaCcaTGcaAgGgAT hsa-miR-190 MIMAT0000458 SEQ ID NO 132
TaTatCAaaCaTaTC hsa-miR-193a MIMAT0000459 SEQ ID NO 133
AcTttGTagGcCaGT hsa-miR-194 MIMAT0000460 SEQ ID NO 134
TgGagTTgcTgTtAC hsa-miR-195 MIMAT0000461 SEQ ID NO 135
TaTttCTgtGcTgCT hsa-miR-206 MIMAT0000462 SEQ ID NO 136
AcTtcCTtaCaTtCC hsa-miR-320 MIMAT0000510 SEQ ID NO 137
TcTcaACccAgCtTT hsa-miR-200c MIMAT0000617 SEQ ID NO 138
TtAccCGgcAgTaTT hsa-miR-155 MIMAT0000646 SEQ ID NO 139
TcAcgATtaGcAtTA hsa-miR-128b MIMAT0000676 SEQ ID NO 140
GaCcgGTtcAcTgTG hsa-miR-106b MIMAT0000680 SEQ ID NO 141
CaCtgTCagCaCtTT hsa-miR-29c MIMAT0000681 SEQ ID NO 142
AtTtcAAatGgTgCT hsa-miR-200a MIMAT0000682 SEQ ID NO 143
TtAccAGacAgTgTT hsa-miR-302a* MIMAT0000683 SEQ ID NO 144
AgTacATccAcGtTT hsa-miR-302a MIMAT0000684 SEQ ID NO 145
AaCatGGaaGcAcTT hsa-miR-34b MIMAT0000685 SEQ ID NO 146
CtAatGAcaCtGcCT hsa-miR-34c MIMAT0000686 SEQ ID NO 147
GcTaaCTacAcTgCC hsa-miR-299-3p MIMAT0000687 SEQ ID NO 148
TtTacCAtcCcAcAT hsa-miR-301 MIMAT0000688 SEQ ID NO 149
CaAtaCTatTgCaCT hsa-miR-99b MIMAT0000689 SEQ ID NO 150
GtCggTTctAcGgGT hsa-miR-296 MIMAT0000690 SEQ ID NO 151
AtTgaGGggGgGcCC hsa-miR-130b MIMAT0000691 SEQ ID NO 152
TtTcaTCatTgCaCT hsa-miR-30e-5p MIMAT0000692 SEQ ID NO 153
GtCaaGGatGtTtAC hsa-miR-30e-3p MIMAT0000693 SEQ ID NO 154
AaCatCCgaCtGaAA hsa-miR-361 MIMAT0000703 SEQ ID NO 155
CtGgaGAttCtGaTA hsa-miR-362 MIMAT0000705 SEQ ID NO 156
CtAggTTccAaGgAT hsa-miR-363 MIMAT0000707 SEQ ID NO 157
TgGatACcgTgCaAT hsa-miR-365 MIMAT0000710 SEQ ID NO 158
AtTttTAggGgCaTT hsa-miR-302b* MIMAT0000714 SEQ ID NO 159
AcTtcCAtgTtAaAG hsa-miR-302b MIMAT0000715 SEQ ID NO 160
AaCatGGaaGcAcTT hsa-miR-302c* MIMAT0000716 SEQ ID NO 161
GtAccCCcaTgTtAA hsa-miR-302c MIMAT0000717 SEQ ID NO 162
AaCatGGaaGcAcTT hsa-miR-302d MIMAT0000718 SEQ ID NO 163
AaCatGGaaGcAcTT hsa-miR-367 MIMAT0000719 SEQ ID NO 164
TtGctAAagTgCaAT hsa-miR-368 MIMAT0000720 SEQ ID NO 165
GgAatTTccTcTaTG hsa-miR-369-3p MIMAT0000721 SEQ ID NO 166
TcAacCAtgTaTtAT hsa-miR-370 MIMAT0000722 SEQ ID NO 167
TtCcaCCccAgCaGG hsa-miR-371 MIMAT0000723 SEQ ID NO 168
CaAaaGAtgGcGgCA hsa-miR-372 MIMAT0000724 SEQ ID NO 169
AaTgtCGcaGcAcTT hsa-miR-373* MIMAT0000725 SEQ ID NO 170
CgCccCCatTtTgAG hsa-miR-373 MIMAT0000726 SEQ ID NO 171
AaAatCGaaGcAcTT hsa-miR-374 MIMAT0000727 SEQ ID NO 172
TcAggTTgtAtTaTA hsa-miR-375 MIMAT0000728 SEQ ID NO 173
GaGccGAacGaAcAA hsa-miR-376a MIMAT0000729 SEQ ID NO 174
GaTttTCctCtAtGA hsa-miR-377 MIMAT0000730 SEQ ID NO 175
GtTgcCTttGtGtGA hsa-miR-378 MIMAT0000731 SEQ ID NO 176
GaCctGGagTcAgGA hsa-miR-422b MIMAT0000732 SEQ ID NO 177
CtGacTCcaAgTcCA hsa-miR-379 MIMAT0000733 SEQ ID NO 178
GtTccATagTcTaCC hsa-miR-380-5p MIMAT0000734 SEQ ID NO 179
GtTctATggTcAaCC hsa-miR-380-3p MIMAT0000735 SEQ ID NO 180
TgGacCAtaTtAcAT hsa-miR-381 MIMAT0000736 SEQ ID NO 181
AgCttGCccTtGtAT hsa-miR-382 MIMAT0000737 SEQ ID NO 182
CaCcaCGaaCaAcTT hsa-miR-383 MIMAT0000738 SEQ ID NO 183
AaTcaCCttCtGaTC hsa-miR-340 MIMAT0000750 SEQ ID NO 184
AaGtaACtgAgAcGG hsa-miR-330 MIMAT0000751 SEQ ID NO 185
AgGccGTgtGcTtTG hsa-miR-328 MIMAT0000752 SEQ ID NO 186
GgGcaGAgaGgGcCA hsa-miR-342 MIMAT0000753 SEQ ID NO 187
CgAttTCtgTgTgAG hsa-miR-337 MIMAT0000754 SEQ ID NO 188
TcAtaTAggAgCtGG hsa-miR-323 MIMAT0000755 SEQ ID NO 189
CgAccGTgtAaTgTG hsa-miR-326 MIMAT0000756 SEQ ID NO 190
AgGaaGGgcCcAgAG hsa-miR-151 MIMAT0000757 SEQ ID NO 191
GgAgcTTcaGtCtAG hsa-miR-135b MIMAT0000758 SEQ ID NO 192
GgAatGAaaAgCcAT hsa-miR-148b MIMAT0000759 SEQ ID NO 193
TtCtgTGatGcAcTG hsa-miR-331 MIMAT0000760 SEQ ID NO 194
GgAtaGGccCaGgGG hsa-miR-324-5p MIMAT0000761 SEQ ID NO 195
TgCccTAggGgAtGC hsa-miR-324-3p MIMAT0000762 SEQ ID NO 196
GcAccTGggGcAgTG hsa-miR-338 MIMAT0000763 SEQ ID NO 197
AaTcaCTgaTgCtGG hsa-miR-339 MIMAT0000764 SEQ ID NO 198
TcCtgGAggAcAgGG hsa-miR-335 MIMAT0000765 SEQ ID NO 199
TcGttATtgCtCtTG hsa-miR-133b MIMAT0000770 SEQ ID NO 200
GgTtgAAggGgAcCA hsa-miR-325 MIMAT0000771 SEQ ID NO 201
CtGgaCAccTaCtAG hsa-miR-345 MIMAT0000772 SEQ ID NO 202
GgActAGgaGtCaGC hsa-miR-346 MIMAT0000773 SEQ ID NO 203
GgCatGCggGcAgAC ebv-miR-BHRF1-1 MIMAT0000995 SEQ ID NO 204
GgGgcTGatCaGgTT ebv-miR-BHRF1-2* MIMAT0000996 SEQ ID NO 205
TgCtgCAacAgAaTT ebv-miR-BHRF1-2 MIMAT0000997 SEQ ID NO 206
TcTgcCGcaAaAgAT ebv-miR-BHRF1-3 MIMAT0000998 SEQ ID NO 207
TaCacACttCcCgTT ebv-miR-BART1-5p MIMAT0000999 SEQ ID NO 208
GtCacTTccAcTaAG ebv-miR-BART2 MIMAT0001000 SEQ ID NO 209
GcGaaTGcaGaAaAT hsa-miR-384 MIMAT0001075 SEQ ID NO 210
AaCaaTTtcTaGgAA hsa-miR-196b MIMAT0001080 SEQ ID NO 211
AaCagGAaaCtAcCT hsa-miR-422a MIMAT0001339 SEQ ID NO 212
CtGacCCtaAgTcCA hsa-miR-423 MIMAT0001340 SEQ ID NO 213
GgCctCAgaCcGaGC hsa-miR-424 MIMAT0001341 SEQ ID NO 214
AcAtgAAttGcTgCT hsa-miR-425-3p MIMAT0001343 SEQ ID NO 215
AcAcgACatTcCcGA hsa-miR-18b MIMAT0001412 SEQ ID NO 216
CaCtaGAtgCaCcTT hsa-miR-20b MIMAT0001413 SEQ ID NO 217
CaCtaTGagCaCtTT hsa-miR-448 MIMAT0001532 SEQ ID NO 218
CaTccTAcaTaTgCA hsa-miR-429 MIMAT0001536 SEQ ID NO 219
TtAccAGacAgTaTT hsa-miR-449 MIMAT0001541 SEQ ID NO 220
TaAcaATacAcTgCC hsa-miR-450 MIMAT0001545 SEQ ID NO 221
GaAcaCAtcGcAaAA hcmv-miR-UL22A MIMAT0001574 SEQ ID NO 222
AcGggAAggCtAgTT hcmv-miR-UL22A* MIMAT0001575 SEQ ID NO 223
AcTagCAttCtGgTG hcmv-miR-UL36 MIMAT0001576 SEQ ID NO 224
CaGgtGTctTcAaCG hcmv-miR-UL112 MIMAT0001577 SEQ ID NO 225
GaTctCAccGtCaCT hcmv-miR-UL148D MIMAT0001578 SEQ ID NO 226
AaGaaGGggAgGaCG hcmv-miR-US5-1 MIMAT0001579 SEQ ID NO 227
CtCgtCAggCtTgTC hcmv-miR-US5-2 MIMAT0001580 SEQ ID NO 228
GtCacACctAtCaTA hcmv-miR-US25-1 MIMAT0001581 SEQ ID NO 229
GaGccACtgAgCgGT hcmv-miR-US25-2-5p MIMAT0001582 SEQ ID NO 230
AcCtgAAcaGaCcGC hcmv-miR-US25-2-3p MIMAT0001583 SEQ ID NO 231
AgCtcTCcaAgTgGA hcmv-miR-US33 MIMAT0001584 SEQ ID NO 232
CgGtcCGggCaCaAT hsa-miR-191* MIMAT0001618 SEQ ID NO 233
GaAatCCaaGcGcAG hsa-miR-200a* MIMAT0001620 SEQ ID NO 234
AcTgtCCggTaAgAT hsa-miR-369-5p MIMAT0001621 SEQ ID NO 235
AtAacACggTcGaTC hsa-miR-431 MIMAT0001625 SEQ ID NO 236
GaCggCCtgCaAgAC hsa-miR-433 MIMAT0001627 SEQ ID NO 237
AgGagCCcaTcAtGA hsa-miR-329 MIMAT0001629 SEQ ID NO 238
GtTaaCCagGtGtGT hsa-miR-453 MIMAT0001630 SEQ ID NO 239
CaCcaCGgaCaAcCT hsa-miR-451 MIMAT0001631 SEQ ID NO 240
GtAatGGtaAcGgTT hsa-miR-452 MIMAT0001635 SEQ ID NO 241
GtTtcCTctGcAaAC hsa-miR-452* MIMAT0001636 SEQ ID NO 242
TtGcaGAtgAgAcTG hsa-miR-409-5p MIMAT0001638 SEQ ID NO 243
GtTgcTCggGtAaCC hsa-miR-409-3p MIMAT0001639 SEQ ID NO 244
CaCcgAGcaAcAtTC hsa-miR-412 MIMAT0002170 SEQ ID NO 245
GtGgaCCagGtGaAG hsa-miR-410 MIMAT0002171 SEQ ID NO 246
CcAtcTGtgTtAtAT hsa-miR-376b MIMAT0002172 SEQ ID NO 247
GaTttTCctCtAtGA hsa-miR-483 MIMAT0002173 SEQ ID NO 248
GgGagGAgaGgAgTG
hsa-miR-484 MIMAT0002174 SEQ ID NO 249 AgGggACtgAgCcTG
hsa-miR-485-5p MIMAT0002175 SEQ ID NO 250 AtCacGGccAgCcTC
hsa-miR-485-3p MIMAT0002176 SEQ ID NO 251 GaGagCCgtGtAtGA
hsa-miR-486 MIMAT0002177 SEQ ID NO 252 GcAgcTCagTaCaGG hsa-miR-487a
MIMAT0002178 SEQ ID NO 253 AtGtcCCtgTaTgAT kshv-miR-K12-10a
MIMAT0002179 SEQ ID NO 254 CgGggGGacAaCaCT kshv-miR-K12-10b
MIMAT0002180 SEQ ID NO 255 CgGggGGacAaCaCC kshv-miR-K12-11
MIMAT0002181 SEQ ID NO 256 AcAggCTaaGcAtTA kshv-miR-K12-1
MIMAT0002182 SEQ ID NO 257 CcCagTTtcCtGtAA kshv-miR-K12-2
MIMAT0002183 SEQ ID NO 258 GaCccGGacTaCaGT kshv-miR-K12-9*
MIMAT0002184 SEQ ID NO 259 GtTtaCGcaGcTgGG kshv-miR-K12-9
MIMAT0002185 SEQ ID NO 260 AgCtgCGtaTaCcCA kshv-miR-K12-8
MIMAT0002186 SEQ ID NO 261 CtCtcAGtcGcGcCT kshv-miR-K12-7
MIMAT0002187 SEQ ID NO 262 CaGcaACatGgGaTC kshv-miR-K12-6-5p
MIMAT0002188 SEQ ID NO 263 GaTtaGGtgCtGcTG kshv-miR-K12-6-3p
MIMAT0002189 SEQ ID NO 264 AgCccGAaaAcCaTC kshv-miR-K12-5
MIMAT0002190 SEQ ID NO 265 AgTtcCAggCaTcCT kshv-miR-K12-4-5p
MIMAT0002191 SEQ ID NO 266 GtActGCggTtTaGC kshv-miR-K12-4-3p
MIMAT0002192 SEQ ID NO 267 AgGccTCagTaTtCT kshv-miR-K12-3
MIMAT0002193 SEQ ID NO 268 CgTccTCagAaTgTG kshv-miR-K12-3*
MIMAT0002194 SEQ ID NO 269 CaTtcTGtgAcCgCG hsa-miR-488 MIMAT0002804
SEQ ID NO 270 AgTgcCAttAtCtGG hsa-miR-489 MIMAT0002805 SEQ ID NO
271 TaTatGTgaTgTcAC hsa-miR-490 MIMAT0002806 SEQ ID NO 272
GgAgtCCtcCaGgTT hsa-miR-491 MIMAT0002807 SEQ ID NO 273
GgAagGGttCcCcAC hsa-miR-511 MIMAT0002808 SEQ ID NO 274
GcAgaGCaaAaGaCA hsa-miR-146b MIMAT0002809 SEQ ID NO 275
TgGaaTTcaGtTcTC hsa-miR-202* MIMAT0002810 SEQ ID NO 276
GtAtaTGcaTaGgAA hsa-miR-202 MIMAT0002811 SEQ ID NO 277
CaTgcCCtaTaCcTC hsa-miR-492 MIMAT0002812 SEQ ID NO 278
TtGtcCCgcAgGtCC hsa-miR-493-5p MIMAT0002813 SEQ ID NO 279
AgCctACcaTgTaCA hsa-miR-432 MIMAT0002814 SEQ ID NO 280
AtGacCTacTcCaAG hsa-miR-432* MIMAT0002815 SEQ ID NO 281
TgGagGAgcCaTcCA hsa-miR-494 MIMAT0002816 SEQ ID NO 282
TcCcgTGtaTgTtTC hsa-miR-495 MIMAT0002817 SEQ ID NO 283
TgCacCAtgTtTgTT hsa-miR-496 MIMAT0002818 SEQ ID NO 284
AgAttGGccAtGtAA hsa-miR-193b MIMAT0002819 SEQ ID NO 285
AcTttGAggGcCaGT hsa-miR-497 MIMAT0002820 SEQ ID NO 286
CcAcaGTgtGcTgCT hsa-miR-181d MIMAT0002821 SEQ ID NO 287
GaCaaCAatGaAtGT hsa-miR-512-5p MIMAT0002822 SEQ ID NO 288
CcCtcAAggCtGaGT hsa-miR-512-3p MIMAT0002823 SEQ ID NO 289
AgCtaTGacAgCaCT hsa-miR-498 MIMAT0002824 SEQ ID NO 290
GcCccCTggCtTgAA hsa-miR-520e MIMAT0002825 SEQ ID NO 291
AaAaaGGaaGcAcTT hsa-miR-515-5p MIMAT0002826 SEQ ID NO 292
GcTttCTttTgGaGA hsa-miR-515-3p MIMAT0002827 SEQ ID NO 293
CcAaaAGaaGgCaCT hsa-miR-519e* MIMAT0002828 SEQ ID NO 294
GcTccCTttTgGaGA hsa-miR-519e MIMAT0002829 SEQ ID NO 295
TaAaaGGagGcAcTT hsa-miR-520f MIMAT0002830 SEQ ID NO 296
CtAaaAGgaAgCaCT hsa-miR-526c MIMAT0002831 SEQ ID NO 297
GcGctTCccTcTaGA hsa-miR-519c MIMAT0002832 SEQ ID NO 298
TaAaaAGatGcAcTT hsa-miR-520a* MIMAT0002833 SEQ ID NO 299
GtActTCccTcTgGA hsa-miR-520a MIMAT0002834 SEQ ID NO 300
CaAagGGaaGcAcTT hsa-miR-526b MIMAT0002835 SEQ ID NO 301
GtGctTCccTcAaGA hsa-miR-526b* MIMAT0002836 SEQ ID NO 302
TaAaaGGaaGcAcTT hsa-miR-519b MIMAT0002837 SEQ ID NO 303
TaAaaGGatGcAcTT hsa-miR-525 MIMAT0002838 SEQ ID NO 304
GtGcaTCccTcTgGA hsa-miR-525* MIMAT0002839 SEQ ID NO 305
AaAggGAagCgCcTT hsa-miR-523 MIMAT0002840 SEQ ID NO 306
TaTagGGaaGcGcGT hsa-miR-518f* MIMAT0002841 SEQ ID NO 307
GtGctTCccTcTaGA hsa-miR-518f MIMAT0002842 SEQ ID NO 308
TaAagAGaaGcGcTT hsa-miR-520b MIMAT0002843 SEQ ID NO 309
TaAaaGGaaGcAcTT hsa-miR-518b MIMAT0002844 SEQ ID NO 310
AaAggGGagCgCtTT hsa-miR-526a MIMAT0002845 SEQ ID NO 311
GtGctTCccTcTaGA hsa-miR-520c MIMAT0002846 SEQ ID NO 312
TaAaaGGaaGcAcTT hsa-miR-518c* MIMAT0002847 SEQ ID NO 313
TgCttCCctCcAgAG hsa-miR-518c MIMAT0002848 SEQ ID NO 314
AaAgaGAagCgCtTT hsa-miR-524* MIMAT0002849 SEQ ID NO 315
GtGctTCccTtTgTA hsa-miR-524 MIMAT0002850 SEQ ID NO 316
AaAggGAagCgCcTT hsa-miR-517* MIMAT0002851 SEQ ID NO 317
TgCttCCatCtAgAG hsa-miR-517a MIMAT0002852 SEQ ID NO 318
TaAagGGatGcAcGA hsa-miR-519d MIMAT0002853 SEQ ID NO 319
AaAggGAggCaCtTT hsa-miR-521 MIMAT0002854 SEQ ID NO 320
TaAagGGaaGtGcGT hsa-miR-520d* MIMAT0002855 SEQ ID NO 321
GgCttCCctTtGtAG hsa-miR-520d MIMAT0002856 SEQ ID NO 322
CaAagAGaaGcAcTT hsa-miR-517b MIMAT0002857 SEQ ID NO 323
CtAaaGGgaTgCaCG hsa-miR-520g MIMAT0002858 SEQ ID NO 324
AaGggAAgcAcTtTG hsa-miR-516-5p MIMAT0002859 SEQ ID NO 325
TtCttACctCcAgAT hsa-miR-516-3p MIMAT0002860 SEQ ID NO 326
CcTctGAaaGgAaGC hsa-miR-518e MIMAT0002861 SEQ ID NO 327
TgAagGGaaGcGcTT hsa-miR-527 MIMAT0002862 SEQ ID NO 328
GgGctTCccTtTgCA hsa-miR-518a MIMAT0002863 SEQ ID NO 329
CaAagGGaaGcGcTT hsa-miR-518d MIMAT0002864 SEQ ID NO 330
AaAggGAagCgCtTT hsa-miR-517c MIMAT0002866 SEQ ID NO 331
TaAaaGGatGcAcGA hsa-miR-520h MIMAT0002867 SEQ ID NO 332
AaGggAAgcAcTtTG hsa-miR-522 MIMAT0002868 SEQ ID NO 333
TaAagGGaaCcAtTT hsa-miR-519a MIMAT0002869 SEQ ID NO 334
TaAaaGGatGcAcTT hsa-miR-499 MIMAT0002870 SEQ ID NO 335
TcActGCaaGtCtTA hsa-miR-500 MIMAT0002871 SEQ ID NO 336
CcTtgCCcaGgTgCA hsa-miR-501 MIMAT0002872 SEQ ID NO 337
CcAggGAcaAaGgAT hsa-miR-502 MIMAT0002873 SEQ ID NO 338
CcCagATagCaAgGA hsa-miR-503 MIMAT0002874 SEQ ID NO 339
AcTgtTCccGcTgCT hsa-miR-504 MIMAT0002875 SEQ ID NO 340
GtGcaGAccAgGgTC hsa-miR-505 MIMAT0002876 SEQ ID NO 341
AcCagCAagTgTtGA hsa-miR-513 MIMAT0002877 SEQ ID NO 342
GaCacCTccCtGtGA hsa-miR-506 MIMAT0002878 SEQ ID NO 343
TcAgaAGggTgCcTT hsa-miR-507 MIMAT0002879 SEQ ID NO 344
TcCaaAAggTgCaAA hsa-miR-508 MIMAT0002880 SEQ ID NO 345
CaAaaGGctAcAaTC hsa-miR-509 MIMAT0002881 SEQ ID NO 346
AcAgaCGtaCcAaTC hsa-miR-510 MIMAT0002882 SEQ ID NO 347
GcCacTCtcCtGaGT hsa-miR-514 MIMAT0002883 SEQ ID NO 348
TcAcaGAagTgTcAA hsa-miR-532 MIMAT0002888 SEQ ID NO 349
CtAcaCTcaAgGcAT hsa-miR-299-5p MIMAT0002890 SEQ ID NO 350
GtGggACggTaAaCC hsa-miR-18a* MIMAT0002891 SEQ ID NO 351
GaGcaCTtaGgGcAG hsa-miR-455 MIMAT0003150 SEQ ID NO 352
AgTccAAagGcAcAT hsa-miR-493-3p MIMAT0003161 SEQ ID NO 353
AcAcaGTagAcCtTC hsa-miR-539 MIMAT0003163 SEQ ID NO 354
CaAggATaaTtTcTC hsa-miR-544 MIMAT0003164 SEQ ID NO 355
GcTaaAAatGcAgAA hsa-miR-545 MIMAT0003165 SEQ ID NO 356
AtAaaTGttTgCtGA hsa-miR-487b MIMAT0003180 SEQ ID NO 357
AtGacCCtgTaCgAT hsa-miR-551a MIMAT0003214 SEQ ID NO 358
AcCaaGAgtGgGtCG hsa-miR-552 MIMAT0003215 SEQ ID NO 359
TaAccAGtcAcCtGT hsa-miR-553 MIMAT0003216 SEQ ID NO 360
AaAatCTcaCcGtTT hsa-miR-554 MIMAT0003217 SEQ ID NO 361
CtGagTCagGaCtAG hsa-miR-92b MIMAT0003218 SEQ ID NO 362
CgGgaCGagTgCaAT hsa-miR-555 MIMAT0003219 SEQ ID NO 363
AgGttCAgcTtAcCC hsa-miR-556 MIMAT0003220 SEQ ID NO 364
TtAcaATgaGcTcAT hsa-miR-557 MIMAT0003221 SEQ ID NO 365
GcCcaCCcgTgCaAA hsa-miR-558 MIMAT0003222 SEQ ID NO 366
TtGgtACagCaGcTC hsa-miR-559 MIMAT0003223 SEQ ID NO 367
GtGcaTAttTaCtTT hsa-miR-560 MIMAT0003224 SEQ ID NO 368
GcCggCCggCgCaCG hsa-miR-561 MIMAT0003225 SEQ ID NO 369
AgGatCTtaAaCtTT hsa-miR-562 MIMAT0003226 SEQ ID NO 370
AtGgtACagCtAcTT hsa-miR-563 MIMAT0003227 SEQ ID NO 371
AaAcgTAtgTcAaCC hsa-miR-564 MIMAT0003228 SEQ ID NO 372
TgCtgACacCgTgCC hsa-miR-565 MIMAT0003229 SEQ ID NO 373
AcAtcGCgaGcCaGC hsa-miR-566 MIMAT0003230 SEQ ID NO 374
GgGatCAcaGgCgCC
hsa-miR-567 MIMAT0003231 SEQ ID NO 375 CcTggAAgaAcAtAC hsa-miR-568
MIMAT0003232 SEQ ID NO 376 GtAtaCAttTaTaCA hsa-miR-551b
MIMAT0003233 SEQ ID NO 377 AcCaaGTatGgGtCG hsa-miR-569 MIMAT0003234
SEQ ID NO 378 CcAggATtcAtTaAC hsa-miR-570 MIMAT0003235 SEQ ID NO
379 GgTaaTTgcTgTtTT hsa-miR-571 MIMAT0003236 SEQ ID NO 380
TcAgaTGgcCaAcTC hsa-miR-572 MIMAT0003237 SEQ ID NO 381
CcAccGCcgAgCgGA hsa-miR-573 MIMAT0003238 SEQ ID NO 382
TtAcaCAtcAcTtCA hsa-miR-574 MIMAT0003239 SEQ ID NO 383
TgTgtGCatGaGcGT hsa-miR-575 MIMAT0003240 SEQ ID NO 384
CcTgtCCaaCtGgCT hsa-miR-576 MIMAT0003241 SEQ ID NO 385
GtGgaGAaaTtAgAA hsa-miR-577 MIMAT0003242 SEQ ID NO 386
AcCaaTAttTtAtCT hsa-miR-578 MIMAT0003243 SEQ ID NO 387
CcTagAGcaCaAgAA hsa-miR-579 MIMAT0003244 SEQ ID NO 388
TtTatACcaAaTgAA hsa-miR-580 MIMAT0003245 SEQ ID NO 389
GaTtcATcaTtCtCA hsa-miR-581 MIMAT0003246 SEQ ID NO 390
TcTagAGaaCaCaAG hsa-miR-582 MIMAT0003247 SEQ ID NO 391
GgTtgAAcaAcTgTA hsa-miR-583 MIMAT0003248 SEQ ID NO 392
GgGacCTtcCtCtTT hsa-miR-584 MIMAT0003249 SEQ ID NO 393
CcCagGCaaAcCaTA hsa-miR-585 MIMAT0003250 SEQ ID NO 394
CaTacAGatAcGcCC hsa-miR-548a MIMAT0003251 SEQ ID NO 395
GtAatTGccAgTtTT hsa-miR-586 MIMAT0003252 SEQ ID NO 396
AaAaaTAcaAtGcAT hsa-miR-587 MIMAT0003253 SEQ ID NO 397
TcAtcACctAtGgAA hsa-miR-548b MIMAT0003254 SEQ ID NO 398
GcAacTGagGtTcTT hsa-miR-588 MIMAT0003255 SEQ ID NO 399
AaCccATtgTgGcCA hsa-miR-589 MIMAT0003256 SEQ ID NO 400
CcGgcATttGtTcTG hsa-miR-550 MIMAT0003257 SEQ ID NO 401
CtGagGGagTaAgAC hsa-miR-590 MIMAT0003258 SEQ ID NO 402
TtTtaTGaaTaAgCT hsa-miR-591 MIMAT0003259 SEQ ID NO 403
TgAgaACccAtGgTC hsa-miR-592 MIMAT0003260 SEQ ID NO 404
TcGcaTAttGaCaCA hsa-miR-593 MIMAT0003261 SEQ ID NO 405
TgCctGGctGgTgCC hsa-miR-595 MIMAT0003263 SEQ ID NO 406
CaCcaCGgcAcAcTT hsa-miR-596 MIMAT0003264 SEQ ID NO 407
GgAgcCGggCaGgCT hsa-miR-597 MIMAT0003265 SEQ ID NO 408
GtCatCGagTgAcAC hsa-miR-598 MIMAT0003266 SEQ ID NO 409
TgAcaACgaTgAcGT hsa-miR-599 MIMAT0003267 SEQ ID NO 410
GaTaaACtgAcAcAA hsa-miR-600 MIMAT0003268 SEQ ID NO 411
GcTctTGtcTgTaAG hsa-miR-601 MIMAT0003269 SEQ ID NO 412
CaAcaATccTaGaCC hsa-miR-602 MIMAT0003270 SEQ ID NO 413
AgCtgTCgcCcGtGT hsa-miR-603 MIMAT0003271 SEQ ID NO 414
GtAatTGcaGtGtGT hsa-miR-604 MIMAT0003272 SEQ ID NO 415
CtGaaTTccGcAgCC hsa-miR-605 MIMAT0003273 SEQ ID NO 416
GgCacCAtgGgAtTT hsa-miR-606 MIMAT0003274 SEQ ID NO 417
TgAttTTcaGtAgTT hsa-miR-607 MIMAT0003275 SEQ ID NO 418
AgAtcTGgaTtTgAA hsa-miR-608 MIMAT0003276 SEQ ID NO 419
TcCcaACacCaCcCC hsa-miR-609 MIMAT0003277 SEQ ID NO 420
AtGagAGaaAcAcCC hsa-miR-610 MIMAT0003278 SEQ ID NO 421
GcAcaCAttTaGcTC hsa-miR-611 MIMAT0003279 SEQ ID NO 422
CcCgaGGggTcCtCG hsa-miR-612 MIMAT0003280 SEQ ID NO 423
AgAagCCctGcCcAG hsa-miR-613 MIMAT0003281 SEQ ID NO 424
AaGaaGGaaCaTtCC hsa-miR-614 MIMAT0003282 SEQ ID NO 425
GcAagAAcaGgCgTT hsa-miR-615 MIMAT0003283 SEQ ID NO 426
GaGacCCagGcTcGG hsa-miR-616 MIMAT0003284 SEQ ID NO 427
CtGaaGGgtTtTgAG hsa-miR-548c MIMAT0003285 SEQ ID NO 428
GtAatTGagAtTtTT hsa-miR-617 MIMAT0003286 SEQ ID NO 429
TtCaaATggGaAgTC hsa-miR-618 MIMAT0003287 SEQ ID NO 430
AgGacAAgtAgAgTT hsa-miR-619 MIMAT0003288 SEQ ID NO 431
CaAacATgtCcAgGT hsa-miR-620 MIMAT0003289 SEQ ID NO 432
CtAtaTCtaTcTcCA hsa-miR-621 MIMAT0003290 SEQ ID NO 433
AgCgcTGttGcTaGC hsa-miR-622 MIMAT0003291 SEQ ID NO 434
AaCctCAgcAgAcTG hsa-miR-623 MIMAT0003292 SEQ ID NO 435
AgCccCTgcAaGgGA hsa-miR-624 MIMAT0003293 SEQ ID NO 436
CaAggTActGgTaCT hsa-miR-625 MIMAT0003294 SEQ ID NO 437
AtAgaACttTcCcCC hsa-miR-626 MIMAT0003295 SEQ ID NO 438
AcAttTTcaGaCaGC hsa-miR-627 MIMAT0003296 SEQ ID NO 439
TtTctTAgaGaCtCA hsa-miR-628 MIMAT0003297 SEQ ID NO 440
TgCcaCTctTaCtAG hsa-miR-629 MIMAT0003298 SEQ ID NO 441
CtTacGTtgGgAgAA hsa-miR-630 MIMAT0003299 SEQ ID NO 442
CcTggTAcaGaAtAC hsa-miR-631 MIMAT0003300 SEQ ID NO 443
GgTctGGgcCaGgTC hsa-miR-33b MIMAT0003301 SEQ ID NO 444
TgCaaCAgcAaTgCA hsa-miR-632 MIMAT0003302 SEQ ID NO 445
CaCagGAagCaGaCA hsa-miR-633 MIMAT0003303 SEQ ID NO 446
TgGtaGAtaCtAtTA hsa-miR-634 MIMAT0003304 SEQ ID NO 447
AgTtgGGgtGcTgGT hsa-miR-635 MIMAT0003305 SEQ ID NO 448
GtTtcAGtgCcCaAG hsa-miR-636 MIMAT0003306 SEQ ID NO 449
GgGacGAgcAaGcAC hsa-miR-637 MIMAT0003307 SEQ ID NO 450
CcCgaAAgcCcCcAG hsa-miR-638 MIMAT0003308 SEQ ID NO 451
CcCgcCCgcGaTcCC hsa-miR-639 MIMAT0003309 SEQ ID NO 452
TcGcaACcgCaGcGA hsa-miR-640 MIMAT0003310 SEQ ID NO 453
CaGgtTCctGgAtCA hsa-miR-641 MIMAT0003311 SEQ ID NO 454
TcTatCCtaTgTcTT hsa-miR-642 MIMAT0003312 SEQ ID NO 455
AcAttTGgaGaGgGA hsa-miR-643 MIMAT0003313 SEQ ID NO 456
GaGctAGcaTaCaAG hsa-miR-644 MIMAT0003314 SEQ ID NO 457
CtAagAAagCcAcAC hsa-miR-645 MIMAT0003315 SEQ ID NO 458
GcAgtACcaGcCtAG hsa-miR-646 MIMAT0003316 SEQ ID NO 459
TcAgaGGcaGcTgCT hsa-miR-647 MIMAT0003317 SEQ ID NO 460
AaGtgAGtgCaGcCA hsa-miR-648 MIMAT0003318 SEQ ID NO 461
AgTgcCCtgCaCaCT hsa-miR-649 MIMAT0003319 SEQ ID NO 462
TgAacAAcaCaGgTT hsa-miR-650 MIMAT0003320 SEQ ID NO 463
GaGagCGctGcCtCC hsa-miR-651 MIMAT0003321 SEQ ID NO 464
TcAagCTtaTcCtAA hsa-miR-652 MIMAT0003322 SEQ ID NO 465
CcCtaGTggCgCcAT hsa-miR-548d MIMAT0003323 SEQ ID NO 466
GaAacTGtgGtTtTT hsa-miR-661 MIMAT0003324 SEQ ID NO 467
GcCagAGacCcAgGC hsa-miR-662 MIMAT0003325 SEQ ID NO 468
GgGccACaaCgTgGG hsa-miR-663 MIMAT0003326 SEQ ID NO 469
CcGcgGCgcCcCgCC hsa-miR-449b MIMAT0003327 SEQ ID NO 470
TaAcaATacAcTgCC hsa-miR-653 MIMAT0003328 SEQ ID NO 471
GtAgaGAttGtTtCA hsa-miR-411 MIMAT0003329 SEQ ID NO 472
GcTatACggTcTaCT hsa-miR-654 MIMAT0003330 SEQ ID NO 473
GtTctGCggCcCaCC hsa-miR-655 MIMAT0003331 SEQ ID NO 474
GtTaaCCatGtAtTA hsa-miR-656 MIMAT0003332 SEQ ID NO 475
TtGacTGtaTaAtAT hsa-miR-549 MIMAT0003333 SEQ ID NO 476
TcAtcCAtaGtTgTC hsa-miR-657 MIMAT0003335 SEQ ID NO 477
AgGgtGAgaAcCtGC hsa-miR-658 MIMAT0003336 SEQ ID NO 478
CcTacTTccCtCcGC hsa-miR-659 MIMAT0003337 SEQ ID NO 479
CcCtcCCtgAaCcAA hsa-miR-660 MIMAT0003338 SEQ ID NO 480
CgAtaTGcaAtGgGT hsa-miR-421 MIMAT0003339 SEQ ID NO 481
AtTaaTGtcTgTtGA hsa-miR-542-5p MIMAT0003340 SEQ ID NO 482
AcAtgATgaTcCcCG hcmv-miR-US4 MIMAT0003341 SEQ ID NO 483
CtGcaCGtcCaTgTC hcmv-miR-UL70-5p MIMAT0003342 SEQ ID NO 484
AcGagGCcgAgAcGC hcmv-miR-UL70-3p MIMAT0003343 SEQ ID NO 485
GcGccAGccCaTcCC hsa-miR-363* MIMAT0003385 SEQ ID NO 486
CaTcgTGatCcAcCC hsa-miR-376a* MIMAT0003386 SEQ ID NO 487
AgAagGAgaAtCtAC hsa-miR-542-3p MIMAT0003389 SEQ ID NO 488
TtAtcAAtcTgTcAC ebv-miR-BART1-3p MIMAT0003390 SEQ ID NO 489
GtGgaTAgcGgTgCT hsa-miR-425-5p MIMAT0003393 SEQ ID NO 490
GaGtgATcgTgTcAT ebv-miR-BART3-5p MIMAT0003410 SEQ ID NO 491
AcActAAcaCtAgGT ebv-miR-BART3-3p MIMAT0003411 SEQ ID NO 492
GgTgaCTagTgGtGC ebv-miR-BART4 MIMAT0003412 SEQ ID NO 493
CcAgcAGcaTcAgGT ebv-miR-BART5 MIMAT0003413 SEQ ID NO 494
AgCtaTAttCaCcTT ebv-miR-BART6-5p MIMAT0003414 SEQ ID NO 495
AtGgaTTggAcCaAC ebv-miR-BART6-3p MIMAT0003415 SEQ ID NO 496
GcTagTCcgAtCcCC ebv-miR-BART7 MIMAT0003416 SEQ ID NO 497
AcActGGacTaTgAT ebv-miR-BART8-5p MIMAT0003417 SEQ ID NO 498
AaTctAGgaAaCcGT ebv-miR-BART8-3p MIMAT0003418 SEQ ID NO 499
CcCcaTAgaTtGtGA
ebv-miR-BART9 MIMAT0003419 SEQ ID NO 500 GaCccATgaAgTgTT
ebv-miR-BART10 MIMAT0003420 SEQ ID NO 501 AaCtcCAtgGtTaTG
ebv-miR-BART11-5p MIMAT0003421 SEQ ID NO 502 AgCgcACcaAaCtGT
ebv-miR-BART11-3p MIMAT0003422 SEQ ID NO 503 TcAgcCTggTgTgCG
ebv-miR-BART12 MIMAT0003423 SEQ ID NO 504 AcCaaACacCaCaGG
ebv-miR-BART13 MIMAT0003424 SEQ ID NO 505 TcCctGGcaAgTtAC
ebv-miR-BART14-5p MIMAT0003425 SEQ ID NO 506 TcGgcAGcgTaGgGT
ebv-miR-BART14-3p MIMAT0003426 SEQ ID NO 507 AcTacTGcaGcAtTT
kshv-miR-K12-12 MIMAT0003712 SEQ ID NO 508 GgAatGGtgGcCtGG
ebv-miR-BART15 MIMAT0003713 SEQ ID NO 509 AgGaaACaaAaCcAC
ebv-miR-BART16 MIMAT0003714 SEQ ID NO 510 CaCacACccAcTcTA
ebv-miR-BART17-5p MIMAT0003715 SEQ ID NO 511 AtGccTGcgTcCtCT
ebv-miR-BART17-3p MIMAT0003716 SEQ ID NO 512 GaCacCAggCaTaCA
ebv-miR-BART18 MIMAT0003717 SEQ ID NO 513 AgGaaGTgcGaAcTT
ebv-miR-BART19 MIMAT0003718 SEQ ID NO 514 CcAagCAaaCaAaAC
ebv-miR-BART20-5p MIMAT0003719 SEQ ID NO 515 AaGacATgcCtGcTA
ebv-miR-BART20-3p MIMAT0003720 SEQ ID NO 516 AgGctGTgcCtTcAT
hsv1-miR-H1 MIMAT0003744 SEQ ID NO 517 AcTtcCCgtCcTtCC hsa-miR-758
MIMAT0003879 SEQ ID NO 518 TgGacCAggTcAcAA hsa-miR-671 MIMAT0003880
SEQ ID NO 519 CcCtcCAggGcTtCC hsa-miR-668 MIMAT0003881 SEQ ID NO
520 GcCgaGCcgAgTgAC hsa-miR-767-5p MIMAT0003882 SEQ ID NO 521
AgAcaACcaTgGtGC hsa-miR-767-3p MIMAT0003883 SEQ ID NO 522
AtGggGTatGaGcAG hsa-miR-454-5p MIMAT0003884 SEQ ID NO 523
AcAatATtgAtAgGG hsa-miR-454-3p MIMAT0003885 SEQ ID NO 524
AaGcaATatTgCaCT hsa-miR-769-5p MIMAT0003886 SEQ ID NO 525
GaAccCAgaGgTcTC hsa-miR-769-3p MIMAT0003887 SEQ ID NO 526
AcCccGGagAtCcCA hsa-miR-766 MIMAT0003888 SEQ ID NO 527
GcTgtGGggCtGgAG hsa-miR-765 MIMAT0003945 SEQ ID NO 528
CcTtcCTtcTcCtCC hsa-miR-768-5p MIMAT0003946 SEQ ID NO 529
AcTttCAtcCtCcAA hsa-miR-768-3p MIMAT0003947 SEQ ID NO 530
AgTgtCAgcAtTgTG hsa-miR-770-5p MIMAT0003948 SEQ ID NO 531
GaCacGTggTaCtGG hsa-miR-802 MIMAT0004185 SEQ ID NO 532
TgAatCTttGtTaCT hsa-miR-801 MIMAT0004209 SEQ ID NO 533
CgCacGCagAgCaAT hsa-miR-675 MIMAT0004284 SEQ ID NO 534
GgCccTCtcCgCaCC (SEQ ID refers to Example antimiR)
Sequence CWU 1
1
598115DNAArtificial SequenceHomo sapiens 1acaacctact acctc
15215DNAArtificial SequenceHomo sapiens 2acaacctact acctc
15315DNAArtificial SequenceHomo sapiens 3acaacctact acctc
15415DNAArtificial SequenceHomo sapiens 4gcaacctact acctc
15515DNAArtificial SequenceSynthetic oligonucleotide 5acaacctcct
acctc 15615DNAArtificial SequenceSynthetic oligonucleotide
6acaatctact acctc 15715DNAArtificial SequenceSynthetic
oligonucleotide 7ccattatgtg ctgct 15815DNAArtificial
SequenceSynthetic oligonucleotide 8tatttacgtg ctgct
15915DNAArtificial SequenceSynthetic oligonucleotide 9cactgtaagc
acttt 151015DNAArtificial SequenceSynthetic oligonucleotide
10gtgccttcac tgcag 151115DNAArtificial SequenceSynthetic
oligonucleotide 11cactagatgc acctt 151215DNAArtificial
SequenceSynthetic oligonucleotide 12tgcatagatt tgcac
151315DNAArtificial SequenceSynthetic oligonucleotide 13tgcatggatt
tgcac 151415DNAArtificial SequenceSynthetic oligonucleotide
14cactataagc acttt 151515DNAArtificial SequenceSynthetic
oligonucleotide 15tcagtctgat aagct 151615DNAArtificial
SequenceSynthetic oligonucleotide 16cttcaactgg cagct
151715DNAArtificial SequenceSynthetic oligonucleotide 17tccctggcaa
tgtga 151815DNAArtificial SequenceSynthetic oligonucleotide
18tcagctcagt aggca 151915DNAArtificial SequenceSynthetic
oligonucleotide 19ctgctgaact gagcc 152015DNAArtificial
SequenceSynthetic oligonucleotide 20cgagacaagt gcaat
152115DNAArtificial SequenceSynthetic oligonucleotide 21tcctggatta
cttga 152215DNAArtificial SequenceSynthetic oligonucleotide
22tcctgaatta cttga 152315DNAArtificial SequenceSynthetic
oligonucleotide 23acttagccac tgtga 152415DNAArtificial
SequenceSynthetic oligonucleotide 24agactgtgag ctcct
152515DNAArtificial SequenceSynthetic oligonucleotide 25atttcagatg
gtgct 152615DNAArtificial SequenceSynthetic oligonucleotide
26gtcgaggatg tttac 152715DNAArtificial SequenceSynthetic
oligonucleotide 27aacatccgac tgaaa 152815DNAArtificial
SequenceSynthetic oligonucleotide 28atgccagcat cttgc
152915DNAArtificial SequenceSynthetic oligonucleotide 29ttagtaatgt
gcaat 153015DNAArtificial SequenceSynthetic oligonucleotide
30tgcaactaca atgca 153115DNAArtificial SequenceSynthetic
oligonucleotide 31cgggacaagt gcaat 153215DNAArtificial
SequenceSynthetic oligonucleotide 32gcacgaacag cactt
153315DNAArtificial SequenceSynthetic oligonucleotide 33ataaataccc
gttga 153415DNAArtificial SequenceSynthetic oligonucleotide
34atgtgctagt gccaa 153515DNAArtificial SequenceSynthetic
oligonucleotide 35acaacttact acctc 153615DNAArtificial
SequenceSynthetic oligonucleotide 36atcggatcta cgggt
153715DNAArtificial SequenceSynthetic oligonucleotide 37ttcggatcta
cgggt 153815DNAArtificial SequenceSynthetic oligonucleotide
38ttatcacagt actgt 153915DNAArtificial SequenceSynthetic
oligonucleotide 39atttcaaatg gtgct 154015DNAArtificial
SequenceSynthetic oligonucleotide 40cctgtacaat gctgc
154115DNAArtificial SequenceSynthetic oligonucleotide 41gagtctgagc
atttg 154215DNAArtificial SequenceSynthetic oligonucleotide
42cactgtaagc acttt 154315DNAArtificial SequenceSynthetic
oligonucleotide 43cctgtacaat gctgc 154415DNAArtificial
SequenceSynthetic oligonucleotide 44tcaattcata ggtca
154515DNAArtificial SequenceSynthetic oligonucleotide 45aacatgaaac
tacct 154615DNAArtificial SequenceSynthetic oligonucleotide
46tggagaaggt ggtga 154715DNAArtificial SequenceSynthetic
oligonucleotide 47atctcccctc tggac 154815DNAArtificial
SequenceSynthetic oligonucleotide 48tagtctgaac actgg
154915DNAArtificial SequenceSynthetic oligonucleotide 49tgtgcagact
actgt 155015DNAArtificial SequenceSynthetic oligonucleotide
50tttttgctcg tctta 155115DNAArtificial SequenceSynthetic
oligonucleotide 51cccagaccgc aaaaa 155215DNAArtificial
SequenceSynthetic oligonucleotide 52ttctgtagtg cactg
155315DNAArtificial SequenceSynthetic oligonucleotide 53gtgtaggatg
tttac 155415DNAArtificial SequenceSynthetic oligonucleotide
54gtcggggatg tttac 155515DNAArtificial SequenceSynthetic
oligonucleotide 55acacgtgcac tgtag 155615DNAArtificial
SequenceSynthetic oligonucleotide 56aagcatttcc acaca
155715DNAArtificial SequenceSynthetic oligonucleotide 57aatcactagt
cttcc 155815DNAArtificial SequenceSynthetic oligonucleotide
58tcggatctac agggt 155915DNAArtificial SequenceSynthetic
oligonucleotide 59tcggttctac agggt 156015DNAArtificial
SequenceSynthetic oligonucleotide 60agctaagaca ctgcc
156115DNAArtificial SequenceSynthetic oligonucleotide 61gacagcgttg
aatgt 156215DNAArtificial SequenceSynthetic oligonucleotide
62gacagcaatg aatgt 156315DNAArtificial SequenceSynthetic
oligonucleotide 63cgacaggttg aatgt 156415DNAArtificial
SequenceSynthetic oligonucleotide 64ttctaccatt gccaa
156515DNAArtificial SequenceSynthetic oligonucleotide 65ggcaagtcta
gaacc 156615DNAArtificial SequenceSynthetic oligonucleotide
66ttctaccagt gccat 156715DNAArtificial SequenceSynthetic
oligonucleotide 67gcaacacaag acacg 156815DNAArtificial
SequenceSynthetic oligonucleotide 68tagtctaaac actgg
156915DNAArtificial SequenceSynthetic oligonucleotide 69gtcctaaaca
tttca 157015DNAArtificial SequenceSynthetic oligonucleotide
70aggatgacaa aggga 157115DNAArtificial SequenceSynthetic
oligonucleotide 71ccggtggaat gaagg 157215DNAArtificial
SequenceSynthetic oligonucleotide 72gctgtcacac gcaca
157315DNAArtificial SequenceSynthetic oligonucleotide 73aggatgacaa
aggga 157415DNAArtificial SequenceSynthetic oligonucleotide
74tgactggaga ctgtt 157515DNAArtificial SequenceSynthetic
oligonucleotide 75atcaacggtc gatgg 157615DNAArtificial
SequenceSynthetic oligonucleotide 76tgtctgtgcc tgctg
157715DNAArtificial SequenceSynthetic oligonucleotide 77tcaattcata
ggtca 157815DNAArtificial SequenceSynthetic oligonucleotide
78ttgccagctg agatt 157915DNAArtificial SequenceSynthetic
oligonucleotide 79agttcctgat gcagt 158015DNAArtificial
SequenceSynthetic oligonucleotide 80gttagatcaa gcaca
158115DNAArtificial SequenceSynthetic oligonucleotide 81tgcgtttgga
caatc 158215DNAArtificial SequenceSynthetic oligonucleotide
82gtcagatacg gtgtg 158315DNAArtificial SequenceSynthetic
oligonucleotide 83agcagacaat gtagc 158415DNAArtificial
SequenceSynthetic oligonucleotide 84gtagccagat gtagc
158515DNAArtificial SequenceSynthetic oligonucleotide 85atttgacaaa
ctgac 158615DNAArtificial SequenceSynthetic oligonucleotide
86aaccactagt gactt 158715DNAArtificial SequenceSynthetic
oligonucleotide 87ttaccaggca gtatt 158815DNAArtificial
SequenceSynthetic oligonucleotide 88acaaactact acctc
158915DNAArtificial SequenceSynthetic oligonucleotide 89acaaactact
acctc 159015DNAArtificial SequenceSynthetic oligonucleotide
90acttctttac attcc 159115DNAArtificial SequenceSynthetic
oligonucleotide 91ccatgatgtg ctgct 159215DNAArtificial
SequenceSynthetic oligonucleotide 92tccctggcaa tgtga
159315DNAArtificial SequenceSynthetic oligonucleotide 93acttagccac
tgtga 159415DNAArtificial SequenceSynthetic oligonucleotide
94gtgtaggatg tttac 159515DNAArtificial SequenceSynthetic
oligonucleotide 95ccattgtcac actcc 159615DNAArtificial
SequenceSynthetic oligonucleotide 96tcaccgcgtg cctta
159715DNAArtificial SequenceSynthetic oligonucleotide 97gttagggtct
caggg 159815DNAArtificial SequenceSynthetic oligonucleotide
98gaccggttca ctgtg 159915DNAArtificial SequenceSynthetic
oligonucleotide 99ttttaacatt gcact 1510015DNAArtificial
SequenceSynthetic oligonucleotide 100tggctgtaga ctgtt
1510115DNAArtificial SequenceSynthetic oligonucleotide
101ggttgaaggg gacca 1510215DNAArtificial SequenceSynthetic
oligonucleotide 102ggaataaaaa gccat 1510315DNAArtificial
SequenceSynthetic oligonucleotide 103gtattcttaa gcaat
1510415DNAArtificial SequenceSynthetic oligonucleotide
104attcacaaca ccagc 1510515DNAArtificial SequenceSynthetic
oligonucleotide 105atagggtaaa accac 1510615DNAArtificial
SequenceSynthetic oligonucleotide 106ttaccagaca gtgtt
1510715DNAArtificial SequenceSynthetic oligonucleotide
107tgctttctac tttat 1510815DNAArtificial SequenceSynthetic
oligonucleotide 108agtaggaaac actac 1510915DNAArtificial
SequenceSynthetic oligonucleotide 109acagtgcttc atctc
1511015DNAArtificial SequenceSynthetic oligonucleotide
110catcatctat actgt 1511115DNAArtificial SequenceSynthetic
oligonucleotide 111cctgggaaaa ctgga 1511215DNAArtificial
SequenceSynthetic oligonucleotide 112ttctgtcatg cactg
1511315DNAArtificial SequenceSynthetic oligonucleotide
113ttttgtgact atgca 1511415DNAArtificial SequenceSynthetic
oligonucleotide 114ttttgggatt ccgtt 1511515DNAArtificial
SequenceSynthetic oligonucleotide 115gctagataac caaag
1511615DNAArtificial SequenceSynthetic oligonucleotide
116cggttatcta gcttt 1511715DNAArtificial SequenceSynthetic
oligonucleotide 117taaagggtct caggg 1511815DNAArtificial
SequenceSynthetic oligonucleotide 118accaaaagta ataat
1511915DNAArtificial SequenceSynthetic oligonucleotide
119attactcacg gtacg 1512015DNAArtificial SequenceSynthetic
oligonucleotide 120gctcagacgg atccg 1512115DNAArtificial
SequenceSynthetic oligonucleotide 121tggtcaacca gtcac
1512215DNAArtificial SequenceSynthetic oligonucleotide
122tcaaaacaaa tggag 1512315DNAArtificial SequenceSynthetic
oligonucleotide 123tggaattcag ttctc 1512415DNAArtificial
SequenceSynthetic oligonucleotide 124aagacacgga gccag
1512515DNAArtificial SequenceSynthetic oligonucleotide
125tacaagggtt gggag 1512615DNAArtificial SequenceSynthetic
oligonucleotide 126caacacggat aacct 1512715DNAArtificial
SequenceSynthetic oligonucleotide 127tcaaccgtgt atgat
1512815DNAArtificial SequenceSynthetic oligonucleotide
128atcagttctc cgtcc 1512915DNAArtificial SequenceSynthetic
oligonucleotide 129actgcctttc tctcc 1513015DNAArtificial
SequenceSynthetic oligonucleotide 130aaaggagaat tcttt
1513115DNAArtificial SequenceSynthetic oligonucleotide
131caccatgcaa gggat 1513215DNAArtificial SequenceSynthetic
oligonucleotide 132tatatcaaac atatc 1513315DNAArtificial
SequenceSynthetic oligonucleotide 133actttgtagg ccagt
1513415DNAArtificial SequenceSynthetic oligonucleotide
134tggagttgct gttac 1513515DNAArtificial SequenceSynthetic
oligonucleotide 135tatttctgtg ctgct 1513615DNAArtificial
SequenceSynthetic oligonucleotide 136acttccttac attcc
1513715DNAArtificial SequenceSynthetic oligonucleotide
137tctcaaccca gcttt 1513815DNAArtificial SequenceSynthetic
oligonucleotide 138ttacccggca gtatt 1513915DNAArtificial
SequenceSynthetic oligonucleotide 139tcacgattag
catta 1514015DNAArtificial SequenceSynthetic oligonucleotide
140gaccggttca ctgtg 1514115DNAArtificial SequenceSynthetic
oligonucleotide 141cactgtcagc acttt 1514215DNAArtificial
SequenceSynthetic oligonucleotide 142atttcaaatg gtgct
1514315DNAArtificial SequenceSynthetic oligonucleotide
143ttaccagaca gtgtt 1514415DNAArtificial SequenceSynthetic
oligonucleotide 144agtacatcca cgttt 1514515DNAArtificial
SequenceSynthetic oligonucleotide 145aacatggaag cactt
1514615DNAArtificial SequenceSynthetic oligonucleotide
146ctaatgacac tgcct 1514715DNAArtificial SequenceSynthetic
oligonucleotide 147gctaactaca ctgcc 1514815DNAArtificial
SequenceSynthetic oligonucleotide 148tttaccatcc cacat
1514915DNAArtificial SequenceSynthetic oligonucleotide
149caatactatt gcact 1515015DNAArtificial SequenceSynthetic
oligonucleotide 150gtcggttcta cgggt 1515115DNAArtificial
SequenceSynthetic oligonucleotide 151attgaggggg ggccc
1515215DNAArtificial SequenceSynthetic oligonucleotide
152tttcatcatt gcact 1515315DNAArtificial SequenceSynthetic
oligonucleotide 153gtcaaggatg tttac 1515415DNAArtificial
SequenceSynthetic oligonucleotide 154aacatccgac tgaaa
1515515DNAArtificial SequenceSynthetic oligonucleotide
155ctggagattc tgata 1515615DNAArtificial SequenceSynthetic
oligonucleotide 156ctaggttcca aggat 1515715DNAArtificial
SequenceSynthetic oligonucleotide 157tggataccgt gcaat
1515815DNAArtificial SequenceSynthetic oligonucleotide
158atttttaggg gcatt 1515915DNAArtificial SequenceSynthetic
oligonucleotide 159acttccatgt taaag 1516015DNAArtificial
SequenceSynthetic oligonucleotide 160aacatggaag cactt
1516115DNAArtificial SequenceSynthetic oligonucleotide
161gtacccccat gttaa 1516215DNAArtificial SequenceSynthetic
oligonucleotide 162aacatggaag cactt 1516315DNAArtificial
SequenceSynthetic oligonucleotide 163aacatggaag cactt
1516415DNAArtificial SequenceSynthetic oligonucleotide
164ttgctaaagt gcaat 1516515DNAArtificial SequenceSynthetic
oligonucleotide 165ggaatttcct ctatg 1516615DNAArtificial
SequenceSynthetic oligonucleotide 166tcaaccatgt attat
1516715DNAArtificial SequenceSynthetic oligonucleotide
167ttccacccca gcagg 1516815DNAArtificial SequenceSynthetic
oligonucleotide 168caaaagatgg cggca 1516915DNAArtificial
SequenceSynthetic oligonucleotide 169aatgtcgcag cactt
1517015DNAArtificial SequenceSynthetic oligonucleotide
170cgcccccatt ttgag 1517115DNAArtificial SequenceSynthetic
oligonucleotide 171aaaatcgaag cactt 1517215DNAArtificial
SequenceSynthetic oligonucleotide 172tcaggttgta ttata
1517315DNAArtificial SequenceSynthetic oligonucleotide
173gagccgaacg aacaa 1517415DNAArtificial SequenceSynthetic
oligonucleotide 174gattttcctc tatga 1517515DNAArtificial
SequenceSynthetic oligonucleotide 175gttgcctttg tgtga
1517615DNAArtificial SequenceSynthetic oligonucleotide
176gacctggagt cagga 1517715DNAArtificial SequenceSynthetic
oligonucleotide 177ctgactccaa gtcca 1517815DNAArtificial
SequenceSynthetic oligonucleotide 178gttccatagt ctacc
1517915DNAArtificial SequenceSynthetic oligonucleotide
179gttctatggt caacc 1518015DNAArtificial SequenceSynthetic
oligonucleotide 180tggaccatat tacat 1518115DNAArtificial
SequenceSynthetic oligonucleotide 181agcttgccct tgtat
1518215DNAArtificial SequenceSynthetic oligonucleotide
182caccacgaac aactt 1518315DNAArtificial SequenceSynthetic
oligonucleotide 183aatcaccttc tgatc 1518415DNAArtificial
SequenceSynthetic oligonucleotide 184aagtaactga gacgg
1518515DNAArtificial SequenceSynthetic oligonucleotide
185aggccgtgtg ctttg 1518615DNAArtificial SequenceSynthetic
oligonucleotide 186gggcagagag ggcca 1518715DNAArtificial
SequenceSynthetic oligonucleotide 187cgatttctgt gtgag
1518815DNAArtificial SequenceSynthetic oligonucleotide
188tcatatagga gctgg 1518915DNAArtificial SequenceSynthetic
oligonucleotide 189cgaccgtgta atgtg 1519015DNAArtificial
SequenceSynthetic oligonucleotide 190aggaagggcc cagag
1519115DNAArtificial SequenceSynthetic oligonucleotide
191ggagcttcag tctag 1519215DNAArtificial SequenceSynthetic
oligonucleotide 192ggaatgaaaa gccat 1519315DNAArtificial
SequenceSynthetic oligonucleotide 193ttctgtgatg cactg
1519415DNAArtificial SequenceSynthetic oligonucleotide
194ggataggccc agggg 1519515DNAArtificial SequenceSynthetic
oligonucleotide 195tgccctaggg gatgc 1519615DNAArtificial
SequenceSynthetic oligonucleotide 196gcacctgggg cagtg
1519715DNAArtificial SequenceSynthetic oligonucleotide
197aatcactgat gctgg 1519815DNAArtificial SequenceSynthetic
oligonucleotide 198tcctggagga caggg 1519915DNAArtificial
SequenceSynthetic oligonucleotide 199tcgttattgc tcttg
1520015DNAArtificial SequenceSynthetic oligonucleotide
200ggttgaaggg gacca 1520115DNAArtificial SequenceSynthetic
oligonucleotide 201ctggacacct actag 1520215DNAArtificial
SequenceSynthetic oligonucleotide 202ggactaggag tcagc
1520315DNAArtificial SequenceSynthetic oligonucleotide
203ggcatgcggg cagac 1520415DNAArtificial SequenceSynthetic
oligonucleotide 204ggggctgatc aggtt 1520515DNAArtificial
SequenceSynthetic oligonucleotide 205tgctgcaaca gaatt
1520615DNAArtificial SequenceSynthetic oligonucleotide
206tctgccgcaa aagat 1520715DNAArtificial SequenceSynthetic
oligonucleotide 207tacacacttc ccgtt 1520815DNAArtificial
SequenceSynthetic oligonucleotide 208gtcacttcca ctaag
1520915DNAArtificial SequenceSynthetic oligonucleotide
209gcgaatgcag aaaat 1521015DNAArtificial SequenceSynthetic
oligonucleotide 210aacaatttct aggaa 1521115DNAArtificial
SequenceSynthetic oligonucleotide 211aacaggaaac tacct
1521215DNAArtificial SequenceSynthetic oligonucleotide
212ctgaccctaa gtcca 1521315DNAArtificial SequenceSynthetic
oligonucleotide 213ggcctcagac cgagc 1521415DNAArtificial
SequenceSynthetic oligonucleotide 214acatgaattg ctgct
1521515DNAArtificial SequenceSynthetic oligonucleotide
215acacgacatt cccga 1521615DNAArtificial SequenceSynthetic
oligonucleotide 216cactagatgc acctt 1521715DNAArtificial
SequenceSynthetic oligonucleotide 217cactatgagc acttt
1521815DNAArtificial SequenceSynthetic oligonucleotide
218catcctacat atgca 1521915DNAArtificial SequenceSynthetic
oligonucleotide 219ttaccagaca gtatt 1522015DNAArtificial
SequenceSynthetic oligonucleotide 220taacaataca ctgcc
1522115DNAArtificial SequenceSynthetic oligonucleotide
221gaacacatcg caaaa 1522215DNAArtificial SequenceSynthetic
oligonucleotide 222acgggaaggc tagtt 1522315DNAArtificial
SequenceSynthetic oligonucleotide 223actagcattc tggtg
1522415DNAArtificial SequenceSynthetic oligonucleotide
224caggtgtctt caacg 1522515DNAArtificial SequenceSynthetic
oligonucleotide 225gatctcaccg tcact 1522615DNAArtificial
SequenceSynthetic oligonucleotide 226aagaagggga ggacg
1522715DNAArtificial SequenceSynthetic oligonucleotide
227ctcgtcaggc ttgtc 1522815DNAArtificial SequenceSynthetic
oligonucleotide 228gtcacaccta tcata 1522915DNAArtificial
SequenceSynthetic oligonucleotide 229gagccactga gcggt
1523015DNAArtificial SequenceSynthetic oligonucleotide
230acctgaacag accgc 1523115DNAArtificial SequenceSynthetic
oligonucleotide 231agctctccaa gtgga 1523215DNAArtificial
SequenceSynthetic oligonucleotide 232cggtccgggc acaat
1523315DNAArtificial SequenceSynthetic oligonucleotide
233gaaatccaag cgcag 1523415DNAArtificial SequenceSynthetic
oligonucleotide 234actgtccggt aagat 1523515DNAArtificial
SequenceSynthetic oligonucleotide 235ataacacggt cgatc
1523615DNAArtificial SequenceSynthetic oligonucleotide
236gacggcctgc aagac 1523715DNAArtificial SequenceSynthetic
oligonucleotide 237aggagcccat catga 1523815DNAArtificial
SequenceSynthetic oligonucleotide 238gttaaccagg tgtgt
1523915DNAArtificial SequenceSynthetic oligonucleotide
239caccacggac aacct 1524015DNAArtificial SequenceSynthetic
oligonucleotide 240gtaatggtaa cggtt 1524115DNAArtificial
SequenceSynthetic oligonucleotide 241gtttcctctg caaac
1524215DNAArtificial SequenceSynthetic oligonucleotide
242ttgcagatga gactg 1524315DNAArtificial SequenceSynthetic
oligonucleotide 243gttgctcggg taacc 1524415DNAArtificial
SequenceSynthetic oligonucleotide 244caccgagcaa cattc
1524515DNAArtificial SequenceSynthetic oligonucleotide
245gtggaccagg tgaag 1524615DNAArtificial SequenceSynthetic
oligonucleotide 246ccatctgtgt tatat 1524715DNAArtificial
SequenceSynthetic oligonucleotide 247gattttcctc tatga
1524815DNAArtificial SequenceSynthetic oligonucleotide
248gggaggagag gagtg 1524915DNAArtificial SequenceSynthetic
oligonucleotide 249aggggactga gcctg 1525015DNAArtificial
SequenceSynthetic oligonucleotide 250atcacggcca gcctc
1525115DNAArtificial SequenceSynthetic oligonucleotide
251gagagccgtg tatga 1525215DNAArtificial SequenceSynthetic
oligonucleotide 252gcagctcagt acagg 1525315DNAArtificial
SequenceSynthetic oligonucleotide 253atgtccctgt atgat
1525415DNAArtificial SequenceSynthetic oligonucleotide
254cggggggaca acact 1525515DNAArtificial SequenceSynthetic
oligonucleotide 255cggggggaca acacc 1525615DNAArtificial
SequenceSynthetic oligonucleotide 256acaggctaag catta
1525715DNAArtificial SequenceSynthetic oligonucleotide
257cccagtttcc tgtaa 1525815DNAArtificial SequenceSynthetic
oligonucleotide 258gacccggact acagt 1525915DNAArtificial
SequenceSynthetic oligonucleotide 259gtttacgcag ctggg
1526015DNAArtificial SequenceSynthetic oligonucleotide
260agctgcgtat accca 1526115DNAArtificial SequenceSynthetic
oligonucleotide 261ctctcagtcg cgcct 1526215DNAArtificial
SequenceSynthetic oligonucleotide 262cagcaacatg ggatc
1526315DNAArtificial SequenceSynthetic oligonucleotide
263gattaggtgc tgctg 1526415DNAArtificial SequenceSynthetic
oligonucleotide 264agcccgaaaa ccatc
1526515DNAArtificial SequenceSynthetic oligonucleotide
265agttccaggc atcct 1526615DNAArtificial SequenceSynthetic
oligonucleotide 266gtactgcggt ttagc 1526715DNAArtificial
SequenceSynthetic oligonucleotide 267aggcctcagt attct
1526815DNAArtificial SequenceSynthetic oligonucleotide
268cgtcctcaga atgtg 1526915DNAArtificial SequenceSynthetic
oligonucleotide 269cattctgtga ccgcg 1527015DNAArtificial
SequenceSynthetic oligonucleotide 270agtgccatta tctgg
1527115DNAArtificial SequenceSynthetic oligonucleotide
271tatatgtgat gtcac 1527215DNAArtificial SequenceSynthetic
oligonucleotide 272ggagtcctcc aggtt 1527315DNAArtificial
SequenceSynthetic oligonucleotide 273ggaagggttc cccac
1527415DNAArtificial SequenceSynthetic oligonucleotide
274gcagagcaaa agaca 1527515DNAArtificial SequenceSynthetic
oligonucleotide 275tggaattcag ttctc 1527615DNAArtificial
SequenceSynthetic oligonucleotide 276gtatatgcat aggaa
1527715DNAArtificial SequenceSynthetic oligonucleotide
277catgccctat acctc 1527815DNAArtificial SequenceSynthetic
oligonucleotide 278ttgtcccgca ggtcc 1527915DNAArtificial
SequenceSynthetic oligonucleotide 279agcctaccat gtaca
1528015DNAArtificial SequenceSynthetic oligonucleotide
280atgacctact ccaag 1528115DNAArtificial SequenceSynthetic
oligonucleotide 281tggaggagcc atcca 1528215DNAArtificial
SequenceSynthetic oligonucleotide 282tcccgtgtat gtttc
1528315DNAArtificial SequenceSynthetic oligonucleotide
283tgcaccatgt ttgtt 1528415DNAArtificial SequenceSynthetic
oligonucleotide 284agattggcca tgtaa 1528515DNAArtificial
SequenceSynthetic oligonucleotide 285actttgaggg ccagt
1528615DNAArtificial SequenceSynthetic oligonucleotide
286ccacagtgtg ctgct 1528715DNAArtificial SequenceSynthetic
oligonucleotide 287gacaacaatg aatgt 1528815DNAArtificial
SequenceSynthetic oligonucleotide 288ccctcaaggc tgagt
1528915DNAArtificial SequenceSynthetic oligonucleotide
289agctatgaca gcact 1529015DNAArtificial SequenceSynthetic
oligonucleotide 290gccccctggc ttgaa 1529115DNAArtificial
SequenceSynthetic oligonucleotide 291aaaaaggaag cactt
1529215DNAArtificial SequenceSynthetic oligonucleotide
292gctttctttt ggaga 1529315DNAArtificial SequenceSynthetic
oligonucleotide 293ccaaaagaag gcact 1529415DNAArtificial
SequenceSynthetic oligonucleotide 294gctccctttt ggaga
1529515DNAArtificial SequenceSynthetic oligonucleotide
295taaaaggagg cactt 1529615DNAArtificial SequenceSynthetic
oligonucleotide 296ctaaaaggaa gcact 1529715DNAArtificial
SequenceSynthetic oligonucleotide 297gcgcttccct ctaga
1529815DNAArtificial SequenceSynthetic oligonucleotide
298taaaaagatg cactt 1529915DNAArtificial SequenceSynthetic
oligonucleotide 299gtacttccct ctgga 1530015DNAArtificial
SequenceSynthetic oligonucleotide 300caaagggaag cactt
1530115DNAArtificial SequenceSynthetic oligonucleotide
301gtgcttccct caaga 1530215DNAArtificial SequenceSynthetic
oligonucleotide 302taaaaggaag cactt 1530315DNAArtificial
SequenceSynthetic oligonucleotide 303taaaaggatg cactt
1530415DNAArtificial SequenceSynthetic oligonucleotide
304gtgcatccct ctgga 1530515DNAArtificial SequenceSynthetic
oligonucleotide 305aaagggaagc gcctt 1530615DNAArtificial
SequenceSynthetic oligonucleotide 306tatagggaag cgcgt
1530715DNAArtificial SequenceSynthetic oligonucleotide
307gtgcttccct ctaga 1530815DNAArtificial SequenceSynthetic
oligonucleotide 308taaagagaag cgctt 1530915DNAArtificial
SequenceSynthetic oligonucleotide 309taaaaggaag cactt
1531015DNAArtificial SequenceSynthetic oligonucleotide
310aaaggggagc gcttt 1531115DNAArtificial SequenceSynthetic
oligonucleotide 311gtgcttccct ctaga 1531215DNAArtificial
SequenceSynthetic oligonucleotide 312taaaaggaag cactt
1531315DNAArtificial SequenceSynthetic oligonucleotide
313tgcttccctc cagag 1531415DNAArtificial SequenceSynthetic
oligonucleotide 314aaagagaagc gcttt 1531515DNAArtificial
SequenceSynthetic oligonucleotide 315gtgcttccct ttgta
1531615DNAArtificial SequenceSynthetic oligonucleotide
316aaagggaagc gcctt 1531715DNAArtificial SequenceSynthetic
oligonucleotide 317tgcttccatc tagag 1531815DNAArtificial
SequenceSynthetic oligonucleotide 318taaagggatg cacga
1531915DNAArtificial SequenceSynthetic oligonucleotide
319aaagggaggc acttt 1532015DNAArtificial SequenceSynthetic
oligonucleotide 320taaagggaag tgcgt 1532115DNAArtificial
SequenceSynthetic oligonucleotide 321ggcttccctt tgtag
1532215DNAArtificial SequenceSynthetic oligonucleotide
322caaagagaag cactt 1532315DNAArtificial SequenceSynthetic
oligonucleotide 323ctaaagggat gcacg 1532415DNAArtificial
SequenceSynthetic oligonucleotide 324aagggaagca ctttg
1532515DNAArtificial SequenceSynthetic oligonucleotide
325ttcttacctc cagat 1532615DNAArtificial SequenceSynthetic
oligonucleotide 326cctctgaaag gaagc 1532715DNAArtificial
SequenceSynthetic oligonucleotide 327tgaagggaag cgctt
1532815DNAArtificial SequenceSynthetic oligonucleotide
328gggcttccct ttgca 1532915DNAArtificial SequenceSynthetic
oligonucleotide 329caaagggaag cgctt 1533015DNAArtificial
SequenceSynthetic oligonucleotide 330aaagggaagc gcttt
1533115DNAArtificial SequenceSynthetic oligonucleotide
331taaaaggatg cacga 1533215DNAArtificial SequenceSynthetic
oligonucleotide 332aagggaagca ctttg 1533315DNAArtificial
SequenceSynthetic oligonucleotide 333taaagggaac cattt
1533415DNAArtificial SequenceSynthetic oligonucleotide
334taaaaggatg cactt 1533515DNAArtificial SequenceSynthetic
oligonucleotide 335tcactgcaag tctta 1533615DNAArtificial
SequenceSynthetic oligonucleotide 336ccttgcccag gtgca
1533715DNAArtificial SequenceSynthetic oligonucleotide
337ccagggacaa aggat 1533815DNAArtificial SequenceSynthetic
oligonucleotide 338cccagatagc aagga 1533915DNAArtificial
SequenceSynthetic oligonucleotide 339actgttcccg ctgct
1534015DNAArtificial SequenceSynthetic oligonucleotide
340gtgcagacca gggtc 1534115DNAArtificial SequenceSynthetic
oligonucleotide 341accagcaagt gttga 1534215DNAArtificial
SequenceSynthetic oligonucleotide 342gacacctccc tgtga
1534315DNAArtificial SequenceSynthetic oligonucleotide
343tcagaagggt gcctt 1534415DNAArtificial SequenceSynthetic
oligonucleotide 344tccaaaaggt gcaaa 1534515DNAArtificial
SequenceSynthetic oligonucleotide 345caaaaggcta caatc
1534615DNAArtificial SequenceSynthetic oligonucleotide
346acagacgtac caatc 1534715DNAArtificial SequenceSynthetic
oligonucleotide 347gccactctcc tgagt 1534815DNAArtificial
SequenceSynthetic oligonucleotide 348tcacagaagt gtcaa
1534915DNAArtificial SequenceSynthetic oligonucleotide
349ctacactcaa ggcat 1535015DNAArtificial SequenceSynthetic
oligonucleotide 350gtgggacggt aaacc 1535115DNAArtificial
SequenceSynthetic oligonucleotide 351gagcacttag ggcag
1535215DNAArtificial SequenceSynthetic oligonucleotide
352agtccaaagg cacat 1535315DNAArtificial SequenceSynthetic
oligonucleotide 353acacagtaga ccttc 1535415DNAArtificial
SequenceSynthetic oligonucleotide 354caaggataat ttctc
1535515DNAArtificial SequenceSynthetic oligonucleotide
355gctaaaaatg cagaa 1535615DNAArtificial SequenceSynthetic
oligonucleotide 356ataaatgttt gctga 1535715DNAArtificial
SequenceSynthetic oligonucleotide 357atgaccctgt acgat
1535815DNAArtificial SequenceSynthetic oligonucleotide
358accaagagtg ggtcg 1535915DNAArtificial SequenceSynthetic
oligonucleotide 359taaccagtca cctgt 1536015DNAArtificial
SequenceSynthetic oligonucleotide 360aaaatctcac cgttt
1536115DNAArtificial SequenceSynthetic oligonucleotide
361ctgagtcagg actag 1536215DNAArtificial SequenceSynthetic
oligonucleotide 362cgggacgagt gcaat 1536315DNAArtificial
SequenceSynthetic oligonucleotide 363aggttcagct taccc
1536415DNAArtificial SequenceSynthetic oligonucleotide
364ttacaatgag ctcat 1536515DNAArtificial SequenceSynthetic
oligonucleotide 365gcccacccgt gcaaa 1536615DNAArtificial
SequenceSynthetic oligonucleotide 366ttggtacagc agctc
1536715DNAArtificial SequenceSynthetic oligonucleotide
367gtgcatattt acttt 1536815DNAArtificial SequenceSynthetic
oligonucleotide 368gccggccggc gcacg 1536915DNAArtificial
SequenceSynthetic oligonucleotide 369aggatcttaa acttt
1537015DNAArtificial SequenceSynthetic oligonucleotide
370atggtacagc tactt 1537115DNAArtificial SequenceSynthetic
oligonucleotide 371aaacgtatgt caacc 1537215DNAArtificial
SequenceSynthetic oligonucleotide 372tgctgacacc gtgcc
1537315DNAArtificial SequenceSynthetic oligonucleotide
373acatcgcgag ccagc 1537415DNAArtificial SequenceSynthetic
oligonucleotide 374gggatcacag gcgcc 1537515DNAArtificial
SequenceSynthetic oligonucleotide 375cctggaagaa catac
1537615DNAArtificial SequenceSynthetic oligonucleotide
376gtatacattt ataca 1537715DNAArtificial SequenceSynthetic
oligonucleotide 377accaagtatg ggtcg 1537815DNAArtificial
SequenceSynthetic oligonucleotide 378ccaggattca ttaac
1537915DNAArtificial SequenceSynthetic oligonucleotide
379ggtaattgct gtttt 1538015DNAArtificial SequenceSynthetic
oligonucleotide 380tcagatggcc aactc 1538115DNAArtificial
SequenceSynthetic oligonucleotide 381ccaccgccga gcgga
1538215DNAArtificial SequenceSynthetic oligonucleotide
382ttacacatca cttca 1538315DNAArtificial SequenceSynthetic
oligonucleotide 383tgtgtgcatg agcgt 1538415DNAArtificial
SequenceSynthetic oligonucleotide 384cctgtccaac tggct
1538515DNAArtificial SequenceSynthetic oligonucleotide
385gtggagaaat tagaa 1538615DNAArtificial SequenceSynthetic
oligonucleotide 386accaatattt tatct 1538715DNAArtificial
SequenceSynthetic oligonucleotide 387cctagagcac aagaa
1538815DNAArtificial SequenceSynthetic oligonucleotide
388tttataccaa atgaa 1538915DNAArtificial SequenceSynthetic
oligonucleotide 389gattcatcat tctca 1539015DNAArtificial
SequenceSynthetic oligonucleotide 390tctagagaac
acaag 1539115DNAArtificial SequenceSynthetic oligonucleotide
391ggttgaacaa ctgta 1539215DNAArtificial SequenceSynthetic
oligonucleotide 392gggaccttcc tcttt 1539315DNAArtificial
SequenceSynthetic oligonucleotide 393cccaggcaaa ccata
1539415DNAArtificial SequenceSynthetic oligonucleotide
394catacagata cgccc 1539515DNAArtificial SequenceSynthetic
oligonucleotide 395gtaattgcca gtttt 1539615DNAArtificial
SequenceSynthetic oligonucleotide 396aaaaatacaa tgcat
1539715DNAArtificial SequenceSynthetic oligonucleotide
397tcatcaccta tggaa 1539815DNAArtificial SequenceSynthetic
oligonucleotide 398gcaactgagg ttctt 1539915DNAArtificial
SequenceSynthetic oligonucleotide 399aacccattgt ggcca
1540015DNAArtificial SequenceSynthetic oligonucleotide
400ccggcatttg ttctg 1540115DNAArtificial SequenceSynthetic
oligonucleotide 401ctgagggagt aagac 1540215DNAArtificial
SequenceSynthetic oligonucleotide 402ttttatgaat aagct
1540315DNAArtificial SequenceSynthetic oligonucleotide
403tgagaaccca tggtc 1540415DNAArtificial SequenceSynthetic
oligonucleotide 404tcgcatattg acaca 1540515DNAArtificial
SequenceSynthetic oligonucleotide 405tgcctggctg gtgcc
1540615DNAArtificial SequenceSynthetic oligonucleotide
406caccacggca cactt 1540715DNAArtificial SequenceSynthetic
oligonucleotide 407ggagccgggc aggct 1540815DNAArtificial
SequenceSynthetic oligonucleotide 408gtcatcgagt gacac
1540915DNAArtificial SequenceSynthetic oligonucleotide
409tgacaacgat gacgt 1541015DNAArtificial SequenceSynthetic
oligonucleotide 410gataaactga cacaa 1541115DNAArtificial
SequenceSynthetic oligonucleotide 411gctcttgtct gtaag
1541215DNAArtificial SequenceSynthetic oligonucleotide
412caacaatcct agacc 1541315DNAArtificial SequenceSynthetic
oligonucleotide 413agctgtcgcc cgtgt 1541415DNAArtificial
SequenceSynthetic oligonucleotide 414gtaattgcag tgtgt
1541515DNAArtificial SequenceSynthetic oligonucleotide
415ctgaattccg cagcc 1541615DNAArtificial SequenceSynthetic
oligonucleotide 416ggcaccatgg gattt 1541715DNAArtificial
SequenceSynthetic oligonucleotide 417tgattttcag tagtt
1541815DNAArtificial SequenceSynthetic oligonucleotide
418agatctggat ttgaa 1541915DNAArtificial SequenceSynthetic
oligonucleotide 419tcccaacacc acccc 1542015DNAArtificial
SequenceSynthetic oligonucleotide 420atgagagaaa caccc
1542115DNAArtificial SequenceSynthetic oligonucleotide
421gcacacattt agctc 1542215DNAArtificial SequenceSynthetic
oligonucleotide 422cccgaggggt cctcg 1542315DNAArtificial
SequenceSynthetic oligonucleotide 423agaagccctg cccag
1542415DNAArtificial SequenceSynthetic oligonucleotide
424aagaaggaac attcc 1542515DNAArtificial SequenceSynthetic
oligonucleotide 425gcaagaacag gcgtt 1542615DNAArtificial
SequenceSynthetic oligonucleotide 426gagacccagg ctcgg
1542715DNAArtificial SequenceSynthetic oligonucleotide
427ctgaagggtt ttgag 1542815DNAArtificial SequenceSynthetic
oligonucleotide 428gtaattgaga ttttt 1542915DNAArtificial
SequenceSynthetic oligonucleotide 429ttcaaatggg aagtc
1543015DNAArtificial SequenceSynthetic oligonucleotide
430aggacaagta gagtt 1543115DNAArtificial SequenceSynthetic
oligonucleotide 431caaacatgtc caggt 1543215DNAArtificial
SequenceSynthetic oligonucleotide 432ctatatctat ctcca
1543315DNAArtificial SequenceSynthetic oligonucleotide
433agcgctgttg ctagc 1543415DNAArtificial SequenceSynthetic
oligonucleotide 434aacctcagca gactg 1543515DNAArtificial
SequenceSynthetic oligonucleotide 435agcccctgca aggga
1543615DNAArtificial SequenceSynthetic oligonucleotide
436caaggtactg gtact 1543715DNAArtificial SequenceSynthetic
oligonucleotide 437atagaacttt ccccc 1543815DNAArtificial
SequenceSynthetic oligonucleotide 438acattttcag acagc
1543915DNAArtificial SequenceSynthetic oligonucleotide
439tttcttagag actca 1544015DNAArtificial SequenceSynthetic
oligonucleotide 440tgccactctt actag 1544115DNAArtificial
SequenceSynthetic oligonucleotide 441cttacgttgg gagaa
1544215DNAArtificial SequenceSynthetic oligonucleotide
442cctggtacag aatac 1544315DNAArtificial SequenceSynthetic
oligonucleotide 443ggtctgggcc aggtc 1544415DNAArtificial
SequenceSynthetic oligonucleotide 444tgcaacagca atgca
1544515DNAArtificial SequenceSynthetic oligonucleotide
445cacaggaagc agaca 1544615DNAArtificial SequenceSynthetic
oligonucleotide 446tggtagatac tatta 1544715DNAArtificial
SequenceSynthetic oligonucleotide 447agttggggtg ctggt
1544815DNAArtificial SequenceSynthetic oligonucleotide
448gtttcagtgc ccaag 1544915DNAArtificial SequenceSynthetic
oligonucleotide 449gggacgagca agcac 1545015DNAArtificial
SequenceSynthetic oligonucleotide 450cccgaaagcc cccag
1545115DNAArtificial SequenceSynthetic oligonucleotide
451cccgcccgcg atccc 1545215DNAArtificial SequenceSynthetic
oligonucleotide 452tcgcaaccgc agcga 1545315DNAArtificial
SequenceSynthetic oligonucleotide 453caggttcctg gatca
1545415DNAArtificial SequenceSynthetic oligonucleotide
454tctatcctat gtctt 1545515DNAArtificial SequenceSynthetic
oligonucleotide 455acatttggag aggga 1545615DNAArtificial
SequenceSynthetic oligonucleotide 456gagctagcat acaag
1545715DNAArtificial SequenceSynthetic oligonucleotide
457ctaagaaagc cacac 1545815DNAArtificial SequenceSynthetic
oligonucleotide 458gcagtaccag cctag 1545915DNAArtificial
SequenceSynthetic oligonucleotide 459tcagaggcag ctgct
1546015DNAArtificial SequenceSynthetic oligonucleotide
460aagtgagtgc agcca 1546115DNAArtificial SequenceSynthetic
oligonucleotide 461agtgccctgc acact 1546215DNAArtificial
SequenceSynthetic oligonucleotide 462tgaacaacac aggtt
1546315DNAArtificial SequenceSynthetic oligonucleotide
463gagagcgctg cctcc 1546415DNAArtificial SequenceSynthetic
oligonucleotide 464tcaagcttat cctaa 1546515DNAArtificial
SequenceSynthetic oligonucleotide 465ccctagtggc gccat
1546615DNAArtificial SequenceSynthetic oligonucleotide
466gaaactgtgg ttttt 1546715DNAArtificial SequenceSynthetic
oligonucleotide 467gccagagacc caggc 1546815DNAArtificial
SequenceSynthetic oligonucleotide 468gggccacaac gtggg
1546915DNAArtificial SequenceSynthetic oligonucleotide
469ccgcggcgcc ccgcc 1547015DNAArtificial SequenceSynthetic
oligonucleotide 470taacaataca ctgcc 1547115DNAArtificial
SequenceSynthetic oligonucleotide 471gtagagattg tttca
1547215DNAArtificial SequenceSynthetic oligonucleotide
472gctatacggt ctact 1547315DNAArtificial SequenceSynthetic
oligonucleotide 473gttctgcggc ccacc 1547415DNAArtificial
SequenceSynthetic oligonucleotide 474gttaaccatg tatta
1547515DNAArtificial SequenceSynthetic oligonucleotide
475ttgactgtat aatat 1547615DNAArtificial SequenceSynthetic
oligonucleotide 476tcatccatag ttgtc 1547715DNAArtificial
SequenceSynthetic oligonucleotide 477agggtgagaa cctgc
1547815DNAArtificial SequenceSynthetic oligonucleotide
478cctacttccc tccgc 1547915DNAArtificial SequenceSynthetic
oligonucleotide 479ccctccctga accaa 1548015DNAArtificial
SequenceSynthetic oligonucleotide 480cgatatgcaa tgggt
1548115DNAArtificial SequenceSynthetic oligonucleotide
481attaatgtct gttga 1548215DNAArtificial SequenceSynthetic
oligonucleotide 482acatgatgat ccccg 1548315DNAArtificial
SequenceSynthetic oligonucleotide 483ctgcacgtcc atgtc
1548415DNAArtificial SequenceSynthetic oligonucleotide
484acgaggccga gacgc 1548515DNAArtificial SequenceSynthetic
oligonucleotide 485gcgccagccc atccc 1548615DNAArtificial
SequenceSynthetic oligonucleotide 486catcgtgatc caccc
1548715DNAArtificial SequenceSynthetic oligonucleotide
487agaaggagaa tctac 1548815DNAArtificial SequenceSynthetic
oligonucleotide 488ttatcaatct gtcac 1548915DNAArtificial
SequenceSynthetic oligonucleotide 489gtggatagcg gtgct
1549015DNAArtificial SequenceSynthetic oligonucleotide
490gagtgatcgt gtcat 1549115DNAArtificial SequenceSynthetic
oligonucleotide 491acactaacac taggt 1549215DNAArtificial
SequenceSynthetic oligonucleotide 492ggtgactagt ggtgc
1549315DNAArtificial SequenceSynthetic oligonucleotide
493ccagcagcat caggt 1549415DNAArtificial SequenceSynthetic
oligonucleotide 494agctatattc acctt 1549515DNAArtificial
SequenceSynthetic oligonucleotide 495atggattgga ccaac
1549615DNAArtificial SequenceSynthetic oligonucleotide
496gctagtccga tcccc 1549715DNAArtificial SequenceSynthetic
oligonucleotide 497acactggact atgat 1549815DNAArtificial
SequenceSynthetic oligonucleotide 498aatctaggaa accgt
1549915DNAArtificial SequenceSynthetic oligonucleotide
499ccccatagat tgtga 1550015DNAArtificial SequenceSynthetic
oligonucleotide 500gacccatgaa gtgtt 1550115DNAArtificial
SequenceSynthetic oligonucleotide 501aactccatgg ttatg
1550215DNAArtificial SequenceSynthetic oligonucleotide
502agcgcaccaa actgt 1550315DNAArtificial SequenceSynthetic
oligonucleotide 503tcagcctggt gtgcg 1550415DNAArtificial
SequenceSynthetic oligonucleotide 504accaaacacc acagg
1550515DNAArtificial SequenceSynthetic oligonucleotide
505tccctggcaa gttac 1550615DNAArtificial SequenceSynthetic
oligonucleotide 506tcggcagcgt agggt 1550715DNAArtificial
SequenceSynthetic oligonucleotide 507actactgcag cattt
1550815DNAArtificial SequenceSynthetic oligonucleotide
508ggaatggtgg cctgg 1550915DNAArtificial SequenceSynthetic
oligonucleotide 509aggaaacaaa accac 1551015DNAArtificial
SequenceSynthetic oligonucleotide 510cacacaccca ctcta
1551115DNAArtificial SequenceSynthetic oligonucleotide
511atgcctgcgt cctct 1551215DNAArtificial SequenceSynthetic
oligonucleotide 512gacaccaggc ataca 1551315DNAArtificial
SequenceSynthetic oligonucleotide 513aggaagtgcg aactt
1551415DNAArtificial SequenceSynthetic oligonucleotide
514ccaagcaaac aaaac 1551515DNAArtificial SequenceSynthetic
oligonucleotide 515aagacatgcc tgcta
1551615DNAArtificial SequenceSynthetic oligonucleotide
516aggctgtgcc ttcat 1551715DNAArtificial SequenceSynthetic
oligonucleotide 517acttcccgtc cttcc 1551815DNAArtificial
SequenceSynthetic oligonucleotide 518tggaccaggt cacaa
1551915DNAArtificial SequenceSynthetic oligonucleotide
519ccctccaggg cttcc 1552015DNAArtificial SequenceSynthetic
oligonucleotide 520gccgagccga gtgac 1552115DNAArtificial
SequenceSynthetic oligonucleotide 521agacaaccat ggtgc
1552215DNAArtificial SequenceSynthetic oligonucleotide
522atggggtatg agcag 1552315DNAArtificial SequenceSynthetic
oligonucleotide 523acaatattga taggg 1552415DNAArtificial
SequenceSynthetic oligonucleotide 524aagcaatatt gcact
1552515DNAArtificial SequenceSynthetic oligonucleotide
525gaacccagag gtctc 1552615DNAArtificial SequenceSynthetic
oligonucleotide 526accccggaga tccca 1552715DNAArtificial
SequenceSynthetic oligonucleotide 527gctgtggggc tggag
1552815DNAArtificial SequenceSynthetic oligonucleotide
528ccttccttct cctcc 1552915DNAArtificial SequenceSynthetic
oligonucleotide 529actttcatcc tccaa 1553015DNAArtificial
SequenceSynthetic oligonucleotide 530agtgtcagca ttgtg
1553115DNAArtificial SequenceSynthetic oligonucleotide
531gacacgtggt actgg 1553215DNAArtificial SequenceSynthetic
oligonucleotide 532tgaatctttg ttact 1553315DNAArtificial
SequenceSynthetic oligonucleotide 533cgcacgcaga gcaat
1553415DNAArtificial SequenceSynthetic oligonucleotide
534ggccctctcc gcacc 1553523RNAHomo sapiens 535uggaguguga caaugguguu
ugu 2353623DNAArtificial SequenceSynthetic oligonucleotide
536acaaacacca ttgtcacact cca 2353723DNAArtificial SequenceSynthetic
oligonucleotide 537acaaacacca ttgtcacact cca 2353823DNAArtificial
SequenceSynthetic oligonucleotide 538acaaacacca ttgtcacact cca
2353915DNAArtificial SequenceSynthetic oligonucleotide
539ccattgtcac actcc 1554015DNAArtificial SequenceSynthetic
oligonucleotide 540ccattgtcac actcc 1554113DNAArtificial
SequenceSynthetic oligonucleotide 541attgtcacac tcc
1354211DNAArtificial SequenceSynthetic oligonucleotide
542tgtcacactc c 1154315DNAArtificial SequenceSynthetic
oligonucleotide 543ccattgtcac actcc 1554415DNAArtificial
SequenceSynthetic oligonucleotide 544ccattgtcac actcc
1554523RNAHomo sapiens 545ugugcaaauc caugcaaaac uga
2354623DNAArtificial SequenceSynthetic oligonucleotide
546tcagttttgc atggatttgc aca 2354723DNAArtificial SequenceSynthetic
oligonucleotide 547tcagttttgc atggatttgc aca 2354823DNAArtificial
SequenceSynthetic oligonucleotide 548tcagttttgc atggatttgc aca
2354915DNAArtificial SequenceSynthetic oligonucleotide
549tgcatggatt tgcac 1555015DNAArtificial SequenceSynthetic
oligonucleotide 550tgcatggatt tgcac 1555113DNAArtificial
SequenceSynthetic oligonucleotide 551catggatttg cac
1355211DNAArtificial SequenceSynthetic oligonucleotide
552tggatttgca c 1155315DNAArtificial SequenceSynthetic
oligonucleotide 553tgcatggatt tgcac 1555415DNAArtificial
SequenceSynthetic oligonucleotide 554tgcatggatt tgcac
1555522RNAHomo sapiens 555uuaaugcuaa ucgugauagg gg
2255622DNAArtificial SequenceSynthetic oligonucleotide
556cccctatcac gattagcatt aa 2255722DNAArtificial SequenceSynthetic
oligonucleotide 557cccctatcac gattagcatt aa 2255822DNAArtificial
SequenceSynthetic oligonucleotide 558cccctatcac gattagcatt aa
2255915DNAArtificial SequenceSynthetic oligonucleotide
559tcacgattag catta 1556015DNAArtificial SequenceSynthetic
oligonucleotide 560tcacgattag catta 1556113DNAArtificial
SequenceSynthetic oligonucleotide 561acgattagca tta
1356211DNAArtificial SequenceSynthetic oligonucleotide
562gattagcatt a 1156315DNAArtificial SequenceSynthetic
oligonucleotide 563tcacgattag catta 1556415DNAArtificial
SequenceSynthetic oligonucleotide 564tcacgattag catta
1556522RNAHomo sapiens 565uagcuuauca gacugauguu ga
2256623DNAArtificial SequenceSynthetic oligonucleotide
566tcatcatcag tctgataagc tta 2356723DNAArtificial SequenceSynthetic
oligonucleotide 567tcatcatcag tctgataagc tta 2356823DNAArtificial
SequenceSynthetic oligonucleotide 568tcatcatcag tctgataagc tta
2356915DNAArtificial SequenceSynthetic oligonucleotide
569tcagtctgat aagct 1557015DNAArtificial SequenceSynthetic
oligonucleotide 570tcagtctgat aagct 1557113DNAArtificial
SequenceSynthetic oligonucleotide 571agtctgataa gct
1357211DNAArtificial SequenceSynthetic oligonucleotide
572tctgataagc t 1157315DNAArtificial SequenceSynthetic
oligonucleotide 573tcagtctgat aagct 1557415DNAArtificial
SequenceSynthetic oligonucleotide 574tcagtctgat aagct
1557522RNAHomo sapiens 575uuuguucguu cggcucgcgu ga
2257622DNAArtificial SequenceSynthetic oligonucleotide
576tctcgcgtgc cgttcgttct tt 2257722DNAArtificial SequenceSynthetic
oligonucleotide 577tctcgcgtgc cgttcgttct tt 2257822DNAArtificial
SequenceSynthetic oligonucleotide 578tctcgcgtgc cgttcgttct tt
2257915DNAArtificial SequenceSynthetic oligonucleotide
579gtgccgttcg ttctt 1558015DNAArtificial SequenceSynthetic
oligonucleotide 580gtgccgttcg ttctt 1558113DNAArtificial
SequenceSynthetic oligonucleotide 581gccgttcgtt ctt
1358211DNAArtificial SequenceSynthetic oligonucleotide
582cgttcgttct t 1158315DNAArtificial SequenceSynthetic
oligonucleotide 583gtgccgttcg ttctt 1558415DNAArtificial
SequenceSynthetic oligonucleotide 584gtgccgttcg ttctt
1558516DNAArtificial SequenceSynthetic oligonucleotide
585ccattgtcac actcca 1658616DNAArtificial SequenceSynthetic
oligonucleotide 586ccattgtcac actcca 1658716DNAArtificial
SequenceSynthetic oligonucleotide 587ccattgtcac actcca
1658815DNAArtificial SequenceSynthetic oligonucleotide
588ccattgtcac actcc 1558915DNAArtificial SequenceSynthetic
oligonucleotide 589ccattctgac cctac 1559016DNAArtificial
SequenceSynthetic oligonucleotide 590ccattgtctc aatcca
1659113DNAArtificial SequenceSynthetic oligonucleotide
591attgtcacac tcc 1359215DNAArtificial SequenceSynthetic
oligonucleotide 592ccattctgac cctac 1559322RNAHomo sapiens
593uagcuuauca gacugauguu ga 2259416DNAArtificial SequenceSynthetic
oligonucleotide 594tcagtctgat aagcta 1659516DNAArtificial
SequenceSynthetic oligonucleotide 595tccgtcttag aagata
1659615DNAArtificial SequenceSynthetic oligonucleotide
596tctgtcagat acgat 1559715DNAArtificial SequenceSynthetic
oligonucleotide 597tcagtctgat aagct 1559815DNAArtificial
SequenceSynthetic oligonucleotide 598tcagtctgat aagct 15
* * * * *
References