# Patent

## Title
MicroRNAs

## Abstract

The present invention relates to very short heavily modified oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.

## Background
RELATED APPLICATIONS
This application claims priority from four applications: U.S. 60/977,497 filed 4 Oct. 2007, U.S. 60/979,217 filed 11 Oct. 2007, U.S. 61/028,062, filed 12 Feb. 2008, and EP08104780, filed 17 Jul. 2008, all of which are hereby incorporated by reference. Furthermore we reference and incorporate by reference WO2007/112754 and WO2007/112753 which are earlier applications from the same applicants.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB
The content of the electronically submitted sequence listing (Name: 2763—0130004_sequence_listing_ascii.txt; Size: 567,833 bytes; and Date of Creation: Jun. 4, 2012) filed with the application is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to very short oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.
BACKGROUND OF THE INVENTION
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. 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.
A summary of the role of microRNAs in human diseases, and the inhibition of microRNAs using single stranded oligonucleotides is provided by WO2007/112754 and WO2007/112753, which are both hereby incorporated by reference in its entirety. WO2008046911, hereby incorporated by reference, provides microRNA sequences which are associated with cancer. Numerous microRNAs have been associated with disease phenotypes and it is therefore desirable to provide substances capable of modulating the availability of microRNAs in vivo. WO2007/112754 and WO2007/112753 disclose short single stranded oligonucleotides which are considered to form a strong duplex with their target miRNA. SEQ ID NOs 1-45 are examples of anti microRNA oligonucleotides as disclosed in WO2007/112754 and WO2007/112753.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that the use of very short oligonucleotides which target microRNAs and which have a high proportion of nucleotide analogue nucleotides, such as LNA nucleotides, are highly effective in alleviating the repression of RNAs, such as an mRNA, by the targeted microRNAs in vivo.
The present invention provides an oligomer a contiguous sequence of 7, 8, 9 or 10 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70%, such as at least 80% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2′ substituted nucleotide analogues.
The present invention provides an oligomer a contiguous sequence of 7, 8, 9 or 10 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA units and 2′ substituted nucleotide analogues, and wherein at least 50%, such as at least 60%, such as at least 70% of the nucleotide units of the oligomer are LNA units.
The invention provides oligomers of between 7-10 nucleotides in length which comprises a contiguous nucleotide sequence of a total of between 7-10 nucleotides, such as 7, 8, 9, nucleotide units, wherein at least 50% of the nucleotide units of the oligomer are nucleotide analogues.
The invention further provides for an oligomer of between 7-10 nucleotides in length which comprises a contiguous nucleotide sequence of a total of between 7-10 nucleotides, such as 7, 8, 9, or 10, nucleotide units, wherein the nucleotide sequence is complementary to a corresponding nucleotide sequence found in mammalian or viral microRNA, and wherein at least 50% of the nucleotide units of the oligomer are nucleotide analogues.
The present invention provides olgiomers according to the invention as a medicament.
The present invention provides pharmaceutical compositions comprising the oligomer of the invention and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
The invention provides for a conjugate comprising an oligomer according to the invention, conjugated to at least one non-nucleotide or polynucleotide entity, such as a sterol, such as cholesterol.
The invention provides for the use of an oligomer or a conjugate according to the invention, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression of a microRNA, such as one or more of the microRNAs referred to herein.
The invention provides for the treatment of a disease or medical disorder associated with the presence or overexpression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) comprising an oligomer or conjugate according to the invention to a patient suffering from or likely to suffer from said disease or medical disorder.
The invention provides for a method for reducing the effective amount of a microRNA target in a cell or an organism, comprising administering the oligomer of the invention, or a composition (such as a pharmaceutical composition) comprising the oligomer or conjugate according to the invention to the cell or organism.
The invention provides for a method for reducing the effective amount of a microRNA target in a cell or an organism, comprising administering the oligomer or conjugate or pharmaceutical composition according to the invention to the cell or organism.
The invention provides for a method for de-repression of a target mRNA (or one or more RNAs) in a cell or an organism, comprising administering an oligomer or conjugate according to the invention, or a composition comprising said oligomer or conjugate, to said cell or organism.
The invention provides for the use of an oligomer or a conjugate according to the invention, for inhibiting the mircoRNA in a cell which comprises said microRNA, such as a human cell. The use may be in vivo or in vitro.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Schematic presentation of the miR-21, miR-155 and miR-122 8-mer LNA-antimiRs (Compound 3205 (SEQ ID NO: 2); Compound 3207 (SEQ ID NO: 4); Compound 3208 (SEQ ID NO: 6), indicating the targeting positions with the fully LNA-modified and phosphorothiolated LNA-antimiR. Preferred hybridisation positions for 7-mer, 8-mer, 9-mer and 10-mer LNA oligonucleotides on the mature microRNA are also indicated.
FIG. 2. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2) and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in MCF-7 cells using a luciferase sensor assay. MCF-7 cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 or a mismatch target site (.mm2) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), were all have been normalized against 0 nM psiCHECK2 (=control).
FIG. 3. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2) and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in HeLa cells using a luciferase sensor assay. HeLa cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 (mir-21) or a mismatch target site (mm2) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), were all have been normalized against 0 nM psiCHECK2 (=control).
FIG. 4. Assessment of miR-155 antagonism by Compound 3206 (SEQ ID NO: 3) and Compound 3207 (SEQ ID NO: 4) LNA-antimiRs in LPS-treated mouse RAW cells using a luciferase sensor assay. RAW cells were co-transfected with miR-155 and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly, were all have been normalized against 0 nM psiCHECK2.
FIG. 5. Assessment of miR-122 antagonism by Compound 3208 (SEQ ID NO: 6) and Compound 4 (SEQ ID NO: 5) LNA-antimiRs in HuH-7 cells using a luciferase sensor assay. HuH-7 cells were co-transfected with a miR-122 luciferase sensor containing a perfect match miR-122 target site and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for three separate experiments (bars=s.e.m), where all have been normalized against 0 nM psiCHECK2 (=control).
FIG. 6. Schematic presentation of the miR-21 luciferase reporter constructs.
FIG. 7. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) in PC3 cells using a luciferase reporter assay. PC3 cells were co-transfected with luciferase reporter plasmids containing a perfect match target site for miR-21 or a mismatch target site and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs. LNA nucleotides are indicated by ovals, and DNA residues are indicated by bars.
FIG. 8. Specificity assessment of miR-21 antagonism by an 8-mer LNA-antimiR in HeLa cells using a luciferase reporter assay. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatched target site for miR-21 and LNA-antimiRs Compound 3205 (SEQ ID NO: 2) or an 8-mer LNA mismatch control oligo Compound 3218 (SEQ ID NO: 16) at different concentrations. After 24 hours, cells were harvested and luciferase activity was measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the Renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs. Mismatches are indicated by filled ovals.
FIG. 9. Assessment of the shortest possible length of a fully LNA-modified LNA-antimiR that mediates effective antagonism of miR-21. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and the LNA-antimiRs at different concentrations (Compound 3209=6-mer (SEQ ID NO: 7) and Compound 3210=7-mer (SEQ ID NO: 8)). After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.
FIG. 10. Length assessment of fully LNA-substituted LNA-antimiRs antagonizing miR-21. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and LNA-antimiRs at different concentrations (Compound 3211=9-mer (SEQ ID NO: 9), Compound 3212=10-mer (SEQ ID NO: 10), Compound 3213=12-mer (SEQ ID NO: 11) and Compound 3214=14-mer (SEQ ID NO: 12)). After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.
FIG. 11. Determination of the most optimal position for an 8-mer LNA-antimiR within the miR target recognition sequence. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match or a mismatch target site for miR-21 and the LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs (Compound 3205 (SEQ ID NO: 2; Compound 3215 (SEQ ID NO: 13); Compound 3216 (SEQ ID NO: 14); Compound 3217 (SEQ ID NO: 15).
FIG. 12. Validation of interaction of the Pdcd-4-3′-UTR and miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR. HeLa cells were co-transfected with a luciferase reporter plasmid containing part of the 3′UTR of Pdcd4 gene and LNA-antimiRs at different concentrations (Compound 3205=8-mer, perfect match (SEQ ID NO: 2); Compound 3218=8-mer, mismatch (SEQ ID NO: 16); Compound 3204=15-mer, LNA/DNA mix (SEQ ID NO: 1); Compound 3220=15-mer, gapmer (SEQ ID NO: 18)). After 24 hours, cells were harvested and luciferase activity measured. Shown are renilla/firefly ratios that have been normalized against 0 nM. Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.
FIG. 13. Comparison of an 8-mer LNA-antimiR (Compound 3207 (SEQ ID NO: 4)) with a 15-mer LNA-antimiR (Compound 3206 (SEQ ID NO: 3)) in antagonizing miR-155 in mouse RAW cells. Mouse RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-155 and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without miR-155 target site (=control). Shown is also a schematic presentation of the miR-155 sequence and the design and position of the LNA-antimiRs.
FIG. 14. Assessment of c/EBP□Assessment of c/EBPer LNA-antimiR (Compound 3207 (SEQ ID NO: 4)) with a 15-mer LNA-antimiR (Compound 3206 (SEQ ID NO: 3)) in antagonizing miR-155 in mouse RAW cells. Mouse RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-155 and the diffter 20 hours, cells were harvested and western blot analysis of protein extracts from RAW cells was performed. The different isoforms of c/EBPβ are indicated, and the ratios calculated on c/EBPβ LIP and beta-tubulin are shown below.
FIG. 15. Antagonism of miR-106b by a fully LNA-modified 8-mer (Compound 3221 (SEQ ID NO: 19)) LNA-antimiR or by a 15-mer mixmer (Compound 3228 (SEQ ID NO: 26)) antimiR. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-106b and the different LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values of four replicates where the renilla/firefly ratios have been normalized against 0 nM empty vector without miRNA target site (=control). Shown is also a schematic presentation of the miR-106b sequence and the design and position of the LNA-antimiRs.
FIG. 16. Antagonism of miR-19b by a fully LNA-modified 8-mer (Compound 3222 (SEQ ID NO: 20)) LNA-antimiR and a 15-mer (Compound 3229 (SEQ ID NO: 27)) mixmer antimiR. HeLa cells were co-transfected with luciferase reporter plasmids containing a perfect match for miR-19a and the two LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values of four replicate experiments, where the renilla/firefly ratios have been normalized against 0 nM empty vector without a miR-19a target site (=control). Shown is also a schematic presentation of the miR-19a sequence and the design and position of the LNA-antimiRs.
FIG. 17. Schematic presentation showing the mature human miR-221 (SEQ ID NO: 432) and miR-222 (SEQ ID NO: 434) sequences. Shown in the square is the seed sequence (7-mer) that is conserved in both miRNA sequences.
FIG. 18. Targeting of a microRNA family using short, fully LNA-substituted LNA-antimiR. PC3 cells were co-transfected with luciferase reporter plasmids for miR-221 and miR-222 separately or together and with the different LNA-antimiRs at varying concentrations. When co-transfecting with the LNA-antimiRs (15-mers) Compound 3223 (SEQ ID NO: 21) (against miR-221) and Compound 3224 (SEQ ID NO: 22) (against miR-222), the total concentration was 2 nM (1 nM each), while transfecting the cells with Compound 3225 (SEQ ID NO: 23) (7-mer) the concentrations were 0, 1, 5, 10 or 25 nM. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without a miRNA target site (=control). Shown is also a schematic presentation of the miR-221/222 sequence and the design and position of the LNA-antimiRs.
FIG. 19. Assessment of p27 protein levels as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR. PC3 cells were transfected with the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 at varying concentrations. After 24 hours, cells were harvested and protein levels were measured on a western blot. Shown are the ratios of p27/tubulin.
FIG. 20. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) and an 8-mer with 2 mismatches Compound 3218 (SEQ ID NO: 16) in HepG2 cells using a luciferase reporter assay.
HepG2 cells were co-transfected with luciferase reporter plasmid containing a perfect match target site for miR-21 and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.
FIG. 21. Validation of interaction of the Pdcd4 3′UTR and miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR versus the 15-mer Compound 3204 (SEQ ID NO: 1) and an 8-mer with two mismatches Compound 3218 (SEQ ID NO: 16).
Huh-7 cells were co-transfected with a luciferase reporter plasmid containing part of the 3′UTR of Pdcd4 gene, pre-miR-21 (10 nM) and LNA-antimiRs at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) of three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=control). Shown is also a schematic presentation of the miR-21 sequence and the design and position of the LNA-antimiRs.
FIG. 22. Antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) leads to increased levels of Pdcd4 protein levels.
HeLa cells were transfected with 5 nM LNA-antimiR Compound 3205 (SEQ ID NO: 2) (perfect match), or Compound 3219 (SEQ ID NO: 17) LNA scrambled (8 mer) or Compound 3218 (SEQ ID NO: 16) (8-mer mismatch). Cells were harvested after 24 hours and subjected to Western blot with Pdcd4 antibody.
FIG. 23. ALT and AST levels in mice treated with Compound 3205 (SEQ ID NO: 2) (perfect match) or Compound 3218 (SEQ ID NO: 16) (mismatch control). Mice were sacrificed after 14 days and after receiving 25 mg/kg every other day.
FIG. 24. Assessment of PU.1 protein levels as a functional readout for miR-155 antagonism by short LNA-antimiR Compound 3207 (SEQ ID NO: 4).
THP-1 cells were co-transfected with pre-miR-155 (5 nmol) and different LNA oligonucleotides (5 nM) and 100 ng/ml LPS was added. After 24 hours, cells were harvested and western blot analysis of protein extracts from the THP-1 cells was performed. PU.1 and tubulin are indicated.
FIG. 25. Assessment of p27 protein levels as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR.
PC3 cells were transfected with the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 and a LNA scrambled control at 5 and 25 nM. After 24 hours, cells were harvested and protein levels were measured on a western blot. Shown are the ratios of p27/tubulin.
FIG. 26. Knock-down of miR-221/222 by the 7-mer Compound 3225 (SEQ ID NO: 23) (perfect match) LNA-antimiR reduces colony formation in soft agar in PC3 cells.
PC3 cells were transfected with 25 nM of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 or a 7-mer scrambled control Compound 3231 (SEQ ID NO: 28). After 24 hours, cells were harvested and seeded on soft agar. After 12 days, colonies were counted. One experiment has been done in triplicate.
FIG. 27. Overview of the human let-7 family, and of tested antagonists.
(upper) The sequences represent the mature miRNA for each member and the box depicts nucleotides 2-16, the positions typically antagonized by LNA-antimiRs (let-7a, SEQ ID NO: 96; let-7b, SEQ ID NO: 98; let-7c, SEQ ID NO: 100; let-7d, SEQ ID NO: 102; let-7e, SEQ ID NO: 104; let-7f, SEQ ID NO: 106; let-71, SEQ ID NO: 111; miR-98, SEQ ID NO: 947). Columns to the right show the number of nucleotide differences compared to let-7a, within the seed (S: position 2-8), extended seed (ES; position 2-9), and the remaining sequence typically targeted by LNA-antimiRs (NE; position 9-16), respectively. Nucleotides with inverted colors are altered compared to let-7a. (lower) Summary of tested antagonists against the let-7 family, including information on design, length and perfectly complementary targets. All compounds are fully phoshorothiolated.
FIG. 28. Assessment of let-7 antagonism by six different LNA-antimiRs in Huh-7 cells using a luciferase sensor assay.
Huh-7 cells were co-transfected with luciferase sensor plasmids containing a partial HMGA2 3′UTR (with four let-7 binding sites), with or without let-7a precursor (grey and black bars, respectively), and with 6 different LNA-antimiRs at increasing concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean of renilla/firefly ratios for duplicate measurements and standard deviations for each assay. Within each LNA-antimiR group all ratios have been normalized to the average of wells containing no let-7a precursor (black bars).
FIG. 29. Luciferase results from Huh-7 cells transfected with the HMGA2 3′UTR sensor plasmid, LNA-antimiRs Compound 3226 (SEQ ID NO: 24) (left) and Compound 3227 (SEQ ID NO: 25) (right), and pre-miRs for let-7a (A), let-7d (B), let-7e (C), and let-71 (D). Grey bars indicate the target de-repression after pre-mis inclusion, whereas black control bars represent the equivalent level without pre-miR addition. Each ratio is based on quadruplicate measurements and have been normalized against the average of wells containing no precursor (black bars) within each treatment group.
FIG. 30. Luciferase results from HeLa cells transfected with the HMGA2 3′UTR sensor plasmid or control vector, and the LNA-antimiR Compound 3227 (SEQ ID NO: 25) at various concentrations. Each ratio is based on quadruplicate measurements normalized against untreated (0 nM) empty control vector (psi-CHECK-2; grey bars).
FIG. 31. Assessment of miR-21 antagonism by 8 mer Compound 3205 (SEQ ID NO: 2) in HCT116 cells using a luciferase sensor assay. HCT116 cells were co-transfected with luciferase sensor plasmids containing a perfect match target site for miR-21 (grey bars) and LNA-antimiR and control oigonucleotides at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown is one typical example of two where the renilla/firefly ratios have been normalized against 0 nM empty vector (=black bars).
FIG. 32. Silencing of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR reduces colony formation in soft agar in PC3 cells. PC3 cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvested and seeded on soft agar. After 12 days, colonies were counted. Shown is the mean of three separate experiments, each performed in triplicate, and normalised against 0 nM control (i.e. transfection but with no LNA). p=0.01898 for Compound 3205 (SEQ ID NO: 2).
FIG. 33. Knock-down of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR reduces colony formation in soft agar in HepG2 cells. HepG2 cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvested and seeded on soft agar. After 17 days, colonies were counted. Shown is the mean of three replicates from one experiment (bars=SEM).
FIG. 34. Wound closure in the invasive human prostate cell line PC3 after treatment with Compound 3205 (SEQ ID NO: 2). (A) PC3 cells were transfected at day 3 with LNA-antimiR and control oligonucleotides at 25 nM, Compound 3205 (SEQ ID NO: 2) (8 mer, perfect match) and Compound 3219 (SEQ ID NO: 17) (8 mer, mismatch) and the following day a scratch was made. Pictures were taken after 24 hours in order to monitor the migration. (B) The area in each timepoint has been measured with the software program Image J and normalized against respective 0 h time-point.
FIG. 35. Length assessment of fully LNA-substituted LNA-antimiRs antagonizing miR-155. RAW cells were co-transfected with luciferase reporter plasmids containing a perfect match target site for miR-155 and with LNA-antimiR oligonucleotides at different concentrations. After 24 hours, cells were harvested and luciferase activity measured. Shown are the mean values (bars=s.e.m) for three independent experiments where the renilla/firefly ratios have been normalized against 0 nM empty vector without target site (=mock). Shown is also a schematic presentation of the miR sequence and the design and position of the LNA-antimiRs.
FIG. 36. Binding of 5′-FAM labeled LNA-antimiR-21 Compound 3205 (SEQ ID NO: 2) to mouse plasma protein.
(A) % unbound LNA-antimiR-21 compound as a function of oligonucleotide concentration in mouse plasma. (B) Concentration of unbound LNA-antimiR-21 Compound 3205 (SEQ ID NO: 2) as a function of Compound 3205 (SEQ ID NO: 2) concentration in mouse plasma.
FIG. 37. Quantification Ras protein levels by Western blot analysis. A. Gel image showing Ras and Tubulin (internal standard) protein in treated (anti-let-7; 8-mer) vs. untreated (saline) lung and kidney samples. B. Quantifications of Ras protein levels in the lung and kidney, respectively, of LNA-antimiR-treated mice (black bars), normalized against equivalent saline controls (grey bars), using tubulin as equal-loading control.
FIG. 38. Silencing of miR-21 by Compound 3205 (SEQ ID NO: 2) leads to increased levels of Pdcd4 protein levels in vivo. Mice were injected with saline or 25 mg/kg LNA-antimiR Compound 3205 (SEQ ID NO: 2) over 14 days every other day, with a total of 5 doses. Mice were sacrificed and protein was isolated from kidney and subjected to Western blot analysis with Pdcd4 antibody. A. Gel image showing Pdcd4 and Gapdh (internal standard) protein in treated (antimiR-21; 8-mer) vs. untreated (saline) kidney samples (M1, mouse 1; M2, mouse 2). B. Quantification of Pdcd4 protein levels in kidneys of LNA-antimiR-treated mice (dark grey bars), normalized against the average of equivalent saline controls (light grey bars), using Gapdh as loading control.



DETAILED DESCRIPTION OF THE INVENTION
Short oligonucleotides which incorporate LNA are known from the in vitro reagents area, (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 in vivo or pharmaceutical use. For example, the terminal nucleotides 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 oligonucleotides (referred to herein as oligomers) per se.
The following embodiments refer to certain embodiments of the oligomer of the invention, which may be used in a pharmaceutical composition. Aspects which refer to the oligomer may also refer to the contiguous nucleotide sequence, and vice versa.
The Oligomer
The oligomer of the invention is a single stranded oligonucleotide which comprises nucleotide analogues, such as LNA, which form part of, or the entire contiguous nucleotide sequence of the oligonucleotide. The nucleotide sequence of the oligomer consists of a contiguous nucleotide sequence.
The term “oligonucleotide” (or simply “oligo”), which is used interchangeably with the term “oligomer” refers, in the context of the present invention, to a molecule formed by covalent linkage of two or more nucleotides. 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 have between 7-10 nucleotides, such as in individual embodiments, 7, 8, 9, or 10.
The term ‘nucleotide’ refers to nucleotides, such as DNA and RNA, and nucleotide analogues. It should be recognised that, in some aspects, the term nucleobase may also be used to refer to a nucleotide which may be either naturally occurring or non-naturally occurring—in this respect the term nucleobase and nucleotide may be used interchangeably herein.
In some embodiments, the contiguous nucleotide sequence consists of 7 nucleotide analogues. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues. In some embodiments, the contiguous nucleotide sequence consists of 9 nucleotide analogues.
In one embodiment at least about 50% of the nucleotides of the oligomer are nucleotide analogues, such as at least about 55%, such as at least about 60%, or at least about 65% or at least about 70%, such as at least about 75%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95% or such as 100%. It will also be apparent that the oligonucleotide may comprise of a nucleotide sequence which consists of only nucleotide analogues. Suitably, the oligomer may comprise at least one LNA monomer, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA monomers. As described below, the contiguous nucleotide sequence may consist only of LNA units (including linkage groups, such as phosphorothioate linkages), or may consist of LNA and DNA units, or LNA and other nucleotide analogues. In some embodiments, the contiguous nucleotide sequence comprises either one or two DNA nucleotides, the remainder of the nucleotides being nucleotide analogues, such as LNA unit.
In some embodiments, the contiguous nucleotide sequence consists of 6 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide consists of 7 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 9 nucleotide analogues and a single DNA nucleotide. In some embodiments, the contiguous nucleotide sequence consists of 7 nucleotide analogues and two DNA nucleotides. In some embodiments, the contiguous nucleotide sequence consists of 8 nucleotide analogues and two DNA nucleotides.
The oligomer may consist of the contiguous nucleotide sequence.
In a specially preferred embodiment, all the nucleotide analogues are LNA. In a further preferred embodiment, all nucleotides of the oligomer are LNA. In a further preferred embodiment, all nucleotides of the oligomer are LNA and all internucleoside linkage groups are phosphothioate.
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 (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 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 MeC.
It should be recognised that the term in ‘one embodiment’ should not necessarily be limited to refer to one specific embodiment, but may refer to a feature which may be present in ‘some embodiments’, or even as a generic feature of the invention. Likewise, the use of the term ‘some embodiments’ may be used to describe a feature of one specific embodiment, or a collection of embodiments, or even as a generic feature of the invention.
The terms “corresponding to” and “corresponds to” refer to the comparison between the nucleotide sequence of the oligomer or contiguous nucleotide sequence (a first sequence) and the equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the microRNA nucleic acid target (such as a microRNA target selected from SEQ ID NO:40 to SEQ ID NO:976, and/or ii) the sequence of 9-mer, 8-mer and 7-mer nucleotides provided herein in Table 1. Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides. A first sequence which corresponds to a further sequence under i) or ii) typically is identical to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence).
When referring to the length of a nucleotide molecule as referred to herein, the length corresponds to the number of monomer units, i.e. nucleotides, irrespective as to whether those monomer units are nucleotides or nucleotide analogues. With respect to nucleotides or nucleobases, the terms monomer and unit are used interchangeably herein.
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.
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.
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. However, in one preferred embodiment the term complementary shall mean 100% complementary or fully complementary.
In a preferred example the oligonucleotide of the invention is 100% complementary to a miRNA sequence, such as a human microRNA sequence, or one of the microRNA sequences referred to herein.
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.
Preferably, 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.
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.
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.
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 oligomer of 5 or more, such as 6, 7, 8, 9, or 10 consecutive nucleotides, such as eight or more.
Length
Surprisingly we have found that such short ‘antimiRs’ provide an improved specific inhibition of microRNAs in vivo, whilst retaining remarkable specificity for the microRNA target. A further benefit has been found to be the ability to inhibit several microRNAs simultaneously due to the conservation of homologous short sequences between microRNA species—such as the seed regions as described herein. According to the present invention, it has been found that it is particularly advantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides, such as 7, 8 or 9 nucleotides.
Sequences
The contiguous nucleotide sequence is complementary (such as 100% complementary—i.e. perfectly complementary) to a corresponding region of a mammalian, human or viral microRNA (miRNA) sequence, preferably a human or viral miRNA sequence.
The microRNA sequence may suitably be a mature microRNA. In some embodiments the microRNA may be a microRNA precursor.
The human microRNA sequence may be selected from SEQ ID NO:1 to SEQ ID NO: 558 as disclosed in WO2008/046911, which are all hereby and specifically incorporated by reference. As described in WO2008/046911, these microRNAs are associated with cancer.
The viral microRNA sequence may, in some embodiments, be selected from the group consisting of Herpes simplex virus 1, Kaposi sarcoma-associated herpesvirus, Epstein Barr virus and Human cytomegalovirus.
In one embodiment, the contiguous nucleotide sequence is complementary (such as 100% complementary) to a corresponding region of a miRNA sequence selected from the group of miRNAs listed in table 1. Table 1 provides 7-mer, 8-mer and 9-mer oligomers which target human and viral microRNAs published in miRBase (Release 12.0-http://microrna.sanger.ac.uk/sequences/).
In some embodiments, the oligomers according to the invention may consist of or comprise a contiguous nucleotide sequence which is complementary to a corresponding microRNA sequence selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, miR-375.
Therefore, in one embodiment, the miRNA (i.e target miRNA) is selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, and miR-375.
In one embodiment, the miRNA target is a member of the miR 17-92 cluster, such as miR 17, miR 106a, miR 106b, miR 18, miR 19a, miR 19b/1, miR 19b/2, miR 20/93, miR 92/1, miR 92/2 and miR 25.
In some embodiments the contiguous nucleotide sequence is complementary to a corresponding region of a microRNA (miRNA) sequence selected from the group consisting of miR-21, miR-155, miR-221, mir-222, and mir-122.
In some embodiments said miRNA is selected from the group consisting of miR-1, miR-10 miR-29, miR-125b, miR-126, miR-133, miR-141, miR-143, miR-200b, miR-206, miR-208, miR-302, miR-372, miR-373, miR-375, and miR-520c/e.
In some embodiments the contiguous nucleotide sequence is complementary to a corresponding region of a microRNA (miRNA) sequence present in the miR 17-92 cluster, such as a microRNA selected from the group consisting of miR-17-5p, miR-20a/b, miR-93, miR-106a/b, miR-18a/b, miR-19a/b, miR-25, miR-92a, miR-363.
In one embodiment, the miRNA (i.e target miRNA) is miR-21, such as hsa-miR-21 (SEQ ID NO: 410). In one embodiment, the miRNA (i.e target miRNA) is miR-122, such as hsa-miR-122 (SEQ ID NO: 150). In one embodiment, the miRNA (i.e target miRNA) is miR-19b, such as hsa-miR-19b (SEQ ID NO: 389). In one embodiment, the miRNA (i.e target miRNA) is miR-155, such as hsa-miR-155 (SEQ ID NO: 355). In one embodiment, the miRNA (i.e target miRNA) is miR-375, such as hsa-miR-375 (SEQ ID NO: 561). In one embodiment, the miRNA (i.e target miRNA) is miR-375, such as hsa-miR-106b (SEQ ID NO: 124).
Suitably, the contiguous nucleotide sequence may be complementary to a corresponding region of the microRNA, such as a hsa-miR selected from the group consisting of 19b (SEQ ID NO: 389), 21 (SEQ ID NO: 410), 122 (SEQ ID NO: 150), 155 (SEQ ID NO: 355) and 375 (SEQ ID NO: 561).
The Seed Region and Seedmers
The inventors have found that carefully designed short single stranded oligonucleotides comprising or consisting of nucleotide analogues, such as high affinity nucleotide analogues such as locked nucleic acid (LNA) units, 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, typically nucleotides 2 to 8 or 2 to 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.
The inventors have 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 at least two LNA units in the 3′ end of the oligonucleotides according to the present invention made said oligonucleotides highly nuclease resistant.
In one embodiment, the first or second 3′ nucleotide of the oligomer corresponds to the second 5′ nucleotide of the microRNA sequence, and may be a nucleotide analogue, such as LNA.
In one embodiment, nucleotide units 1 to 6 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.
In one embodiment, nucleotide units 1 to 7 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.
In one embodiment, nucleotide units 2 to 7 (inclusive) of the oligomer as measured from the 3′ end the region of the oligomer are complementary to the microRNA seed region sequence, and may all be nucleotide analogues, such as LNA.
In one embodiment, the oligomer 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 oligomer 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.
In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence which is complementary (such as 100% complementary) to the seed sequence of said microRNA.
In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence selected from any one of the seedmer sequences listed in table 1.
In one embodiment, the 3′ nucleotide of the seedmer forms the 3′ most nucleotide of the contiguous nucleotide sequence, wherein the contiguous nucleotide sequence may, optionally, comprise one or two further nucleotide 5′ to the seedmer sequence.
In one embodiment, the oligomer does not comprise a nucleotide which corresponds to the first nucleotide present in the microRNA sequence counted from the 5′ end.
In one embodiment, the oligonucleotide according to the invention does not comprise a nucleotide at the 3′ end that corresponds to the first 5′ end nucleotide of the target microRNA.
Nucleotide Analogues
According to the present invention, it has been found that it is particularly advantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides, such as 7, 8 or 9 nucleotides, wherein at least 50%, such as 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of the nucleotide units of the oligomer are (preferably high affinity) nucleotide analogues, such as a Locked Nucleic Acid (LNA) nucleotide unit.
In some embodiments, the oligonucleotide of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% of the nucleotides are Locked Nucleic Acid (LNA) nucleotide units.
In such oligomers, in some embodiments, the linkage groups are other than phosphodiester linkages, such as are phosphorothioate linkages.
In one embodiment, all of the nucleotide units of the contiguous nucleotide sequence are LNA nucleotide units.
In one embodiment, the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleotide units.
In a further preferred embodiment, the oligonucleotide of the invention is 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotide sequence which is complementary to a seed region of a human or viral microRNA, and wherein at least 80% of the nucleotides are LNA, and wherein at least 80%, such as 85%, such as 90%, such as 95%, such as 100% of the internucleotide bonds are phosphorothioate bonds. It will be recognised that the contiguous nucleotide sequence of the oligmer (a seedmer) may extend beyond the seed region.
In some embodiments, the oligonucleotide of the invention is 7 nucleotides long, which are all LNA.
In some embodiments, the oligonucleotide of the invention is 8 nucleotides long, of which up to 1 nucleotide may be other than LNA. In some embodiments, the oligonucleotide of the invention is 9 nucleotides long, of which up to 1 or 2 nucleotides may be other than LNA. In some embodiments, the oligonucleotide of the invention is 10 nucleotides long, of which 1, 2 or 3 nucleotides may be other than LNA. The nucleotides ‘other than LNA, may for example, be DNA, or a 2’ substituted nucleotide analogues.
High affinity nucleotide analogues are nucleotide analogues which result in oligonucleotides which has a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This may be determined by measuring the Tm.
In some embodiments, the nucleotide analogue units present in the contiguous nucleotide sequence are selected, optionally independently, 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, and a 2′MOE RNA unit.
In some embodiments, the nucleotide analogue units present in the contiguous nucleotide sequence are selected, optionally independently, from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, and a 2′MOE RNA unit.
The term 2′ fluoro-DNA refers to a DNA analogue with a substitution to fluorine at the 2′ position (2′F). 2′ fluoro-DNA is a preferred form of 2′ fluoro-nucleotide.
In some embodiments, the oligomer comprises 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 10, nucleotide analogue units.
In one embodiment, the oligomer comprises 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 10 LNA.
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.
In one embodiment at least two of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least three of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least four of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least five of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least six of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least seven of the nucleotide analogues such as LNA units are either cytosine or guanine. In one embodiment at least eight of the nucleotide analogues such as LNA units are either cytosine or guanine.
In a preferred embodiment the nucleotide analogues have a higher thermal duplex stability for a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide to said complementary RNA nucleotide.
In one embodiment, the nucleotide analogues confer enhanced serum stability to the single stranded oligonucleotide.
Whilst the specific SEQ IDs in the sequence listing and table 1 refer to oligomers of LNA monomers with phosphorothioate (PS) backbone, it will be recognised that the invention also encompasses the use of other nucleotide analogues and/or linkages, either as an alternative to, or in combination with LNA. As such, the sequence of nucleotides (bases) shown in the sequence listings may be of LNA such as LNA/PS, LNA or may be oligomers containing alternative backbone chemistry, such as sugar/linkage chemistry, whilst retaining the same base sequence (A, T, C or G).
Whilst it is envisaged that other nucleotide analogues, such as 2′-MOE RNA or 2′-fluoro nucleotides may be useful in the oligomers according to the invention, it is preferred that the oligomers have a high proportion, such as at least 50%, LNA. nucleotides. The nucleotide analogue may be a DNA analogue such as a DNA analogue where the 2′-H group is substituted with a substitution other than —OH(RNA) e.g. by substitution with —O—CH3, —O—CH2—CH2—O—CH3, —O—CH2—CH2—CH2—NH2, —O—CH2—CH2—CH2—OH or —F. The nucleotide analogue may be a RNA analogues such as a RNA analogue which have been modified in its 2′-OH group, e.g. by substitution with a group other than —H (DNA), for example —O—CH3, —O—CH2—CH2—O—CH3, —O—CH2—CH2—CH2—NH2, —O—CH2—CH2—CH2—OH or —F. In one embodiment the nucleotide analogue is “ENA”.
LNA
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:




wherein

    
    
        X is selected from the group consisting of O, S and NRH, where RH is H or C1-4-alkyl; Y is (—CH2)r, where r is an integer of 1-4; and B is a nitrogenous base.
    
    


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:




wherein X and B are as defined above.

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.

Thus, the thio-LNA unit may have the chemical structure shown in Scheme 3 below:





wherein B is as defined above.

Preferably, the thio-LNA unit is in its beta-D-form, i.e. having the structure shown in 3A above. likewise, the amino-LNA unit may have the chemical structure shown in Scheme 4 below:





wherein B and RH are as defined above.

Preferably, the amino-LNA unit is in its beta-D-form, i.e. having the structure shown in 4A above.

The oxy-LNA unit may have the chemical structure shown in Scheme 5 below:





wherein B is as defined above.

Preferably, the oxy-LNA unit is in its beta-D-form, i.e. having the structure shown in 5A above. 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 (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.
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.
The term “amino-LNA unit” refers to an LNA unit in which X in Scheme 1 is NH or NRH, where RH is hydrogen or C1-4-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.
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.
In the present context, the term “C1-6-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 C1-6-alkyl substituted at any carbon with a hydrocarbon chain.
In the present context, the term “C1-4-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 C1-4-alkyl substituted at any carbon with a hydrocarbon chain.
When used herein the term “C1-6-alkoxy” is intended to mean C1-6-alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy and hexoxy.
In the present context, the term “C2-6-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 C2-6-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.
In the present context the term “C2-6-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 C2-6-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 “C2-6-alkynyl” is a di-yne or enedi-yne as is known to the person skilled in the art.
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 MeC, preferably MeC.
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.
The terms “unit”, “residue” and “monomer” are used interchangeably herein.
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.
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.
Internucleoside Linkages
The term “internucleoside linkage group” is intended to mean a group capable of covalently coupling together two nucleotides, 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. Examples include phosphate, phosphodiester groups and phosphorothioate groups.
In some embodiments, at least one of, such as all of the internucleoside linkage in the oligomer is phosphodiester. However for in vivo use, phosphorothioate linkages may be preferred.
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.
Specific examples of internucleoside linkage groups which differ from phosphate
(—O—P(O)2—O—) include —O—P(O,S)—O—, —O—P(S)2—O—, —S—P(O)2—O—, —S—P(O,S)—O—, —S—P(S)2—O—, —O—P(O)2—S—, —O—P(O,S)—S—, —S—P(O)2—S—, —O—PO(RH)—O—, O—PO(OCH3)—O—, —O—PO(NRH)—O—, —O—PO(OCH2CH2S—R)—O—, —O—PO(BH3)—O—, —O—PO(NHRH)—O—, —O—P(O)2—NRH—, —NRH—P(O)2—O—, —NRH—CO—O—, —NRH—CO—NRH—, —O—CO—O—, —O—CO—NRH—, —NRH—CO—CH2—, —O—CH2—CO—NRH—, —O—CH2—CH2—NRH—, —CO—NRH—CH2—, —CH2—NRH—CO—, —O—CH2—CH2—S—, —S—CH2—CH2—O—, —S—CH2—CH2—S—, —CH2—SO2—CH2—, —CH2—CO—NRH—, —O—CH2—CH2—NRH—CO —, —CH2—NCH3—O—CH2—, where RH is hydrogen or C1-4-alkyl.
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 internucleoside linkage may be selected form the group consisting of: —O—P(O)2—O—, —O—P(O,S)—O—, —O—P(S)2—O—, —S—P(O)2—O—, —S—P(O,S)—O—, —S—P(S)2—O—, —O—P(O)2—S—, —O—P(O,S)—S—, —S—P(O)2—S—, —O—PO(RH)—O—, O—PO(OCH3)—O—, —O—PO(NRH)—O—, —O—PO(OCH2CH2S—R)—O—, —O—PO(BH3)—O—, —O—PO(NHRH)—O—, —O—P(O)2—NRH—, —NRH—P(O)2—O—, —NRH—CO—O—, —NRH—CO—NRH—, and/or the internucleoside linkage may be selected form the group consisting of: —O—CO—O—, —O—CO—NRH—, —NRH—CO—CH2—, —O—CH2—CO—NRH—, —O—CH2—CH2—NRH—, —CO—NRH—CH2—, —CH2—NRH—CO—, —O—CH2—CH2—S—, —S—CH2—CH2—O—, —S—CH2—CH2—S—, —CH2—SO2—CH2—, —CH2—CO—NRH—, —O—CH2—CH2—NRH—CO —, —CH2—NCH3—O—CH2—, where RH is selected from hydrogen and C1-4-alkyl. Suitably, in some embodiments, sulphur (S) containing internucleoside linkages as provided above may be preferred. The internucleoside linkages may be independently selected, or all be the same, such as phosphorothioate linkages.
In one embodiment, at least 75%, such as 80% or 85% or 90% or 95% or all of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
MicromiR Oligonucleotides Targeting More than One MicroRNA
In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding sequence of at least two miRNA sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequence. The use of a single universal base may allow a single oligomer of the invention to target two independant microRNAs which either one or both have a single mismatch in the region which corresponds to oligomer at the position where the universal nucleotide is positioned.
In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence which is complementary to the sequence of at least two miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA seed region sequences.
In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding region of both miR-221 and miR-222.
In one embodiment, the contiguous nucleotide sequence is complementary to the corresponding region of more than one member of the miR-17-92 cluster—such as two or more or all of miR-17-5p, miR-20a/b, miR-93, miR-106a/b; or two or more or all of miR-25, miR-92a and miR-363.
In one embodiment, the contiguous nucleotide sequence consists of or comprises a sequence that is complementary to 5′GCTACAT3′.
Oligomer Design
In one embodiment, the first nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit. In one embodiment, which may be the same or different, the last nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.
In one embodiment, the second nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.
In one embodiment, the ninth and/or the tenth nucleotide of the oligomer according to the invention, counting from the 3′ end, is a nucleotide analogue, such as an LNA unit.
In one embodiment, the ninth nucleotide of the oligomer according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
In one embodiment, the tenth nucleotide of the oligomer according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
In one embodiment, both the ninth and the tenth nucleotide of the oligomer according to the invention, calculated from the 3′ end is a nucleotide analogue, such as an LNA unit.
In one embodiment, the oligomer according to the invention does not comprise a region of more than 3 consecutive DNA nucleotide units. In one embodiment, the oligomer according to the invention does not comprise a region of more than 2 consecutive DNA nucleotide units.
In one embodiment, the oligomer comprises at least a region consisting of at least two consecutive nucleotide analogue units, such as at least two consecutive LNA units.
In one embodiment, the oligomer comprises at least a region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNA units.
Other Patterns of Nucleotide Analogues such as LNA in the Oligomer
Whilst it is envisaged that oligomers containing at least 6 LNA, such as at least 7 nucleotide units may be preferable, the discovery that such short oligomers are highly effective at targeting microRNAs in vivo can be used to prepare shorter oligomers of the invention which comprise other nucleotide analogues, such as high affinity nucleotide analogues. Indeed, the combination of LNA with other high affinity nucleotide analogues are considered as part of the present invention.
Modification of nucleotides in positions 1 to 2, counting from the 3′ end. The nucleotide at positions 1 and/or 2 may be a nucleotide analogue, such as a high affinity nucleotide analogue, such as LNA, or a nucleotide analogue selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. The two 3′ nucleotide may therefore be
Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA or another nucleotide analogue, such as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.
The above modification at the 2 3′ terminal nucleotides may be combined with modification of nucleotides in positions 3-8 counting from the 3′ end, as described below. In this respect nucleotides designated as X and x may be the same throughout the oligomer. It will be noted that when the oligomer is only 7 nucleotides in length the 8th nucleotide counting from the 3′ end should be discarded. In the following embodiments which refer to the modification of nucleotides in positions 3 to 8, counting from the 3′ end, the LNA units, in one embodiment, may be replaced with other nucleotide anlogues, 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, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. “x” is preferably DNA or RNA, most preferably DNA. However, it is preferred that X is LNA.
In one 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.
Alternatively defined, in an 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 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 an 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.
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.
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.
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.
In yet a further embodiment, the oligonucleotide according to the present invention comprises at least six LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises at from three to six LNA units in positions three to eight, counting from the 3′ end, and in addition from none to three other high affinity nucleotide analogues in the same region, such that the total amount of high affinity nucleotide analogues (including the LNA units) amount to six in the region from positions three to eight, counting from the 3′ end.
In some embodiments, such as when X is LNA, said non-LNA unit (x) is another nucleotide analogue unit, such as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA.
For oligomers which have 9 or 10 nucleotides, the nucleotide at positions 9 and/or 10 may be a nucleotide analogue, such as a high affinity nucleotide analogue, such as LNA, or a nucleotide analogue selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. The two 5′ nucleotides may therefore be
Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA or another nucleotide analogue, such as a 2′ substituted nucleotide analogue selected from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOE RNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or 2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.
The above modification at the 2 5′ terminal nucleotides may be combined with modification of nucleotides in positions 3-8 counting from the 3′ end, and/or the 2 3′ nucleotitides as described above. In this respect nucleotides designated as X and x may be the same throughout the oligomer.
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.
In one embodiment, the invention further provides for an oligomer as described in the context of the pharmaceutical composition of the invention, or for use in vivo in an organism, such as a medicament, wherein said oligomer (or contiguous nucleotide sequence) comprises either
i) at least one phosphorothioate linkage and/or
ii) at least one 3′ terminal LNA unit, and/or iii) at least one 5′ terminal LNA unit.
The oligomer may therefore contain at least one phosphorothioate linkage, such as all linkages being phosphorthioates, and at least one 3′ terminal LNA unit, and at least one 5′ terminal LNA unit.
It is preferable for most therapeutic uses that the oligonucleotide is fully phosphorothiolated—an 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 in one aspect of the oligonucleotide according to the invention is that the second 3′ nucleotide, and/or the 9th and 10th (from the 3′ end), if present, may also be LNA.
In one embodiment, the oligomer comprises at least five nucleotide analogue units, such as at least five LNA units, in positions which are complementary to the miRNA seed region.
In one embodiment, the nucleotide sequence of the oligomer 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.
In one embodiment, the oligomer comprises six or seven nucleotide analogue units, such as six or seven LNA units, in positions which are complementary to the miRNA seed region.
In one embodiment, the nucleotide sequence of the oligomer 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.
In one embodiment, the two nucleotide motif at position 7 to 8, counting from the 3′ end of the oligomer 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.
In one embodiment, the two nucleotide motif at position 7 to 8, counting from the 3′ end of the oligomer 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.
In one embodiment, the oligomer comprises at 12 nucleotides and wherein the two nucleotide motif at position 11 to 12, counting from the 3′ end of the oligomer 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.
In one embodiment, the oligomer comprises 12 nucleotides and wherein the two nucleotide motif at position 11 to 12, counting from the 3′ end of the oligomer 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, such as a DNA unit.
In one embodiment, the oligomer comprises a nucleotide analogue unit, such as an LNA unit, at the 5′ end.
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, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit.
In one embodiment, all the nucleotides of the oligomer of the invention are nucleotide analogue units.
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,
In one embodiment, the oligomer comprises said at least one LNA analogue unit and at least one further nucleotide analogue unit other than LNA.
In one embodiment, the non-LNA nucleotide analogue unit or units are independently selected from 2′-OMe RNA units and 2′-fluoro DNA units.
In one embodiment, the oligomer 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.
In one embodiment, the sequence of nucleotides of the oligomer consists of alternative X and Y units.
In one embodiment, the oligomer comprises alternating LNA and DNA units (Xx) or (xX).
In one embodiment, the oligomer comprises a motif of alternating LNA followed by 2 DNA units (Xxx), xXx or xxX.
In one embodiment, at least one of the DNA or non-LNA nucleotide analogue units are replaced with a LNA nucleotide in a position selected from the positions identified as LNA nucleotide units in any one of the embodiments referred to above. In one embodiment, “X” donates an LNA unit.
Further Designs for Oligomers of the Invention
Table 1 below provides non-limiting examples of short microRNA sequences that could advantageously be targeted with an oligonucleotide of the present invention.
The oligonucleotides according to the invention, such as those disclosed in table 1 may, in one embodiment, have a sequence of 7, 8, 9 or 10 LNA nucleotides 5′-3′ LLLLLLL(L)(L)(L)(L), or have a sequence of nucleotides selected form the group consisting of, the first 7, 8, 9 or 10 nucleotides of the following motifs:
LdLddL(L)(d)(d)(L)(d)(L)(d)(L)(L), LdLdLL(L)(d)(d)(L)(L)(L)(d)(L)(L), LMLMML(L)(M)(M)(L)(M)(L)(M)(L)(L), LMLMLL(L)(M)(M)(L)(L)(L)(M)(L)(L), LFLFFL(L)(F)(F)(L)(F)(L)(F)(L)(L), LFLFLL(L)(F)(F)(L)(L)(L)(F)(L)(L), and every third designs such as; LddLdd(L)(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)′dLddLd(d)(L)(d)(d)(L)(d)(d)(L)(d)(d)(L), ddLddL(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)(d), LMMLMM(L)(M)(M)(L)(M)(M)(L)(M)(M)(L)(M), MLMMLM(M)(L)(M)(M)(L)(M)(M)(L)(M)(M)(L), MMLMML(M)(M)(L)(M)(M)(L)(M)(M)(L)(M)(M), LFFLFF(L)(F)(F)(L)(F)(F)(L)(F)(F)(L)(F), FLFFLF(F)(L)(F)(F)(L)(F)(F)(L)(F)(F)(L), FFLFFL(F)(F)(L)(F)(F)(L)(F)(F)(L)(F)(F), and dLdLdL(d)(L)(d)(L)(d)(L)(d)(L)(d)(L)(d) and an every second design, such as; LdLdLd(L)(d)(L)(d)(L)(d)(L)(d)(L)(d)(L), MLMLML(M)(L)(M)(L)(M)(L)(M)(L)(M)(L)(M), LMLMLM(L)(M)(L)(M)(L)(M)(L)(M)(L)(M)(L), FLFLFL(F)(L)(F)(L)(F)(L)(F)(L)(F)(L)(F), and LFLFLF(L)(F)(L)(F)(L)(F)(L)(F)(L)(F)(L); wherein L=LNA unit, d=DNA units, M=2′MOE RNA, F=2′Fluoro and residues in brackets are optional.
Pharmaceutical Composition and Medical Application
The invention provides for a pharmaceutical composition comprising the oligomer according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
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.
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.
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 oligomer 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. The reduction of the effective amount of the miRNA in a cell may, in one embodiment, be measured by detecting the level of de-repression of the miRNA's target in the cell.
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 oligomer according to the invention to the cell or the organism.
The invention further provides for the use of a oligomer of between 7-10 such as 7, 8, 9, or 10 nucleotides 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.
In one embodiment the medical condition (or disease) is hepatitis C(HCV), and the miRNA is miR-122.
In one embodiment, the pharmaceutical composition according to the invention is for use in the treatment of a medical disorder or disease selected from the group consisting of: hepatitis C virus infection and hypercholesterolemia and related disorders, and cancers.
In one embodiment the medical disorder or disease is a CNS disease, such as a CNS disease where one or more microRNAs are known to be indicated.
In the context of hypercholesterolemia related disorders refers to diseases such as atherosclerosis or hyperlipidemia. Further examples of related diseases also include different types of HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia, statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD), atherosclerosis.
In one embodiment, the pharmaceutical composition according to the invention further comprises a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor (such as those disclosed in U.S. 60/977,497, hereby incorporated by reference).
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 oligomer of between 7-10 such as 7, 8, 9, or 10 nucleotides in length, to a person in need of treatment.
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 oligomer of between 6 7-10 such as 7, 8, 9, or 10 nucleotides in length, to the cell or the organism.
It should be recognised that “reducing the effective amount” of one or more microRNAs in a cell or organism, refers to the inhibition of the microRNA function in the call or organism. The cell is preferably amammalain cell or a human cell which expresses the microRNA or microRNAs.
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 oligomer of 7-10 such as 7, 8, 9, or 10 nucleotides in length, or (or a composition comprising said oligonucleotide) to the cell or the organism.
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, fragile X mental retardation, DiGeorge syndrome and cancer, such as in non limiting example, chronic lymphocytic leukemia, breast cancer, lung cancer and colon cancer, in particular cancer.
Methods of Synthesis
The invention further provides for a method for the synthesis of an oligomer targeted against a human microRNA, such as an oligomer described herein, said method comprising the steps of:


    a. Optionally selecting a first nucleotide, counting from the 3′ end, which is a nucleotide analogue, such as an LNA nucleotide.
    b. Optionally selecting a second nucleotide, counting from the 3′ end, which is a nucleotide analogue, such as an LNA nucleotide.
    c. Selecting a region of the oligomer which corresponds to the miRNA seed region, wherein said region is as defined herein.
    d. Selecting a seventh and optionally an eight nucleotideas defined herein.


e. Optionally selecting one or two further 5′ terminal of the oligomer is as defined herein;
wherein the synthesis is performed by sequential synthesis of the regions defined in steps a-e, wherein said synthesis may be performed in either the 3′-5′ (a to f) or 5′-3′ (e to a) direction, and wherein said oligomer is complementary to a sequence of the miRNA target.
The invention further provides for a method for the preparation of an oligomer (such as an oligomer according to the invention), said method comprising the steps of a) comparing the sequences of two or more miRNA sequences to identify two or more miRNA sequences which comprise a common contiguous nucleotide sequence of at least 7 nucleotides in length, such as 7, 8, 9 or 10 nucleotides in length (i.e. a sequence found in both non-identical miRNAs), b) preparing an oligomer sequence which consists or comprises of a contiguous nucleotide sequence with is complementary to said common contiguous nucleotide sequence, wherein said oligomer is, as according to the oligomer of the invention. In a preferred example, the common contiguous nucleotide sequence consists or comprises of the seed region of each of said two or more miRNA sequences (which comprise a common contiguous nucleotide sequence of at least 6 nucleotides in length). In one embodiment, the seed regions of the two or more miRNAs are identical. Suitably the oligomer consists or comprises a seedmer sequence of 7 or 8 nucleotides in length which comprises of a sequence which is complementary to said two or more miRNAs. This method may be used in conjunction with step c of the above method.
The method for the synthesis of the oligomer according to the invention may be performed using standard solid phase oligonucleotide systhesis.
In one embodiment, the method for the synthesis of a oligomer targeted against a human microRNA, is performed in the 3′ to 5′ direction a-e. 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 (i) is complementary to the target microRNA sequence (ii) does not contain a nucleotide at the 3′ end that corresponds to the first 5′ end nucleotide of the target microRNA.
Duplex Stability and Tm 
In one embodiment, the oligomer 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 Tm of between 30° C. and 70° C. or 80° C., such as between 30° C. and 60° C. of 70° C., or between 30° C. and 50° C. or 60° C. In one embodiment the Tm is at least 40° C. Tm may be determined by determining the Tm of the oligomer and a complementary RNA target in the following buffer conditions: 100 mM NaCl, 0.1 mM EDTA, 10 mM Na-phosphate, pH 7.0 (see examples for a detailed protocol). A high affinity analogue may be defined as an analogue which, when used in the oligomer of the invention, results in an increase in the Tm of the oligomer as compared to an identical oligomer which has contains only DNA bases.

Conjugates

In one embodiment, said oligomer is conjugated with one or more non-nucleotide (or poly-nucleotide) compounds.
In the context the term “conjugate” is intended to indicate a heterogenous molecule formed by the covalent attachment (“conjugation”) of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non-polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.
Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide components, such as a conjugate component.
In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.
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 various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, 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.
Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
The oligomers 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 certain embodiments the conjugated moiety is a sterol, such as cholesterol.
In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example between 1-50, such as 2-20 such as 3-10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol—see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.
By way of example, the following conjugate moieties may be used in the conjugates of the invention:




Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described. Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3′-hydroxyl group or the exocyclic NH2 group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH2 group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in “Protective Groups in Organic Synthesis” by Theodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self-cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which is incorporated by reference herein in its entirety.
In some embodiments, oligomers of the invention are functionalized at the 5′ end in order to allow covalent attachment of the conjugated moiety to the 5′ end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3′ end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5′ end, the 3′ end, the backbone and the base.
In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis. In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH2)w, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (—O—C(O)—(CH2)wNH).
In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH2)w-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (—O—C(O)—(CH2)wSH).
In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).
Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.
In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a functionalizing reagent coupled to a 5′-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3′-hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and 4,914,210.
In some embodiments, the 5′-terminus of a solid-phase bound oligomer is functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.
In various embodiments, the incorporation of monomers containing 2′-sugar modifications, such as a 2′-carbamate substituted sugar or a 2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2′-position of one or more monomers is prepared using a reagent such as, for example, 5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.
In still further embodiments, the oligomers of the invention may have amine-containing functional moieties on the nucleotide, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.
Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, Ill.). Other commercially available linking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5′-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).
Therapy and Pharmaceutical Compositions—Formulation and Administration
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.
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 or buffered saline.
In a still further aspect the present invention relates to an oligonucleotide according to the present invention for use as a medicament.
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 μg to 1 g 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.
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.
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.
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.
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.
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 Pharmacol 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 carboxymethylcellulose, 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.
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.
Examples of therapeutic indications which may be treated by the pharmaceutical compositions of the invention:











microRNA
Possible medical indications







miR-1
Cardiac arythmia


miR-21
Glioblastoma, breast cancer, hepatocellular



carcinoma, colorectal cancer, sensitization



of gliomas to cytotoxic drugs, cardiac



hypertrophy


miR-21, miR-200b
Response to chemotherapy and regulation of


and miR-141
cholangiocarcinoma growth


miR-122
hypercholesterolemia, hepatitis C infection,



hemochromatosis


miR-19b
lymphoma and other tumour types


miR-26a
Osteoblast differentiation of human stem



cells


miR-155
lymphoma, pancreatic tumor development,



breast and lung cancer


miR-203
Psoriasis


miR-375
diabetes, metabolic disorders, glucose-



induced insulin secretion from pancreatic



endocrine cells


miR-181
myoblast differentiation, auto immune



disorders


miR-10b
Breast cancer cell invasion and metastasis


miR-125b-1
Breast, lung, ovarian and cervical cancer


miR-221 and 222
Prostate carcinoma, human thyroid papillary



car, human hepatocellular carcinoma


miRNA-372 and -373
testicular germ cell tumors.


miR-142
B-cell leukemia


miR-17-19b
B-cell lymphomas, lung cancer, hepatocellular


cluster
carcinoma









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.
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.
The invention further refers to 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.
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.
The invention further provides for a kit comprising a pharmaceutical composition according to the invention, and a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor.
Cancer
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.
Such cancers may include lymphoreticular neoplasia, lymphoblastic leukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma, leukemia, connective tissue tumors, lymphomas, and solid tumors.
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.
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.
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.
Alternatively, the cancer may suitably be a glioma.
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.
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.
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
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.
Hsa-miR 122 (SEQ ID NO: 150) is indicated in hepatitis C infection and as such oligonucleotides according to the invention which target miR-122 may be used to treat Hepatitus C infection.
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.
In a preferred embodiment, the invention provides for a combination treatment providing an anti miR-122 oligomer in combination with an inhibitor of VLDL assembly, such as an inhibitor of apoB, or of MTP.
Inflammatory Diseases
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.
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.
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.
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
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.
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.
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
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.
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
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
We have demonstrated that a LNA-antimiR, 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 (or hypercholesterolemia and related disorders). The skilled man will appreciate that increased plasma cholesterol levels is undesirable as it increases the risk of various conditions, e.g. atherosclerosis.
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.
EMBODIMENTS
The following embodiments of the present invention may be used in combination with the other embodiments described herein.
1. A pharmaceutical composition comprising an oligomer of between 6-12 nucleotides in length, wherein said oligomer comprises a contiguous nucleotide sequence of a total of between 6-12 nucleotides, such as 6, 7, 8, 9, 10, 11 or 12 nucleotide units, wherein at least 50% of the nucleobase units of the oligomer are high affinity nucleotide analogue units, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

2. The pharmaceutical composition according to embodiment 1, wherein the contiguous nucleotide sequence is complementary to a corresponding region of a mammalian, human or viral microRNA (miRNA) sequence.

3. The pharmaceutical composition according to embodiment 2, wherein the contiguous nucleotide sequence is complementary to a corresponding region of a miRNA sequence selected from the group of miRNAs listed in any one of tables 3, 4 or 5.

4. The pharmaceutical composition according to embodiment 2 or 3, wherein the contiguous nucleotide sequence consists of or comprises a sequence which is complementary to the seed sequence of said microRNA.

5. The pharmaceutical composition according to any one of embodiments 2-4, wherein the contiguous nucleotide sequence consists of or comprises a sequence selected from any one of the sequences listed in table 3 or 4.

6. The pharmaceutical composition according to embodiment 4 or 5, wherein the 3′ nucleobase of the seedmer forms the 3′ most nucleobase of the contiguous nucleotide sequence, wherein the contiguous nucleotide sequence may, optionally, comprise one or two further 5′ nucleobases.

7. The pharmaceutical composition according to any one of embodiments 1-6, wherein said contiguous nucleotide sequence does not comprise a nucleotide which corresponds to the first nucleotide present in the micro RNA sequence counted from the 5′ end.

8. The pharmaceutical composition according to any one of embodiments 1-7, wherein the contiguous nucleotide sequence is complementary to a corresponding nucleotide sequence present in a miRNA selected from those shown in table 3 or 4 or 5.

9. The pharmaceutical composition according to embodiment 8, wherein said miRNA is selected from the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, and miR-375.

10. The pharmaceutical composition according to any one of embodiments 1-9, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of the nucleobase units of the contiguous nucleotide sequence are nucleotide analogue units.

11. The pharmaceutical composition according to embodiment 10, wherein the nucleotide analogue units are 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, and a 2′MOE RNA unit.

12. The pharmaceutical composition according to embodiment 10 or 11, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of the nucleobase units of the contiguous nucleotide sequence are Locked Nucleic Acid (LNA) nucleobase units.

13. The pharmaceutical composition according to embodiment 12, wherein all of the nucleobase units of the contiguous nucleotide sequence are LNA nucleobase units.

14. The pharmaceutical composition according to any one of embodiments 1-13, wherein the contiguous nucleotide sequence comprises or consists of 7, 8, 9 or 10, preferably contiguous, LNA nucleobase units.

15. The pharmaceutical composition according to any one of embodiments 1-14, wherein the oligomer consist of 7, 8, 9 or 10 contiguous nucleobase units and wherein at least 7 nucleobase units are nucleotide analogue units.

16. The pharmaceutical composition according to embodiment 15, wherein the nucleotide analogue units are Locked Nucleic Acid (LNA) nucleobase units.

17. The pharmaceutical composition according to embodiment 15, wherein the nucleotide analogue units in the molecule consists of a mixture of at least 50% LNA units and up to 50% other nucleotide analogue units.

18. The pharmaceutical composition according to any one of embodiments 1-17, wherein at least 75%, such as 80% or 85% or 90% or 95% or all of the internucleoside linkages present between the nucleobase units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

19. The pharmaceutical composition according to any one of embodiments 1-18, wherein said oligomer is conjugated with one or more non-nucleobase compounds.

20. The pharmaceutical composition according to any one of embodiments 1-19, wherein the contiguous nucleotide sequence is complementary to the corresponding sequence of at least two miRNA sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequences.

21. The pharmaceutical composition according to any one of embodiments 1-20, wherein the contiguous nucleotide sequence consists or comprises of a sequence which is complementary to the sequence of at least two miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA seed region sequences.

22. The pharmaceutical composition according to any one of embodiments 20 or 21, wherein the contiguous nucleotide sequence is complementary to the corresponding region of both miR-221 and miR-222.

23. The pharmaceutical composition according to embodiment 22, wherein the contiguous nucleotide sequence consists or comprises of a sequence that is complementary to 5′GCUACAU3′.

24. The pharmaceutical composition according to any one of embodiments 1-23, wherein the oligomer is constituted as a prodrug.

25. The pharmaceutical composition according to any one of embodiments 1-24, wherein the contiguous nucleotide sequence is complementary to a corresponding region of has-miR-122.

26. The pharmaceutical composition according to embodiment 25, for use in the treatment of a medical disorder or disease selected from the group consisting of: hepatitis C virus infection and hypercholesterolemia and related disorders.

27. The pharmaceutical composition according to embodiment 25 or 26, wherein the composition further comprises a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor.

28. A kit comprising a pharmaceutical composition according to embodiment 25 or 26, and a second independent active ingredient that is an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, or an MTP inhibitor.

29. A method for the treatment of a disease or medical disorder associated with the presence or overexpression of a microRNA, comprising the step of administering a the pharmaceutical composition) according to any one of embodiments 1-28 to a patient who is suffering from, or is likely to siffer from said disease or medical disorder.

30. An oligomer, as defined according to anyone of embodiments 1-25.

31. A conjugate comprising the oligomer according to embodiment 30, and at least one non-nucleobase compounds.

32. The use of an oligomer or a conjugate as defined in any one of embodiments 30-31, 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.

33. A method for reducing the amount, or effective amount, of a miRNA in a cell, comprising administering an oligomer, a conjugate or a pharmaceutical composition, according to any one of the preceeding embodiments to the cell which is expressing said miRNA so as to reduce the amount, or effective amount of the miRNA in the cell.

34. A method for de-repression of a mRNA whose expression is repressed by a miRNA in a cell comprising administering an oligomer, a conjugate or a pharmaceutical composition, according to any one of the preceeding embodiments to the cell to the cell which expressed both said mRNA and said miRNA, in order to de-repress the expression of the mRNA.

References: Details of the reference are provided in the priority documents.

EXAMPLES
LNA Monomer and oligonucleotide synthesis were performed using the methodology referred to in Examples 1 and 2 of WO2007/112754. The stability of LNA oligonucletides in human or rat plasma is performed using the methodology referred to in Example 4 of WO2007/112754. The treatment of in vitro cells with LNA anti-miR antisense oligonucleotide (targeting miR-122) is performed using the methodology referred to in Example 6 of WO2007/112754. The analysis of Oligonucleotide Inhibition of miR expression by microRNA specific quantitative PCR in both an in vitro and in vivo model is performed using the methodology referred to in Example 7 of WO2007/112754. The assessment of LNA antimiR knock-down specificity using miRNA microarray expression profiling is performed using the methodology referred to in Example 8 of WO2007/112754. The detection of microRNAs by in situ hybridization is performed using the methodology referred to in Example 9 of WO2007/112754. The Isolation and analysis of mRNA expression (total RNA isolation and cDNA synthesis for mRNA analysis) in both an in vitro and in vivo model is performed using the methodology referred to in Example 10 of WO2007/112754. In vivo Experiments using Oligomers of the invention targeting microRNA-122. and subsequent analysis are performed using the methods disclosed in Examples 11-27 of WO2007/112754. The above mentioned examples of WO2007/112754 are hereby specifically incorporated by reference.
Example 1
Design of the LNA AntimiR Oligonucleotides and Melting Temperatures







TABLE 2







Oligomers used in the examples and figures. The


Compound number is an identifier used throughout  


the examples and figures - the SEQ ID NO which 


is used in the sequence listing is also provided.











SEQ





ID
Compound



Compound
NO
Sequence
Comment





3204
  1
TcAGtCTGaTaAgCT



 


3205
  2
GATAAGCT



 


3206
  3
TcAcAATtaGCAtTA



 


3207
  4
TAGCATTA



 


4
  5
CcAttGTcaCaCtCC



 


3208
  6
CACACTCC



 


3209
  7
TAAGCT



 


3210
  8
ATAAGCT



 


3211
  9
TGATAAGCT



 


3212
 10
CTGATAAGCT



 


3213
 11
GTCTGATAAGCT



 


2114
 12
CAGTCTGATAAGCT



 


3215
 13
TCTGATAA



 


3216
 14
ATCAGTCT



 


3217
 15
TCAACATC



 


3218 or 
 16
GGTAAACT
Underline =


3230


mismatch


 


3219
 17
CGTAATGA
Underline =





mismatch


 


3220
 18
TCAgtctgataaGCTa
5′ fluorescent





label (FAM)


 


3221
 19
AGCACTTT



 


3222
 20
ATTTGCAC



 


3223
 21
AgCagACaaTgTaGC
5′ fluorescent





label (FAM)


 


3224
 22
GtAgcCAgaTgTaGC
5′ fluorescent





label (FAM)


 


3225
 23
ATGTAGC



 


3226
 24
ACaAcCTacTaCcTC



 


3227
 25
ACTACCTC



 


3228
 26
CaCtgTCagCaCtTT



 


3229
 27
TgCatAGatTtGcAC



 


3231
 28
GTAGACT



 


3232
 29
TACCTC



 


3233
 30
CTACCTC



 


3234
 31
TNCTACCTC
N = universal base.


 


3235
 32
TNCTACCTC
N = universal base.


 


3236
 33
GCaAcCTacTaCcTC



 


3237
 34
ACaAcCTccTaCcTC



 


3238
 35
ACaAaCTacTaCcTC



 


3239
 36
CTACCTC



 


3240
 37
CTAACTC



 


3241
 38
TTAGCATTA



 


3242
 39
CGATTAGCATTA



 


3243
977
CACGATTAGCATTA



 


3244
978
GCATTA



 


3245
979
AGCATTA



 


3246
980
ATTAGCATTA





Capital and lower case letters denote LNA and DNA, respectively.


LNA cytosines are preferably methyl cytosine/5′methyl-cytosine*


All internucleoside linkages are preferably phosphorothioate*


All LNA may, for example, be beta-D-oxy LNA*


*Used in the specific examples.






Example 2
In Vitro Model: Cell Culture
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.
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.
Cells were cultured in the appropriate medium as described below and maintained at 37° C. at 95-98% humidity and 5% CO2. Cells were routinely passaged 2-3 times weekly.
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.
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.
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.
HeLa: The cervical carcinoma cell line HeLa was cultured in MEM (Sigma) containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and 5% CO2.
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.
DU-145: The human prostate cancer cell line DU-145 was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS.
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.
786-0: The human renal cell carcinoma cell line 786-0 was purchased from ATCC and maintained according to manufacturers instructions
HUVEC: The human umbilical vein endothelial cell line HUVEC was purchased from Camcrex and maintained in EGM-2 medium.
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.
B16: The murine melanoma cell line B16 was purchased from ATCC and maintained according to the manufacturers instructions.
LNCap: The human prostate cancer cell line LNCap was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS
Huh-7: Human liver, epithelial like cultivated in Eagles MEM with 10% FBS, 2 mM Glutamax I, 1× non-essential amino acids, Gentamicin 25 μg/ml
L428: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
L1236: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany)): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
Example 3
Design of a LNA AntimiR Library for all Human MicroRNA Sequences in miRBase microRNA Database
The miRBase version used was version 12, as reported in 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, and available via http://microrna.sanger.ac.uk/sequences/index.shtml.
Table 1 shows 7-, 8- and 9-mer nucleotide sequences comprising the seedmer sequence of micro RNA's according to the miRBase micro RNA database. The seedmer sequence comprises the reverse complement of the microRNA seed region. In some embodiments the oligomer of the invention has a contiguous nucleotide sequence selected from the 7-mer, 8-mer or 9-mer sequences. With respect to the 7-mer, 8-mer and 9-mer sequences, in some embodiments, all the internucleoside linkages are phosphorothioate. The 7-mer, 8-mer and 9-mer nucleotide sequences may consist of sequence of nucleotide analogues as described herein, such as LNA nucleotide analogues. LNA cytosines may be methyl-cytosine (5′ methyl-cytosine). In some embodiments, the LNA is beta-D-oxy-LNA.
Table 3 provides a list of microRNAs grouped into those which can be targeted by the same seedmer oligomers, such as the 7-, 8- or 9-mers provided herein (see table 1).







TABLE 3







hsa-let-7a* (SEQ ID NO: 97), hsa-let-7f-1* (SEQ ID NO: 107)


hsa-let-7a (SEQ ID NO: 96), hsa-let-7b (SEQ ID NO: 98), 


hsa-let-7c (SEQ ID NO: 100), hsa-let-7d (SEQ ID NO: 102), 


hsa-let-7f (SEQ ID NO: 106), hsa-miR-98 (SEQ ID NO: 947),


hsa-let-7g (SEQ ID NO: 109), hsa-let-7i (SEQ ID NO: 111)


hsa-miR-1 (SEQ ID NO: 113), hsa-miR-206 (SEQ ID NO: 403)


hsa-miR-103 (SEQ ID NO: 118), hsa-miR-107 (SEQ ID NO: 126)


hsa-miR-10a (SEQ ID NO: 127), hsa-miR-10b (SEQ ID NO: 129)


hsa-miR-125b (SEQ ID NO: 190), hsa-miR-125a-5p (SEQ ID NO: 189)


hsa-miR-129* (SEQ ID NO: 229), hsa-miR-129-3p (SEQ ID NO: 230)


hsa-miR-130a (SEQ ID NO: 251), hsa-miR-301a (SEQ ID NO: 473), 


hsa-miR-130b (SEQ ID NO: 253), hsa-miR-454 (SEQ ID NO: 608), 


hsa-miR-301b (SEQ ID NO: 474)


hsa-miR-133a (SEQ ID NO: 261), hsa-miR-133b (SEQ ID NO: 262)


hsa-miR-135a (SEQ ID NO: 264), hsa-miR-135b (SEQ ID NO: 266)


hsa-miR-141 (SEQ ID NO: 278), hsa-miR-200a (SEQ ID NO: 392)


hsa-miR-146a (SEQ ID NO: 290), hsa-miR-146b-5p (SEQ ID NO: 293)


hsa-miR-152 (SEQ ID NO: 308), hsa-miR-148b (SEQ ID NO: 300)


hsa-miR-154* (SEQ ID NO: 314), hsa-miR-487a (SEQ ID NO: 619)


hsa-miR-15a (SEQ ID NO: 317), hsa-miR-16 (SEQ ID NO: 321), 


hsa-miR-15b (SEQ ID NO: 319), hsa-miR-195 (SEQ ID NO: 377),


hsa-miR-497 (SEQ ID NO: 634)


hsa-miR-17 (SEQ ID NO: 324), hsa-miR-20a (SEQ ID NO: 406), 


hsa-miR-93 (SEQ ID NO: 930), hsa-miR-106a (SEQ ID NO: 122),


hsa-miR-106b (SEQ ID NO: 124), hsa-miR-20b (SEQ ID NO: 408), 


hsa-miR-526b* (SEQ ID NO: 708)


hsa-miR-181a (SEQ ID NO: 326), hsa-miR-181c (SEQ ID NO: 330)


hsa-miR-181b (SEQ ID NO: 329), hsa-miR-181d (SEQ ID NO: 332)


hsa-miR-18a (SEQ ID NO: 349), hsa-miR-18b (SEQ ID NO: 351)


hsa-miR-190 (SEQ ID NO: 353), hsa-miR-190b (SEQ ID NO: 357)


hsa-miR-192 (SEQ ID NO: 369), hsa-miR-215 (SEQ ID NO: 417)


hsa-miR-196a (SEQ ID NO: 379), hsa-miR-196b (SEQ ID NO: 381)


hsa-miR-199a-3p, hsa-miR-199b-3p (SEQ ID NO: 385)


hsa-miR-199a-5p (SEQ ID NO: 384), hsa-miR-199b-5p (SEQ ID NO: 386)


hsa-miR-19a* (SEQ ID NO: 388), hsa-miR-19b-1* (SEQ ID NO: 390),


hsa-miR-19b-2* (SEQ ID NO: 391)


hsa-miR-19a (SEQ ID NO: 387), hsa-miR-19b (SEQ ID NO: 389)


hsa-miR-200b (SEQ ID NO: 394), hsa-miR-200c (SEQ ID NO: 396)


hsa-miR-204 (SEQ ID NO: 401), hsa-miR-211 (SEQ ID NO: 413)


hsa-miR-208a (SEQ ID NO: 404), hsa-miR-208b (SEQ ID NO: 405)


hsa-miR-212 (SEQ ID NO: 414), hsa-miR-132 (SEQ ID NO: 255)


hsa-miR-23a* (SEQ ID NO: 440), hsa-miR-23b* (SEQ ID NO: 442)


hsa-miR-23a (SEQ ID NO: 439), hsa-miR-23b (SEQ ID NO: 441), 


hsa-miR-130a* (SEQ ID NO: 252)


hsa-miR-24-1* (SEQ ID NO: 444), hsa-miR-24-2* (SEQ ID NO: 445)


hsa-miR-25 (SEQ ID NO: 446), hsa-miR-92a (SEQ ID NO: 925), 


hsa-miR-367 (SEQ ID NO: 547), hsa-miR-92b (SEQ ID NO: 928)


hsa-miR-26a (SEQ ID NO: 448), hsa-miR-26b (SEQ ID NO: 451)


hsa-miR-26a-1* (SEQ ID NO: 449), hsa-miR-26a-2* (SEQ ID NO: 450)


hsa-miR-27a (SEQ ID NO: 453), hsa-miR-27b (SEQ ID NO: 455)


hsa-miR-29a (SEQ ID NO: 465), hsa-miR-29b (SEQ ID NO: 467), 


hsa-miR-29c (SEQ ID NO: 470)


hsa-miR-302a (SEQ ID NO: 475), hsa-miR-302b (SEQ ID NO: 477), 


hsa-miR-302c (SEQ ID NO: 479), hsa-miR-302d (SEQ ID NO: 481),


hsa-miR-373 (SEQ ID NO: 555), hsa-miR-520e (SEQ ID NO: 696), 


hsa-miR-520a-3p (SEQ ID NO: 690), hsa-miR-520b (SEQ ID NO: 692),


hsa-miR-520c-3p (SEQ ID NO: 693), hsa-miR-520d-3p (SEQ ID NO: 694)


hsa-miR-302b* (SEQ ID NO: 478), hsa-miR-302d* (SEQ ID NO: 482)


hsa-miR-30a* (SEQ ID NO: 486), hsa-miR-30d* (SEQ ID NO: 493), 


hsa-miR-30e* (SEQ ID NO: 495)


hsa-miR-30a (SEQ ID NO: 485), hsa-miR-30c (SEQ ID NO: 489), 


hsa-miR-30d (SEQ ID NO: 492), hsa-miR-30b (SEQ ID NO: 487), 


hsa-miR-30e (SEQ ID NO: 494)


hsa-miR-330-5p (SEQ ID NO: 513), hsa-miR-326 (SEQ ID NO: 509)


hsa-miR-34a (SEQ ID NO: 534), hsa-miR-34c-5p (SEQ ID NO: 539), 


hsa-miR-449a (SEQ ID NO: 599), hsa-miR-449b (SEQ ID NO: 600)


hsa-miR-362-3p (SEQ ID NO: 542), hsa-miR-329 (SEQ ID NO: 511)


hsa-miR-374a (SEQ ID NO: 557), hsa-miR-374b (SEQ ID NO: 559)


hsa-miR-376a (SEQ ID NO: 562), hsa-miR-376b (SEQ ID NO: 564)


hsa-miR-378 (SEQ ID NO: 568), hsa-miR-422a (SEQ ID NO: 585)


hsa-miR-379* (SEQ ID NO: 571), hsa-miR-411* (SEQ ID NO: 582)


hsa-miR-381 (SEQ ID NO: 574), hsa-miR-300 (SEQ ID NO: 472)


hsa-miR-509-5p (SEQ ID NO: 655), hsa-miR-509-3-5p (SEQ ID NO: 653)


hsa-miR-515-5p (SEQ ID NO: 666), hsa-miR-519e* (SEQ ID NO: 689)


hsa-miR-516b*, hsa-miR-516a-3p (SEQ ID NO: 667)


hsa-miR-517a (SEQ ID NO: 671), hsa-miR-517c (SEQ ID NO: 673)


hsa-miR-518a-5p (SEQ ID NO: 674), hsa-miR-527 (SEQ ID NO: 709)


hsa-miR-518f (SEQ ID NO: 681), hsa-miR-518b (SEQ ID NO: 675), 


hsa-miR-518c (SEQ ID NO: 676), hsa-miR-518a-3p (SEQ ID NO: 674),


hsa-miR-518d-3p (SEQ ID NO: 678)


hsa-miR-519c-3p (SEQ ID NO: 686), hsa-miR-519b-3p (SEQ ID NO: 685), 


hsa-miR-519a (SEQ ID NO: 683)


hsa-miR-519c-5p, hsa-miR-519b-5p, hsa-miR-523*, hsa-miR-518f*


(SEQ ID NO: 682), hsa-miR-526a, hsa-miR-520c-5p, hsa-miR-518e*,


hsa-miR-518d-5p (SEQ ID NO: 679), hsa-miR-522*,


hsa-miR-519a* (SEQ ID NO: 684)


hsa-miR-519e (SEQ ID NO: 688), hsa-miR-33b* (SEQ ID NO: 527)


hsa-miR-520a-5p (SEQ ID NO: 691), hsa-miR-525-5p (SEQ ID NO: 706)


hsa-miR-520g (SEQ ID NO: 698), hsa-miR-520h (SEQ ID NO: 699)


hsa-miR-524-5p (SEQ ID NO: 704), hsa-miR-520d-5p (SEQ ID NO: 695)


hsa-miR-525-3p (SEQ ID NO: 705), hsa-miR-524-3p (SEQ ID NO: 703)


hsa-miR-548b-5p (SEQ ID NO: 724), hsa-miR-548a-5p (SEQ ID NO: 722), 


hsa-miR-548c-5p (SEQ ID NO: 726), hsa-miR-548d-5p (SEQ ID NO: 728)


hsa-miR-7-1* (SEQ ID NO: 880), hsa-miR-7-2* (SEQ ID NO: 881)


hsa-miR-99a (SEQ ID NO: 948), hsa-miR-100 (SEQ ID NO: 114),


hsa-miR-99b (SEQ ID NO: 950)









We have constructed an 8-mer LNA-antimiR against miR-21, miR-155 and miR-122 (designated here as micromiR) that is fully LNA modified and phosphorothiolated (see FIG. 1 and Table 6). Our results from repeated experiments in MCF-7, HeLa, Raw and Huh-7 cells using a luciferase sensor plasmid for miR-21, miR-155 and miR-122 demonstrate that the fully LNA-modified short LNA-antimiRs are highly potent in antagonizing microRNAs.







TABLE 4







LNA_antimiR & MicromiR sequences and predicted


Tms











Com-
SEQ ID


Tm


pound
NO
microRNA
sequence
(° C.)





3204
1
miR-21
T c A G t C T G a T a A g C T
73


 


3205
2

G A T A A G C T
33


 


3206
3
miR-155
T c A c A A T t a G C A t T A
63


 


3207
4

T A G C A T T A
45


 


   4
5
miR-122
C c A t t G T c a C a C t C C
73


 


3208
6

C A C A C T C C
62





Capital letters are LNA units, such as beta-D-oxy LNA. Lower case letters are DNA units. Internucleoside linkages are preferably phosphorothioate. LNA cytosines are all preferably methylated/5-methyl cytosine.






The melting temperatures can be assessed towards the mature microRNA sequence, using a synthetic microRNA oligonucleotide (typically consisting of RNA nucleotides with a phosphodiester backbone). Typically measured Tms are higher than predicted Tms when using LNA oligomers against the RNA target.
Example 4
Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO: 2) LNA-AntimiR in MCF-7 Cells Using a Luciferase Sensor Assay
In order to assess the efficiency of a fully LNA-modified 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2) oligonucleotide in targeting and antagonizing miR-21, luciferase sensor constructs were made containing a perfect match target site for the mature miR-21 and as control, a target site with two mutations in the seed (FIG. 6). In order to monitor microRNA-21 inhibition, the breast carcinoma cell line MCF-7 was transfected with the different luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) at varying concentrations in comparison with a 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) against miR-21. After 24 hours, luciferase activity was measured.
Results: As seen in FIG. 2, the new fully LNA-modified 8-mer LNA-antimiR (Compound 3205 (SEQ ID NO: 2) shows two-fold higher potency compared to Compound 3204 (SEQ ID NO: 1), as shown by de-repression of the luciferase activity. By contrast, the control miR-21 sensor construct with two mismatches in the miR-21 seed did not show any de-repression of the firefly luciferase activity, thereby demonstrating the specificity of the perfect match miR-21 sensor in monitoring miR-21 activity in cells. The de-repression of luciferase activity by the 8-mer LNA-antimiR is clearly dose-dependent, which is not seen with Compound 3204 (SEQ ID NO: 1). Moreover, the new 8-mer is also much more potent at lower doses than Compound 3204 (SEQ ID NO: 1).
To conclude, the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) shows significantly improved potency in inhibition of miR-21 in vitro compared to the 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) targeting miR-21.
Materials and Methods:
Cell line: The breast carcinoma cell line MCF-7 was purchased from ATCC (#HTB-22TM). MCF-7 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 400.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, MCF-7 cells were transfected with 0.8 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector (SDS Promega) together with 1 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and harvested with cell scraper, after which cells were centrifugated for 5 min at 10.000 rpm. The supernatant was discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 5
Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO: 2) LNA-AntimiR in HeLa Cells Using a Luciferase Sensor Assay
To further assess the efficiency of the fully LNA-modified 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) in targeting miR-21, the cervix carcinoma cell line HeLa was also transfected with the previously described miR-21 luciferase sensor constructs alongside Compound 3205 (SEQ ID NO: 2) at varying concentrations as described in the above section (FIG. 3).
Results: Compound 3205 (SEQ ID NO: 2) shows complete de-repression of the miR-21 luciferase sensor construct in HeLa cells already at 5 nM compared to Compound 3204 (SEQ ID NO: 1), which did not show complete de-repression until the highest dose (50 nM). In addition, antagonism of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR is dose-dependent. To demonstrate the specificity of the miR-21 luciferase sensor assay, a mismatched miR-21 target site (2 mismatches in seed) was also transfected into HeLa cells, but did not show any de-repression of the firefly luciferase activity.
To conclude, the fully LNA-modified Compound 3205 (SEQ ID NO: 2) shows significantly improved potency in inhibition of miR-21 in vitro, in both MCF-7 and HeLa cells compared to the 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1).
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24 well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 6
Assessment of miR-155 Antagonism by Compound 3207 (SEQ ID NO: 4) LNA-AntimiR in Mouse RAW Cells Using a Luciferase Sensor Assay
To ask whether a fully LNA-modified 8-mer LNA-antimiR can effectively antagonize miR-155, a perfect match target site for miR-155 was cloned into the same luciferase vector (psiCHECK2) and transfected into the mouse leukaemic monocyte macrophage RAW cell line. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation.
Results: Luciferase measurements showed that the fully LNA-modified 8-mer LNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarly effective in antagonizing miR-155 compared to the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) (FIG. 4). Both LNA-antimiRs showed a >50% de-repression of the miR-155 luciferase sensor at 0.25 nM concentration and inhibited miR-155 in a dose-dependent manner.
Conclusion: These data further support the results from antagonizing miR-21, as shown in examples 1 and 2, demonstrating that a fully thiolated 8-mer LNA-antimiR is highly potent in microRNA targeting.
Materials and Methods:
Cell line: The mouse leukaemic monocyte macrophage RAW 264.7 was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 500.000 cells were seeded per well in a 6 well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, MCF-7 cells were transfected with 0.3 ug miR-155 or empty psiCHECK2 vector together with 10 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and harvested with cell scraper, after which cells were centrifugated for 5 min at 2.500 rpm. The supernatant were discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 7
Assessment of miR-122 Antagonism by Compound 3208 (SEQ ID NO: 6) LNA-AntimiR in HuH-7 Cells Using a Luciferase Sensor Assay
The potency of the fully modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) against miR-122 was assessed in the human hepatoma cell line HuH-7. The HuH-7 cells were transfected with luciferase sensor construct containing a perfect match miR-122 target site. After 24 hours luciferase measurements were performed (FIG. 5).
Results: The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) is more potent than the 15-mer LNA-antimiR Compound 4 (SEQ ID NO:5) at low concentration, as shown by de-repression of the miR-122 luciferase sensor. Both LNA-antimiRs inhibit miR-122 in a dose-dependet manner (FIG. 5).
Conclusion: The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) targeting miR-122 shows improved potency in inhibition of miR-122 in vitro.
Materials and Methods:
Cell line: The human hepatoma cell line HuH-7 was a kind gift from R. Bartenschlager, Heidelberg. Huh-7 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 8.000 cells were seeded per well in a 96 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HuH-7 cells were transfected with 57 ng miR-122 or empty psiCHECK2 vector together with 1 μl Lipofectamine2000 (Invitrogen). After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: 50 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 96 well plate was put on an orbital shaker for 30 min. To each well the Dual-luciferase Reporter assay system (Promega) was added and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 8
Assessment of miR-21 Antagonism by Comparing an 8-mer (Compound 3205, SEQ ID NO: 2) versus a 15-mer (Compound 3204, SEQ ID NO: 1) LNA-AntimiR in Human Prostate Carcinoma Cells (PC3)
We have previously shown (patent application 1051), that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated is able to completely de-repress the miR-21 luciferase reporter levels in the human cervix carcinoma cell line HeLa and partly de-repress the miR-21 luciferase reporter levels in the human breast carcinoma cell line MCF-7. We next extended this screening approach to the human prostate cancer cell line PC3. To assess the efficiency of the different LNA-antimiR oligonucleotides against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 and a target site with two mismatches in the seed were cloned in the 3′UTR of Renilla luciferase gene (FIG. 7). In order to monitor miR-21 inhibition, PC3 cells were transfected with the different luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison with the 15-mer LNA-antimiR perfect match Compound 3204 (SEQ ID NO: 1) at varying concentrations. After 24 hours, luciferase activity was measured.
Results: The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 15-mer LNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However, complete de-repression of the luciferase reporter was not obtained even at the highest concentrations (FIG. 7). In contrast, the cells that were transfected with the 8-mer fully LNA substituted LNA-antimiR showed complete de-repression already at 1 nM, indicating significantly improved potency compared to the 15-mer LNA-antimiR. The luciferase control reporter harboring a mismatch target site for miR-21 was not affected by either LNA-antimiR, demonstrating high specificity of both LNA-antimiRs.
Conclusion: The micromer is far more potent than the 15-mer LNA-antimiR in targeting miR-21 and has so far shown to be most potent in prostate carcinoma cells.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 100.000 cells were seeded per well in a 12-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 0.3 mg miR-21 or empty psiCHECK2 vector together with 1.2 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 20 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 9
Specificity Assessment of miR-21 Antagonism by an 8-mer LNA-AntimiR
To investigate the specificity of our short LNA-antimiR targeting miR-21, we designed an 8-mer mismatch control LNA-antimiR (Compound 3218, SEQ ID NO: 16) containing 2 mismatches in the seed recognition sequence (see FIG. 8). The luciferase reporter constructs described in example 1 were transfected into the human cervix carcinoma cell line HeLa together with the LNA mismatch control oligo Compound 3218 (SEQ ID NO: 16) and its efficacy was compared with the 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2) targeting miR-21. After 24 hours, luciferase activity was measured.
Results: As shown in FIG. 8, transfection of the fully LNA-modified 8-mer LNA-antimiR in HeLa cells resulted in complete de-repression of the luciferase miR-21 reporter already at 5 nM. In contrast, when the cells were transfected with the 8-mer LNA mismatch control oligo, combined with the results obtained with the control miR-21 luciferase reporter having two mismatches in the miR-21 seed, these data demonstrate high specificity of the fully LNA-substituted 8-mer LNA-antimiR in targeting miR-21 in Hela cells.
Analysis of the miRBase microRNA sequence database showed that the miR-21 recognition sequence, of the LNA-antimiR Compound 3205 (SEQ ID NO: 2) is unique for microRNA-21. However, when decreasing the micromer length to 7 nt, it is not specific for only miR-21, since ath-miR-844, mmu-miR-590-3p and has-miR-590-3p are also targeted.
Conclusion: Exhanging two nucleotide positions within the 8-mer LNA-antimiR with two mismatching nucleotides completely abolished the antagonizing activity of the LNA-antimiR for miR-21.
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates were put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 10
Assessment of the Shortest Possible Length of a Fully LNA-Modified LNA-AntimiR that Mediates Effective Antagonism of miR-21
To further investigate the LNA-antimiR length requirements, we designed a 7-mer and a 6-mer LNA-antimiR targeting miR-21, both fully LNA-modified and phosphorothiolated oligonucleotides. The miR-21 luciferase reporter constructs were transfected into HeLa cells along with the LNA-antimiRs at varying concentrations. Luciferase measurements were performed after 24 hours.
Results: As seen in FIG. 9, the 7-mer LNA-antimiR mediates de-repression of the miR-21 luciferase reporter plasmid, but at lower potency compared to the 8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2). Nevertheless, a dose-dependent trend can still be observed. By contrast, the 6-mer LNA-antimiR did not show any inhibitory activity.
Conclusion: To conclude, the shortest possible length of an LNA-antimiR which is able to mediate miR-21 inhibition is 7 nucleotides. However, the 7-mer LNA-antimiR is less potent compared to the 8-mer LNA-antimiR for miR-21.
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24 well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 11
Length Assessment of Fully LNA-Substituted LNA-AntimiRs Antagonizing miR-21
Next, we investigated the effect of increasing the length from a 9-mer to a 14-mer fully LNA substituted LNA-antimiRs on antagonizing miR-21 in HeLa cells. The resulting LNA-antimiRs were transfected into HeLa cells together with the miR-21 luciferase reporter constructs (FIG. 10). Luciferase measurements were performed after 24 hours.
Results: The 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9) (9-mer) showed dose-dependent de-repression of the miR-21 luciferase reporter which did not reach complete de-repression, as demonstrated for the 7-mer LNA-antimiR (Compound 3210, SEQ ID NO: 8). Increasing the length to 10-mer to 14-mer (Compound 3212, (SEQ ID NO: 10); Compound 3213 (SEQ ID NO: 11); and Compound 3214 (SEQ ID NO: 12)) decreased the potency as shown by less efficient de-repression of the miR-21 reporter.
Conclusion: As shown in FIG. 10, the longest fully LNA-modified and phosphorothiolated LNA-antimiR which is still able to mediate miR-21 inhibition is a 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9). However, it is clearly less efficient than the 7-mer and 8-mer LNA-antimiRs.
Materials and Methods: Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 control vector without target site together with 0.7 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates were put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96-well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 12
Determination of the Most Optimal Position for an 8-mer LNA-AntimiR within the miR Target Recognition Sequence
Our experiments have shown that the most potent fully LNA-modified phosphorothiolated LNA-antimiR is 8 nucleotides in length. To assess the most optimal position for an 8-mer LNA-antimiR within the miR target recognition sequence, we designed four different fully LNA-modified 8-mer LNA-antimiRs tiled across the mature miR-21 sequence as shown in FIG. 11. The different LNA-antimiRs were co-transfected together with the miR-21 luciferase reporter constructs into HeLa cells. Luciferase measurements were performed after 24 hours.
Results: The only LNA-antimiR that mediated efficient silencing of miR-21 as measured by the luciferase reporter was Compound 3205 (SEQ ID NO: 2), which targets the seed region of miR-21. Neither Compound 3215 (SEQ ID NO: 13) which was designed to cover the 3′ end of the seed (50% seed targeting) did not show any effect, nor did the other two LNA-antimiRs Compound 3216 (SEQ ID NO: 14) or Compound 3217 (SEQ ID NO: 15), which were positioned to target the central region and the 3′′end of the mature miR-21, respectively.
Conclusion: The only 8-mer LNA-antimiR mediating potent silencing of miR-21 is the one targeting the seed of the miR-21.
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 13
Validation of Interaction of the miR-21 Target Site in the Pdcd-4-3′-UTR and miR-21 Using the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-AntimiR.
The tumour suppressor protein Pdcd4 inhibits TPA-induced neoplastic transformation, tumour promotion and progression. Pdcd4 has also been shown to be upregulated in apoptosis in response to different inducers. Furthermore, downregulation of Pdcd4 in lung and colorectal cancer has also been associated with a poor patient prognosis. Recently, Asangani et al and Frankel et al showed that the Pdcd-4-3′-UTR contains a conserved target site for miR-21, and transfecting cells with an antimiR-21, resulted in an increase in Pdcd4 protein. We therefore constructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTR region of Pdcd4 encompassing the aforementioned miR-21 target site, which was co-transfected together with different LNA-antimiRs into HeLa cells. The different LNA-antimiRs were; Compound 3205 (SEQ ID NO:2) (8-mer, perfect match) or Compound 3218 (SEQ ID NO: 16) (8-mer, mismatch). Luciferase measurements were performed after 24 hours.
Results: As shown in FIG. 12, in cells transfected with the Pdcd4 3′UTR luciferase reporter and Compound 3205 (SEQ ID NO: 2), an increase in luciferase activity was observed, indicating interaction between the Pdcd4 3′UTR and miR-21. However, transfecting the cells with the mismatch compound, Compound 3218 (SEQ ID NO: 16), no change in luciferase activity was observed, which was expected since the compound does not antagonize miR-21. When comparing the 8-mer LNA-antimiR against two longer designed LNA-antimiRs, the short fully LNA-modified and phosphorothiolated LNA-antimiR was significantly more potent, confirming previous luciferase assay data.
Conclusion: These data conclude that Compound 3205 (SEQ ID NO: 2), which antagonizes miR-21, can regulate the interaction between Pdcd4 3′UTR and miR-21.
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 60.000 cells were seeded per well in a 24-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 0.2 ug Pdcd-4-3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 0.7 Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Varying concentrations of the LNA-antimiR oligonucleotides were also transfected. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to an eppendorf tube and spinned at 10.000 rpm for 30 min after which 10 μl were transferred to a 96 well plate and luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 14
Comparison of an 8-mer LNA-AntimiR (Compound 3207, SEQ ID NO: 4) with a 15-mer LNA-AntimiR (Compound 3206, SEQ ID NO: 3) in Antagonizing miR-155 in Mouse RAW Cells
To ask whether our approach of using short LNA-antimiRs could be adapted to targeting other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-155. A perfect match target site for miR-155 was cloned into the 3′UTR of the luciferase gene in the reporter plasmid psiCHECK2 and transfected into the mouse RAW macrophage cell line together with an 8-mer or a 15-mer LNA-antimiR. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation. After 24 hours, luciferase analysis was performed.
Results: Luciferase measurements showed that the fully LNA-modified 8-mer LNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarly effective in antagonizing miR-155 compared to the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) (FIG. 13). Both LNA-antimiRs showed a >50% de-repression of the miR-155 luciferase sensor at 0.25 nM concentration and inhibited miR-155 in a dose-dependent manner.
Analysis of the miRBase microRNA sequence database showed that the miR-155 recognition sequence, of the LNA-antimiR Compound 3207 (SEQ ID NO: 4) is unique for microRNA-155. However, when decreasing the LNA-antimiR length to 7 nt, it is not specific for only miR-155, mdv1-miR-M4 and kshv-miR-K12-11 (SEQ ID NO: 963) is also targeted.
Conclusion: A fully LNA-modified and phosphorothiolated 8-mer LNA-antimiR is equally potent compared with a 15-mer LNA-antimiR of a mixed LNA/DNA design in antagonizing miR-155. Thus, our approach of using short LNA-antimiRs can be readily adapted to targeting of other miRNAs
Materials and Methods:
Cell line: The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 500.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155 perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and harvested with cell scraper, after which cells were spinned for 5 min at 2.500 rpm. The supernatant was discarded and 50 ml 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 15
Assessment of c/EBPβ Protein Levels as a Functional Readout for miR-155 Antagonism by Short LNA-AntimiR (Compound 3207, SEQ ID NO: 4).
As a functional readout for miR-155 antagonism by short LNA-antimiR (Compound 3207, SEQ ID NO: 4) we determined the protein levels of a novel miR-155 target, c/EBPβ. The mouse macrophage RAW cell line was transfected together with either an 8-mer (Compound 3207, SEQ ID NO: 4) or a 15-mer (Compound 3206, SEQ ID NO: 3) LNA-antimiR in the absence or presence of pre-miR-155. As mismatch controls for the 15-mer, Compound 4 (SEQ ID NO: 5) was used, which targets miR-122 and for the 8-mer Compound 3205 (SEQ ID NO: 2) was used, which targets miR-21. These two control miRNAs do not regulate c/EBPβ expression levels. LPS was used to induce miR-155 accumulation and cells were harvested after 16 hours with LPS. c/EBPβ has three isoforms; LIP, LAP and LAP* that were detected by Western blot analysis and the same membranes were re-probed with beta-tubulin as loading control.
Results: Ratios were calculated for c/EBPβ LIP and beta-tubulin as indicated in FIG. 14. RAW cells that were transfected with the 15-mer LNA-antimiR and no pre-miR-155 all showed equal c/EBPβ LIP/beta-tubulin ratios, due to inhibition of miR-155 increases the c/EBPβ LIP levels (FIG. 14, left panel). By comparison, transfection of pre-miR-155 in RAW cells resulted in decreased c/EBPβ LIP levels as expected, if c/EBPβ was a miR-155 target, as shown in lanes with protein extracts from RAW cells treated with no LNA or a mismatch. However, protein extracts from RAW cells transfected with LNA-antimiR against miR-155, showed an increase of c/EBPβ LIP levels. The same experiments were also carried out with the 8-mer LNA-antimiR-155 (Compound 3207, SEQ ID NO: 4) and as shown in FIG. 14 (right panel) comparable results to those with the 15-mer LNA-antimiR Compound 3206 (SEQ ID NO: 3) were obtained.
Conclusion: Antagonism of miR-155 using either an 8-mer or a 15-mer LNA-antimiR leads to de-repression of the direct target c/EBPβ.
Materials and Methods:
Cell line: The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 500.000 cells were seeded per well in a 6-well plate the day before transfection in order to achieve 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 5 nmol pre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with 10 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After 16 hours, cells were harvested for protein extraction and western blot analysis.
Western blot: Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. The protein concentration was measured with Coomassie Plus according to the manufacturer's instructions and 80 ug was loaded onto a 4-12% BIS-TRIS gel. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody C/EBPβ (Santa Cruz) with a 1:100 concentration. Immunoreactive bands were visualized with ECL Plus (Amersham).
Example 16
Antagonism of miR-106b by a Fully LNA-Modified 8-mer (Compound 3221, SEQ ID NO: 19) LNA-AntimiR
To confirm that our approach of using short LNA-antimiRs could be adapted to targeting of other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-106b. A perfect match target site for miR-106b was cloned into the 3′UTR of the luciferase gene in the vector (psiCHECK2) and transfected into the human cervix carcinoma HeLa cell line together with a short LNA-antimiR (Compound 3221, SEQ ID NO: 19) or with a 15-mer LNA-antimiR (Compound 3228, SEQ ID NO: 26) at varying concentrations. Luciferase measurements were performed after 24 hours.
Results: Transfection of the 8-mer LNA-antimiR Compound 3221 (SEQ ID NO: 19) against miR-106b resulted in dose-dependent inhibition of miR-106b as shown by de-repression of the luciferase reporter, which was completely de-repressed at 1 nM LNA-antimiR concentration (FIG. 15). Comparable results were obtained using the 15-mer LNA-antimiR Compound 3228 (SEQ ID NO: 26) demonstrating that an 8-mer LNA-antimiR is similarly potent to a 15-mer.
Conclusion: Targeting of miR-106b in HeLa cells shows that an 8-mer fully LNA-modified and phosphorotiolated LNA-antimiR is equally potent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 5.200 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 57 ng miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.14 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to eppendorf tubes and spinned at 10.000 rpm for 30 min after which luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 17
Antagonism of miR-19a by a Fully LNA-Modified 8-Mer (Compound 3222, SEQ ID NO: 20) LNA-AntimiR
To further confirm that our approach of using short LNA-antimiRs can be readily adapted to targeting of other miRNAs we designed a fully LNA-modified 8-mer LNA-antimiR against microRNA-19a. A perfect match target site for miR-19a was cloned in the 3′UTR of the luciferase gene in the psiCHECK2 vector. The reporter plasmid was transfected into the human cervix carcinoma HeLa cell line together with a short LNA-antimiR (Compound 3222, SEQ ID NO: 20) or with a 15-mer LNA-antimiR (Compound 3229, SEQ ID NO: 27) targeting miR-19a at varying concentrations. Luciferase measurements were performed after 24 hours.
Results: As shown in FIG. 16, transfection of the 15-mer LNA-antimiR Compound 3229 (SEQ ID NO: 27) into HeLa efficiently antagonizes miR-19a as demonstrated by complete de-repression at 1 nM LNA-antimiR concentration. By comparison, transfection of the 8-mer LNA-antimiR Compound 3222 (SEQ ID NO: 20) resulted in effective miR-19a antagonism already at 0.5 nM concentration, indicating that this 8-mer LNA-antimiR is at least equally potent compared with a 15-mer LNA-antimiR in HeLa cells.
Conclusion: Targeting of miR-19a in HeLa cells shows that an 8-mer fully LNA-modified and phosphorothiolated LNA-antimiR is at least equally potent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.
Materials and Methods: Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 5.200 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 57 ng miR-21 perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector together with 0.14 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 24-well plates was put on an orbital shaker for 30 min. The cells were collected and transferred to eppendorf tubes and spinned at 10.000 rpm for 30 min after which luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 18
Targeting of a MicroRNA Family Using Short, Fully LNA-Substituted LNA-AntimiR
Next, we investigated whether it is possible to target a microRNA family using a single short 7-mer LNA-antimiR complementary to the seed sequence that is common for all family members (see FIG. 17). In this experiment, we focused on miR-221 and miR-222 that are overexpressed in solid tumors of the colon, pancreas, prostate and stomach. It has also been shown that miR-221 and miR-222 are the most significantly upregulated microRNAs in glioblastoma multiforme. Furthermore, overexpression of miR-221 and miR-222 may contribute to the growth and progression of prostate carcinoma, at least in part by blocking the tumor suppressor protein p27. A perfect match target site for both miR-221 and miR-222, respectively, was cloned into the 3′UTR of the luciferase gene resulting in two reporter constructs. These constructs were then transfected either separate or combined into the prostate carcinoma cell line, PC3. In addition to the 7-mer, targeting both miR-221 and miR-222, we also co-transfected a 15-mer LNA-antimiR (15mer) targeting either miR-221 (Compound 3223, SEQ ID NO: 21) or miR-222 (Compound 3224, SEQ ID NO: 22), each transfected separately or together (see FIG. 18 left).
Results: As shown in FIG. 18, transfection of PC3 cells with the LNA-antimiR Compound 3223 (SEQ ID NO: 21) against miR-221 resulted in efficient inhibition of miR-221 at 1 nM LNA-antimiR concentration. An inhibitory effect is also observed when using the luciferase reporter plasmid for miR-222 as well as when co-transfecting both luciferase reporters for miR-221 and miR-222 simultaneously into PC3 cells. This inhibitory effect is most likely due to the shared seed sequence between miR-221 and miR-222. Similarly, transfection of PC3 cells with the LNA-antimiR Compound 3224 (SEQ ID NO: 22) against miR-222 resulted in efficient inhibition of miR-222 at 1 nM LNA-antimiR concentration as shown by complete de-repression of the luciferase reporter for miR-222. An inhibitory effect is also observed when using the luciferase reporter plasmid for miR-222 as well as when co-transfecting both luciferase reporters for miR-221 and miR-222 simultaneously into PC3 cells. Co-tranfection of both LNA-antimiR compounds Compound 3223 (SEQ ID NO: 21) and Compound 3224 (SEQ ID NO: 22) against miR-221 and miR-222, respectively, (see FIG. 18 left), resulted in effective inhibition of both miRNAs as shown by complete de-repression of the luciferase reporter plasmids both when separately transfected and when co-transfected into PC3 cells. Interestingly, transfection of a single fully LNA-modified 7-mer LNA-antimiR (Compound 3225, SEQ ID NO: 23) targeting the seed sequence of miR-221 and miR-222 into PC3 cells resulted in efficient, dose-dependent antagonism of miR-221 and miR-222 simultaneously as shown by complete de-repression of the luciferase reporter plasmids both when separately transfected and when co-transfected into PC3 cells. This demonstrates that a single, short LNA-substituted LNA-antimiR can effectively target seed sequences thereby antagonizing entire microRNA families simultaneously. Analysis of the miRBase microRNA sequence database showed that the miR-221/222 seed recognition sequence, of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) is unique for both miRNAs.
Conclusion: Our results demonstrate that LNA enables design and synthesis of short fully LNA-substituted LNA-antimiR oligonucleotides that can effectively target microRNA seed sequences thereby antagonizing entire microRNA families simultaneously.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714) PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 100.000 cells were seeded per well in a 12-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 0.3 ug of luciferase reporter plasmid for miR-221 or for miR-222 or with empty psiCHECK2 vector without miRNA target site as control together with 1.2 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates was transferred to eppendorf tubes. The cell lysate was spinned for 10 min at 2.500 rpm after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 19
Assessment of P27 Protein Levels as a Functional Readout for Antagonism of the miR-221/222 Family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-AntimiR.
Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) that miR-221 and miR-222 post-transcriptionally regulate the expression of the tumour suppressor gene p27, which is involved in cell cycle regulation. In these studies, down-regulation of miR-221 and miR-222 was shown to increase expression levels of p27. Thus, as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27 after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) into PC3 cells in comparison with an 8-mer LNA mismatch control. After 24 hours the cells were harvested for western blot analysis (FIG. 19).
Results: As shown in FIG. 19, transfection of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting the seed sequence in miR-221 and miR-222 resulted in dose-dependent increase of the p27 protein levels compared to either untransfected or LNA mismatch control transfected PC3 cells. These results clearly demonstrate that the 7-mer LNA-antimiR is able to effectively antagonize the miR-221/222 family leading to de-repression of the direct target p27 at the protein level.
Conclusion: A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence in the miR-221/222 family effectively antagonized both miRNAs leading to de-repression of the direct target p27 at the protein level.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714) PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with LNA-antimiRs at varying concentrations with Lipofectamine2000. Cells were harvested after 24 hours for protein extraction and western blot analysis.
Western blot: Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 250 ml lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min, then spinned at 10.000 rpm for 10 minutes. The protein concentration was measured with Coomassie Plus according to the manufacturer's instructions and 100 ug was loaded onto a 4-12% BIS-TRIS gel. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).
Example 20
Duplex Melting Temperatures (Tm) of the LNA-AntimiRs
As shown in Table 5, Tm values increase with increasing the length of short fully modified LNA-antimiRs (see Tm values for Compound 3205 (SEQ ID NO: 2) and Compounds 3209 to 3214 (SEQ ID NOs: 7 to 12) in Table 7). Most optimal inhibitory effect was achieved with the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) against miR-21, whereas the very low Tm of the 6-mer Compound 3209 (SEQ ID NO: 7) is most likely not sufficient to mediate antagonism of the miR-21 target. On the other hand, increasing the length beyond a 10-mer (Compound 3212, SEQ ID NO: 10) significantly increases the Tm, while simultaneously decreasing the inhibitory activity as measured using the luciferase miR-21 reporter, which is most likely due to high propensity of the fully modified 12- and 14-mer LNA-antimiRs to form homodimers. The experiments using a sliding window of fully LNA-modified 8-mer LNA-antimiRs across the mir-21 recognition sequence clearly demonstrate that in addition to adequate Tm value of the LNA-antimiR, the seed region is most critical for miRNA function and, thus, the most optimal region to be targeted by an LNA-antimiR.







TABLE 5







Tm values for miR-21 LNA-antimiRs, measured


against a complementary RNA oligonucleotide

















Measured


Com-
SEQ ID
micro-
Length

Tm (RNA)


pound
NO:
RNA
(bp)
Sequence
° C.















3205
2
miR-21
 8
5′- GATAAGCT -3′
64.0


 


3209
7
miR-21
 6
5′- TAAGCT -3′
32.0


 


3210
8
miR-21
 7
5′- ATAAGCT -3′
45.0


 


3211
9
miR-21
 9
5′- TGATAAGCT -3′
65.0


 


3212
10
miR-21
10
5′- CTGATAAGCT -3′
63.0


 


3213
11
miR-21
12
5′- GTCTGATAAGCT -3′
86.8


 


3214
12
miR-21
14
5′- CAGTCTGATAAGCT -3′
89.9


 


3215
13
miR-21
 8
5′- TCTGATAA -3′
56.0


 


3216
14
miR-21
 8
5′- ATCAGTCT -3
72.0


 


3217
15
miR-21
 8
5′- TCAACATC -3
48.0









Conclusion: The Tm values along with experimental data obtained with luciferase reporters show that potent antagonism by LNA-antimiR is not only dependent on Tm but also depends on the positioning of the LNA-antimiR within the microRNA recognition sequence.
Materials and Methods:
Tm measurements: The oligonucleotide:miR-21 RNA duplexes were diluted to 3 μM in 500 μl RNase free H2O and mixed with 500 μl 2×Tm-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM Na-phosphate, pH 7.0). The solution was heated to 95° C. for 3 min and then allowed to anneal in RT for 30 min. The duplex melting temperatures (Tm) were measured on a Lambda 40 UV/VIS Spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature was ramped up from 20° C. to 95° C. and then down to 25° C., recording absorption at 260 nm. First derivative and the local maximums of both the melting and annealing were used to assess the duplex melting temperatures.
Example 21
Assessment of miR-21 Antagonism by Comparing an 8-mer (Compound 3205, SEQ ID NO: 2) Versus a 15-mer (Compound 3204, SEQ ID NO: 1) LNA-AntimiR in Human Hepatocytic Cell Line HepG2
We have previously shown in this application, that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated effectively antagonizes miR-21 in the human cervix carcinoma cell line HeLa, the human breast carcinoma cell line MCF-7 and the human prostate cancer cell line PC3. We extended this screening approach to the human hepatocellular cancer cell line HepG2. To assess the efficiency of the 8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene. In order to monitor miR-21 inhibition, HepG2 cells were transfected with the luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison of specificity with the 8-mer LNA-antimiR mismatch (Compound 3218, SEQ ID NO: 16) and for comparison of potency together with the 15-mer (Compound 3204, SEQ ID NO: 1) at varying concentrations. After 24 hours, luciferase activity was measured.
Results: The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 15-mer LNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However, complete de-repression of the luciferase reporter was not obtained, not even at the higher concentrations (FIG. 20). In contrast, the cells that were transfected with the 8-mer fully LNA modified LNA-antimiR (Compound 3205, SEQ ID NO: 2) showed complete de-repression already at 5 nM, indicating significantly improved potency compared to the 15-mer LNA-antimiR. Comparing the specificity of the 8-mer perfect match and the 8-mer mismatch, the mismatch LNA-antimiR (Compound 3218, SEQ ID NO: 16) did not show any de-repression at all, demonstrating high specificity of the LNA-antimiR compound against miR-21.
Conclusion: The 8-mer (Compound 3205, SEQ ID NO: 2) is more potent than the 15-mer LNA-antimiR in targeting miR-21 and antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) is specific.
Materials and Methods:
Cell line: The human hepatocytic HepG2 cell line was purchased from ECACC (#85011430). HepG2 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 650.000 cells were seeded per well in a 6-well plate and reverse transfection were performed. HepG2 cells were transfected with 0.6 μg miR-21 or empty psiCHECK2 vector together with 2.55 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 300 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 50 μl were transferred to a 96 well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 22
Validation of Interaction of the miR-21 Target Site in the Pdcd4 3′UTR and miR-21 Using the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-AntimiR in Human Hepatocellular Cell Line Huh-7
The tumour suppressor protein Pdcd4 inhibits tumour promotion and progression. Furthermore, downregulation of Pdcd4 in lung and colorectal cancer has also been associated with poor patient prognosis. Recently, Asangani et al (Oncogene 2007) and Frankel et al (J Biol Chem 2008) showed that the Pdcd4 3′UTR contains a conserved target site for miR-21, and transfecting cells with an antimiR-21, resulted in an increase in Pdcd4 protein. We therefore constructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTR region of Pdcd4 encompassing the aforementioned miR-21 target site, which was co-transfected together with different LNA-antimiRs and pre-miR-21 (10 nM) into Huh-7 cells. The different LNA-antimiRs were; Compound 3205 (SEQ ID NO: 2) (8-mer, perfect match), Compound 3218 (SEQ ID NO: 16) (8-mer, mismatch) and Compound 3204 (SEQ ID NO: 1) (15-mer, DNA/LNA mixmer). Luciferase measurements were performed after 24 hours.
Results: As shown in FIG. 21, cells transfected with the Pdcd4 3′UTR luciferase reporter and Compound 3205 (SEQ ID NO: 2), an increase in luciferase activity was observed, indicating interaction between the Pdcd4 3′UTR and miR-21. However, transfecting the cells with the mismatch compound, Compound 3218 (SEQ ID NO: 16), no change in luciferase activity was observed, which was expected since the compound does not antagonize miR-21. When comparing the 8-mer LNA-antimiR against the 15-mer LNA-antimiR (Compound 3204, SEQ ID NO: 1), the short fully LNA-modified and phosphorothiolated LNA-antimiR was significantly more potent, confirming previous data.
Materials and Methods:
Cell line: The human hepatoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 11.000 cells were seeded per well in a 96-well plate the day before transfection in order to achieve 50-70% confluency the next day. On the day of transfection, Huh-7 cells were transfected with 20 ng Pdcd4 3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 10 nM pre-miR-21 (Ambion) and 0.14 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Varying concentrations of the LNA-antimiR oligonucleotides were also transfected. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: Cells were washed and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well, after which the 96-well plates was put on an orbital shaker. After 30 min., 50 μl luciferase substrate dissolved in Luciferase Assay Buffer II (Dual-Luciferase Reporter Assay System from Promega, Cat# E1910) was added to the wells with lysated cells and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 23
Assessment of Pdcd4 Protein Levels as a Functional Readout for miR-21 Antagonism by the 8-mer LNA-AntimiR (Compound 3205, SEQ ID NO: 2).
In addition, we also transfected HeLa cells with Compound 3205 (SEQ ID NO: 2) (perfect match), Compound 3218 (SEQ ID NO: 16) (mismatch), Compound 3219 (SEQ ID NO: 17) (scrambled) and analyzed Pdcd4 protein levels after 24 hours with Western blot (FIG. 22). As shown, in the protein extracts from cells where Compound 3205 (SEQ ID NO: 2) had been added, the Pdcd4 protein levels increase, due to antagonism of mir-21 by Compound 3205 (SEQ ID NO: 2) in contrast to the two control LNA oligonucleotides.
Conclusion: Antagonism of miR-21 using an 8-mer (Compound 3205, SEQ ID NO: 2) leads to derepression of the direct target Pdcd4 □ntagorism of miR-21
Materials and Methods:
Cell line: The human cervix carcinoma cell line HeLa was purchased from ECACC (#93021013). HeLa cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 200.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells were transfected with 5 nM LNA oligonucleotides and 2.5 μg/ml Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were harvested for Western blot analysis.
Western blot: Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions. The membrane was incubated overnight at 4° C. with the primary affinity purified rabbit serum antibody Pdcd4 (Rockland) with a 1:2000 concentration. As control, anti-beta tubulin antibodies (Thermo Scientific) were used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).
Example 24
Assessment of Potential Hepatotoxicity of the 8-mer Perfect Match LNA-AntimiR Compound 3205 (SEQ ID NO: 2) and the LNA Mismatch Control Compound 3218 (SEQ ID NO: 16).
Each compound was injected into female NMRI mice, at doses of 25 mg/kg, 5 mg/kg and 1 mg/kg, every other day for 2 weeks. The animals were sacrificed and serum was collected from whole blood for ALT and AST analyses. As seen in FIG. 23, the ALT and AST levels were not elevated for Compound 3205 (SEQ ID NO: 2) compared to saline or Compound 3218 (SEQ ID NO: 16) (mismatch control). However, one mouse showed increased levels (marked red), since the serum samples were contaminated with red blood cells, which contain 6-8 times higher levels of ALT and AST compared to plasma. The mice that received 5 mg/kg and 1 mg/kg were also analyzed for ALT and AST levels and showed no changes compared to saline treated control animals (data not shown).
Materials and Methods:
Experimental Design:


















No.

Conc. at




Gr.
Animal
of
Compound Dose
dose vol.
Adm.



no.
IDno.
mice
level per day
10 ml/kg
Route
Dosing





















1
 1-10
10
NaCl
—
i.v
0, 2, 4, 7, 9





0.9%





2
11-15
5
Compound
2.5 mg/ml
i.v
0, 2, 4, 7, 9





3205








(SEQ ID NO: 2)








25 mg/kg 





3
16-20
5
Compound
0.5 mg/ml
i.v
0, 2, 4, 7, 9





3205








(SEQ ID NO: 2)








5 mg/kg





4
21-25
5
Compound
0.1 mg/ml
i.v
0, 2, 4, 7, 9





3205








(SEQ ID NO: 2)








1 mg/kg





5
26-30
5
Compound
2.5 mg/ml
i.v
0, 2, 4, 7, 9





3230








(SEQ ID NO: 16)








25 mg/kg 





6
31-35
5
Compound
0.5 mg/ml
i.v
0, 2, 4, 7, 9





3230








(SEQ ID NO: 16)








5 mg/kg









Sacrifice; The animals was sacrificed by cervical dislocation.
Sampling of serum for ALT/AST; The animals were anaesthetised with 70% CO2-30% O2 before collection of retro orbital sinus blood. The blood was collected into S-monovette Serum-Gel vials. The serum samples were harvested and stored from each individual mouse. The blood samples were stored at room temperature for two hours and thereafter centrifuged 10 min, 3000 rpm, at room temp. The serum fractions were harvested into Eppendorf tubes on wet ice.
ALT and AST measurements; ALT and AST measurements were performed in 96-well plates using ALT and AST reagents from ABX Pentra (A11A01627-ALT, A11A01629-AST) according to the manufacturer's instructions. In short, serum samples were diluted 2.5 fold with H2O and each sample was assayed in duplicate. After addition of 50 μl diluted sample or standard (multical from ABX Pentra—A11A01652) to each well, 200 μl of 37° 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° C.
Example 25
Assessment of PU.1 Protein Levels as a Functional Readout for miR-155 Antagonism by Short LNA-AntimiR (Compound 3207, SEQ ID NO: 4).
We have previously shown that the 8-mer (Compound 3207, SEQ ID NO: 4) antagonizing miR-155 leads to derepression of the miR-155 target c/EBPbeta in the mouse macrophage RAW cells. To further verify the potency of Compound 3207 (SEQ ID NO: 4) we determined the protein levels of another miR-155 target, PU.1 As a functional readout for miR-155 antagonism by short LNA-antimiR (Compound 3207, SEQ ID NO: 4) we performed Western blot. The antagonism was verified in the human monocytic THP-1 cell line which was transfected together with either an 8-mer (Compound 3207, SEQ ID NO: 4) perfect match or a 8-mer control LNA in the absence or presence of pre-miR-155. LPS was used to induce miR-155 accumulation and cells were harvested after 24 hours.
Results: THP-1 cells that were transfected with pre-miR-155 shows a decrease in PU.1 levels (FIG. 24). Transfecting the cells with the fully LNA-modified and phosphorothiolated Compound 3207 (SEQ ID NO: 4) effectively antagonizes miR-155, leading to unaltered levels of PU.1 protein. By comparison, transfecting the cells with an 8-mer LNA control, PU.1 levels decreased, indicating that antagonism of miR-155 by Compound 3207 (SEQ ID NO: 4) LNA-antimiR is specific.
Conclusion: Antagonism of miR-155 using an 8-mer leads to de-repression of the direct target PU.1 in human THP-1 cells.
Materials and Methods:
Cell line: The human monocytic THP-1 cell line was purchased from ECACC (#88081201). THP-1 cells were cultured in RPMI with L-glutamine, supplemented with 10% fetal bovine serum.
Transfection: 200.000 cells were seeded per well in a 12-well plate the day before. On the day of transfection, THP-1 cells were transfected with 5 nmol pre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. LPS (100 ng/ml) was added to the cells after the 4 hour incubation with the transfection complexes. After 24 hours, cells were harvested for protein extraction and western blot analysis.
Western blot: Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions The membrane was incubated overnight at 4° C. with the rabbit monoclonal PU.1 antibody (Cell Signaling) with a 1:2000 concentration. As equal loading, Tubulin (Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).
Example 26
Assessment of p27 Protein Levels as a Functional Readout for Antagonism of the miR-221/222 Family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-AntimiR.
Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) that miR-221 and miR-222 post-transcriptionally regulate the expression of the tumour suppressor gene p27, which is involved in cell cycle regulation. In these studies, down-regulation of miR-221 and miR-222 was shown to increase expression levels of p27. Thus, as a functional readout for antagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27 after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) into PC3 cells.
Results: As shown in FIG. 25, transfection of the 7-mer LNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting the seed sequence of miR-221 and miR-222 resulted in dose-dependent increase of the p27 protein levels compared to either untransfected or our LNA scrambled control transfected PC3 cells. These results clearly demonstrate that the 7-mer LNA-antimiR is able to effectively antagonize the miR-221/222 family leading to de-repression of the direct target p27 at the protein level at concentrations as low as 5 nM.
Conclusion: A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence in the miR-221/222 family at 5 nM can effectively antagonize both miRNAs leading to de-repression of the direct target p27 at protein level.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with LNA-oligonucleotides at varying concentrations (see FIG. 25) with Lipofectamine2000. Cells were harvested after 24 hours for protein extraction and western blot analysis.
Western blot: Cells were washed with PBS, trypsinated, transferred to eppendorf tubes and 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed on ice for 20 min, then spinned at 10.000 rpm for 10 minutes. Equal amounts (15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to manufacturers instructions. The membrane was incubated overnight at 4° C. with the primary monoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution. As loading control, Tubulin (Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).
Example 27
Knock-Down of miR-221/222 by the 7-mer Compound 3225 (SEQ ID NO: 23) LNA-AntimiR Reduces Colony Formation of PC3 Cells
A hallmark of cellular transformation is the ability for tumour cells to grow in an anchorage-independent way in semisolid medium. We have therefore performed soft agar assay which is a phenotypic assay that is relevant for cancer, given that it measures the decrease of tumour cells. We transfected Compound 3225 (SEQ ID NO: 23) (perfect match) and Compound 3231 (SEQ ID NO: 28) (scrambled) into PC3 cells, and after 24 hours plated cells in soft agar. Colonies were counted after 12 days. We show in FIG. 26 that inhibition of miR-221 and miR-222 by Compound 3225 (SEQ ID NO: 23) can reduce the amount of colonies growing in soft agar compared to the scrambled control LNA-antimiR, indicating decrease of tumour cells.
Conclusion: The 7-mer (Compound 3225, SEQ ID NO: 23) targeting the miR-221/222 family reduces the number of colonies in soft agar, indicating proliferation arrest of PC3 cells.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 25 nM of different LNA oligonucleotides with Lipofectamine2000.
Clonogenic growth in soft agar: 2.5×103 PC3 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO2 in a humified incubator for 12 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.
Example 28
Assessment of let-7 Antagonism by 6-9-mer LNA-AntimiRs in Huh-7 Cells Transfected with let-7a Precursor miRNA, and a Luciferase Sensor Assay
In order to assess the efficiency of fully LNA-modified 6-9-mer oligonucleotides in targeting and antagonizing the let-7 family of miRNAs, a luciferase sensor construct was made, containing some 800 bp of the HMGA2 3′UTR. The sequence cloned into the vector contains four out of seven functional let-7 binding sites (sites 2-5), as previously demonstrated by Mayr et al. (Science, 2007) and Lee and Dutta (Genes Dev, 2007). In order to monitor let-7 inhibition, the hepatocellular carcinoma cell line Huh-7 (with low to non-existing levels of endogenous let-7) was transfected with the luciferase sensor construct, with let-7a precursor miRNA, and with the 6-9 mer let-7 antagonists Compound 3232 (SEQ ID NO: 29), Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32); see FIG. 27) at increasing concentrations. The 6-9-mer LNA-antimiRs were compared with Compound 3226 (SEQ ID NO: 33), a 15-mer against let-7a as a positive control. After 24 hours, luciferase activity was measured.
Results: As seen in FIG. 28, the fully LNA-modified 8- and 9-mer LNA-antimiRs (Compound 3227 (SEQ ID NO: 34), Compound 3234 (SEQ ID NO: 31), and Compound 3235 (SEQ ID NO: 32)) show similar potencies in de-repressing the let-7 targets in the luciferase sensor assay, as the positive control 15-mer Compound 3226 (SEQ ID NO: 24). Full target de-repression for these highly potent compounds is achieved already at 1-5 nM, whereas the 7-mer Compound 3233 (SEQ ID NO: 30) needs to be present at slightly higher concentrations (10 nM) to generate the same effect. However, the 6-mer Compound 3232 (SEQ ID NO: 29) shows no effect even at as high concentrations as 50 nM. The de-repression of luciferase activity by the 7-9- and the 15-mer LNA-antimiRs is dose-dependent, which is particularly clear in the case of the slightly less potent Compound 3233 (SEQ ID NO: 30).
Conclusion: To conclude, the 8-9-mer LNA-antimiRs (Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), and Compound 3235 (SEQ ID NO: 32)) show equal antagonist potencies in inhibition of let-7a in vitro compared to the 15-mer LNA-antimiR Compound 3226 (SEQ ID NO: 24) targeting let-7a. A potent effect, albeit at slightly higher concentrations is also seen for the 7-mer Compound 3233 (SEQ ID NO: 30), whereas a 6-mer has no effect at tested concentrations.
Materials and Methods:
Cell line: The hepatocellular carcinoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 60-80% confluency the next day. On the day of transfection, Huh-7 cells in each well were transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid, let-7a precursor miRNA (Dharmacon; 10 nM end-concentration), LNA-antimiRs Compound 3232 (SEQ ID NO: 29), Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ ID NO: 25), Compound 3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32), Compound 3226 (SEQ ID NO: 24); 0-50 nM end concentrations) together with 0.17 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 29
Assessment of Entire let-7 Family Antagonism by 8-, and 15-mer LNA-AntimiRs in Huh-7 Cells Transfected with a Luciferase Sensor Assay
In order to assess the efficiency of a fully LNA-modified 8-mer oligonucleotide in antagonizing the entire let-7 family of miRNAs, the same luciferase sensor construct as described in the previous example was used. Again, Huh-7 cells (with low to non-existing levels of endogenous let-7) were transfected with the sensor construct, with one of the family-representative let-7a, let-7d, let-7e, or let-71 precursors, and with the antagonist Compound 3227 (SEQ ID NO: 25) at increasing concentrations. The 8-mer LNA-antimiR was compared to Compound 3226 (SEQ ID NO: 24), a 15-mer against let-7a as a positive and potent control. After 24 hours, luciferase activity was measured.
Results: As seen in FIG. 29 the fully LNA-modified 8-mer LNA-antimiRs (Compound 3227) (SEQ ID NO: 25) show similar potencies in de-repressing the various let-7 targets in the luciferase sensor assay, as the positive control 15-mer Compound 3226 (SEQ ID NO: 24). Nearly full target de-repression for the 8-mer is achieved already at 0.5-1 nM, except in the case with let-7e premiR (FIG. 29C), to which only 7 out of 8 nucleotides of Compound 3227 (SEQ ID NO: 25) hybridizes to the target. However, despite the terminal mismatch in this case, Compound 3227 (SEQ ID NO: 25) generates full target de-repression at 5 nM. The positive control 15-mer shows potent antagonism of all precursors and gives nearly full de-repression at 0.5 nM. The de-repression of luciferase activity by both the 8- and the 15-mer LNA-antimiRs is clearly dose-dependent, as seen in all four panels (FIGS. 29A-D).
Conclusion: To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is a potent antagonist against four representative let-7 family members in vitro, and thus likely against the entire family. Compared to a 15-mer positive control antagonist, Compound 3226 (SEQ ID NO: 24), the 8-mer is equally potent for three of four targets, and slightly less potent for the fourth target, let-7e, explained by a terminal mismatch in this case.
Materials and Methods:
Cell line: The hepatocellular carcinoma cell line Huh-7 was a kind gift from R. Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 60-80% confluency the next day. On the day of transfection, Huh-7 cells in each well were transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid, with let-7a, -7d, -7e, or -7i precursor miRNA (Dharmacon; 10 nM end-concentration), and with LNA-antimiRs Compound 3227 (SEQ ID NO: 25) and Compound 3226 (SEQ ID NO: 24); 0-50 nM end concentrations) together with 0.17 μl Lipofectamine-2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: Growth medium was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 30
Assessment of Endogenous Let-7 Antagonism by Compound 3227 (SEQ ID NO: 25), an 8-mer LNA-AntimiRs, in HeLa Cells Transfected with a Luciferase Sensor Assay.
In order to determine the efficiency of a fully LNA-modified 8-mer oligonucleotide in targeting and antagonizing endogenous let-7, the same luciferase sensor construct as described in previous two examples, was co-transfected with Compound 3227 (SEQ ID NO: 25) into the cervical cancer cell line HeLa (that expresses moderate to high levels of let-7 as determined by Q-PCR; data not shown). Empty psiCHECK-2 vector was included as a negative control.
Results: As seen in FIG. 30, the fully LNA-modified 8-mer LNA-antimiR Compound 3227 (SEQ ID NO: 25) shows potent antagonism of endogenous let-7, and gives full target de-repression at concentrations of 5-10 nM. The de-repression of luciferase activity is dose-dependent, starting around 1 nM and reaching a plateau at approximately 10 nM.
Conclusion: To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is a potent antagonist against also endogenous let-7 in vitro, and thus provides definite evidence that entire miRNA families can be successfully targeted by short and fully LNA-modified antagonists.
Materials and Methods:
Cell line: The cervical cancer cell line HeLa was purchased from ATCC (#CCL-2TM). HeLa cells were cultured in Eagle's MEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.
Transfection: 8,000 cells were seeded per well in a 96-well plate the day before transfection in order to receive 50-70% confluency the next day. On the day of transfection, HeLa cells in each well were co-transfected with 20 ng HMGA2 3′UTR/psiCHECK2 plasmid or psiCHECK-2 (empty vector), and with LNA-antimiR Compound 3227 (SEQ ID NO: 25) (0-50 nM, end concentrations) together with 0.17 μl Lipofectamine2000 (Invitrogen) according to manufacturer's instructions. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega) was added to each well. After 15-30 minutes of incubation on an orbital shaker, renilla and firefly luciferase measurements were performed according to manufacturer's instructions (Promega).
Example 31
Assessment of miR-21 Antagonism by an 8-mer LNA-AntimiR-21 (Compound 3205, SEQ ID NO: 2) Versus an 8-mer (Compound 3219, SEQ ID NO: 17) Scrambled Control LNA in the Human Colon Carcinoma Cell Line HCT116
We have previously shown in this application, that an 8-mer LNA-antimiR that is fully LNA-modified and phosphorothiolated effectively antagonizes miR-21 in the human cervix carcinoma cell line HeLa, the human breast carcinoma cell line MCF-7, the human prostate cancer cell line PC3 and human hepatocellular carcinoma HepG2 cell line. We extended this screening approach to the human colon carcinoma cell line HCT116. To assess the efficiency of the 8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporter constructs were generated in which a perfect match target site for the mature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene. In order to monitor miR-21 inhibition, HCT116 cells were transfected with the luciferase constructs together with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer) and for comparison of specificity with the 8-mer LNA scrambled control (Compound 3219, SEQ ID NO: 17). After 24 hours, luciferase activity was measured.
Results: The luciferase reporter experiments showed a dose-dependent de-repression of the luciferase miR-21 reporter activity with the 8-mer LNA-antimiR against miR-21 (Compound 3205, SEQ ID NO: 2) and complete de-repression was obtained at 5 nM (FIG. 31). When comparing the specificity of the 8-mer perfect match and the 8-mer scrambled control, the scrambled control LNA-antimiR (Compound 3219, SEQ ID NO: 17) did not show any de-repression at all, demonstrating high specificity of the LNA-antimiR compound against miR-21.
Conclusion: The 8-mer (Compound 3205, SEQ ID NO: 2) is potent in targeting miR-21 and antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) is specific.
Materials and Methods:
Cell line: The human colon carcinoma HCT116 cell line was purchased from ATCC(CCL-247). HCT116 cells were cultured in RPMI medium, supplemented with 10% fetal bovine serum, and 25 ug/ml Gentamicin.
Transfection: 110.000 cells were seeded per well in a 12-well plate and transfection was performed. HCT116 cells were transfected with 0.3 μg miR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with 1.2 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected were also varying concentrations of LNA-antimiR and control oligonucleotides. After 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer (Promega) was added to the wells. The plates were placed on a shaker for 30 min., after which the cell lysates were transferred to eppendorf tubes. The cell lysate was centrifugated for 10 min at 2.500 rpm after which 50 μl were transferred to a 96 well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 32
Knock-Down of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-AntimiR Reduces Colony Formation of PC3 Cells
A hallmark of cellular transformation is the ability for tumour cells to grow in an anchorage-independent way in semisolid medium. We therefore performed soft agar assay which is a phenotypic assay that is relevant for cancer, given that it measures the decrease of tumour cells. We transfected Compound 3205 (SEQ ID NO: 2) (perfect match LNA-antimiR-21) and Compound 3219 (SEQ ID NO: 17) (LNA scrambled control) into PC3 cells, and after 24 hours plated cells in soft agar. Colonies were counted after 12 days. We show in FIG. 32 that inhibition of miR-21 by Compound 3205 (SEQ ID NO: 2) can reduce the amount of colonies growing in soft agar compared to the scrambled control LNA treated or untreated control (transfected, but with no LNA), demonstrating decrease of tumour cells.
Conclusion: The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 family reduces the number of colonies in soft agar, demonstrating proliferation arrest of PC3 cells.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 250.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, PC3 cells were transfected with 25 nM of different LNA oligonucleotides with Lipofectamine2000.
Clonogenic growth in soft agar: 2.5×103 PC3 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO2 in a humified incubator for 12 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.
Example 33
Silencing of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-AntimiR Reduces Colony Formation of HepG2 Cells
miR-21 is overexpressed in the human hepatocellular carcinoma cell line HepG2 and we have previously shown that we are able to regulate the luciferase activity of a miR-21 sensor plasmid with Compound 3205 (SEQ ID NO: 2) in these cells. HepG2 cells were transfected with Compound 3205 (SEQ ID NO: 2) and Compound 3219 (SEQ ID NO: 17) (scrambled 8-mer), and after 24 hours plated into soft agar. Colonies were counted after 17 days with a microscope.
Results: We show in FIG. 33 that inhibition of miR-21 by Compound 3205 (SEQ ID NO: 2) can reduce the amount of colonies growing in soft agar, showing that proliferation arrest has occurred. In addition, our scrambled 8-mer control, Compound 3219 (SEQ ID NO: 17), had no significant effect on the number of colonies.
Conclusion: The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 reduces the number of colonies in soft agar, indicating proliferation arrest of HepG2 cells.
Materials and Methods:
Cell line: The human hepatocytic HepG2 cell line was purchased from ECACC (#85011430). HepG2 cells were cultured in EMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 650.000 cells were seeded per well in a 6-well plate and reverse transfection was performed. HepG2 cells were transfected with 0.6 mg miR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with 2.55 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected were also LNA-antimiR and control oligonucleotides as varying concentrations. After 24 hours, the cells were harvested for luciferase measurements.
Clonogenic growth in soft agar: 2.0×103 HepG2 cells were seeded in 0.35% agar on the top of a base layer containing 0.5% agar. Cells were plated 24 hours after transfection. Plates were incubated in at 37° C., 5% CO2 in a humified incubator for 17 days and stained with 0.005% crystal violet for 1 h, after which cells were counted. The assay was performed in triplicate.
Example 34
Silencing of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2) LNA-AntimiR Inhibits Cell Migration in PC3 Cells
Cell migration can be monitored by performing a wound healing assay (=scratch assay) where a “scratch” is made in a cell monolayer, and images are captured at the beginning and at regular intervals during cell migration. By comparing the images, quantification of the migration rate of the cells can be determined. This was done in the human prostate cancer cell line PC3. Cells were seeded, and on day 3 the cells were transfected, and the next day, when 100% confluency was reached, a scratch (=wound) was made. When the scratch was made, pictures were taken in order to document the initial wound. Afterwards the area of the wound closure is measured at different time points with the free software program Image J. As shown in FIG. 34A, PC3 cells had been treated with 25 nM Compound 3205 (SEQ ID NO: 2) (perfect match, miR-21), the control Compound 3219 (SEQ ID NO: 17) or left untransfected. Pictures were taken after 24 hours, and the area was calculated for the wound closure at respective time-point. The wound closure for the untransfected cells and for the control, Compound 3219 (SEQ ID NO: 17), was faster as compared to our LNA-antimiR against miR-21, Compound 3205 (SEQ ID NO: 2), indicating that Compound 3205 (SEQ ID NO: 2) inhibits miR-21 and prevents the cells from migrating (FIG. 34B).
Conclusion: The 8-mer (Compound 3205, SEQ ID NO: 2) targeting miR-21 inhibits the cell migration of PC3 cells compared to untransfected and control transfected cells.
Materials and Methods:
Cell line: The human prostate carcinoma PC3 cell line was purchased from ECACC (#90112714). PC3 cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.
Scratch assay: 150.000 cells were seeded per well in a 6-well plate three days before transfection in order to receive 100% confluency the next day. At 24 hours after transfection, a scratch was made in the cell monolayer with a 200 μl tip. Pictures were taken at 0 h and after 24 hours by using a digital camera coupled to a microscope. The software program Image J was used to determine wound closure.
Example 35
Length Assessment of Fully LNA-Substituted LNA-AntimiRs Antagonizing miR-155
We have previously shown a length assessment for miR-21 regarding fully LNA-substituted LNA-antimiRs, and showed that the most potent LNA-antimiRs are 7-, 8- or 9 nt in length. The same experiment was repeated with miR-155. A perfect match target site for miR-155 was cloned into the 3′UTR of the luciferase gene in the reporter plasmid psiCHECK2 and transfected into the mouse RAW macrophage cell line together with fully LNA-substituted LNA-antimiRs of different lengths. Because the endogenous levels of miR-155 are low in the RAW cell line, the cells were treated with 100 ng/ml LPS for 24 hours in order to induce miR-155 accumulation. After 24 hours, luciferase analysis was performed.
Results: As shown in FIG. 35, the most potent LNA-antimiRs are Compound 3207 (SEQ ID NO: 4) (8 nt) and Compound 3241 (SEQ ID NO: 38) (9 nt), reaching almost a 80% de-repression at only 0.25 nM LNA concentration. The 6-mer (Compound 3244, SEQ ID NO: 978) shows no significant de-repression. Increasing the length to 12-mer to 14-mer (Compound 3242 (SEQ ID NO: 39) and Compound 3243 (SEQ ID NO: 977)) decreased the potency as shown by less efficient de-repression of the miR-155 reporter.
Conclusion: The most potent fully LNA-substituted LNA-antimiRs targeting miR-155 were an 8- and 9-mer (Compound 3207 (SEQ ID NO: 4) and Compound 3241 (SEQ ID NO: 38)).
Materials and Methods:
Cell line: The mouse macrophage RAW 264.7 cell line was purchased from ATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.
Transfection: 500.000 cells were seeded per well in a 6-well plate the day before transfection in order to receive 50% confluency the next day. On the day of transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155 perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μl Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected was also varying concentrations of LNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cells after the 4 hour incubation with the transfection complexes. After another 24 hours, cells were harvested for luciferase measurements.
Luciferase assay: The cells were washed with PBS and harvested with cell scraper, after which cells were spinned for 5 min at 2.500 rpm. The supernatant was discarded and 50 ml 1× Passive Lysis Buffer (Promega) was added to the cell pellet, after which cells were put on ice for 30 min. The lysed cells were spinned at 10.000 rpm for 30 min after which 20 μl were transferred to a 96-well plate and luciferase measurements were performed according to the manufacturer's instructions (Promega).
Example 36
Plasma Protein Binding for the Fully LNA-Substituted 8-Mer Compound 3205 (SEQ ID NO: 2) Targeting miR-21 (LNA-AntimiR-21).
The plasma proteins are not saturated with Compound 3205 (SEQ ID NO:2) at the plasma concentrations in the experiment shown in FIG. 36A. In a wide range of Compound 3205 (SEQ ID NO: 2) concentrations in the plasma the protein binding is around 95% of the Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21 in FIG. 36B. At Compound 3205 (SEQ ID NO: 2) concentrations 50.1 μM (174 μg/mL) the binding capacity of plasma proteins for FAM-labeled Compound 3205 (SEQ ID NO: 2) has not been saturated.
Materials and Methods: Mouse plasma (100 μL) was spiked with FAM-labeled Compound 3205 (SEQ ID NO: 2) to 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM concentrations. The solutions were incubated at 37° C. for 30 minutes. The solutions were transferred to a Microcon Ultracel YM-30 filter (regenerated cellulose 30.000 MWCO). The filters were spun for 20 minutes at 2000 g and at room temperature in a microcentrifuge. The filtrate was diluted 5, 10 and 20 times and 100 μL samples were transferred to a microtiter plate (Polystyrene Black NUNC-237108). The fluorescence was detected using a FLUOstar Optima elisa reader with excitation 458 nm and emission 520 nm. The amount of unbound FAM-labeled Compound 3205 (SEQ ID NO: 2) was calculated from a standard curve derived from filtrated plasma spiked with FAM-labeled Compound 3205 (SEQ ID NO: 2) at 12 different (0.45-1000 nM) concentrations. The numbers were corrected with the recovery number established from filtration experiments with Compound 3205 (SEQ ID NO: 2) concentrations 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM in filtrated plasma. The recovery of FAM-labeled Compound 3205 (SEQ ID NO: 2) was 86%.
Example 37
Quantitative Whole Body Autoradiography Study in Female Pigmented Mice After Single Intravenous Administration of 35S-Labelled Compound 3205 (SEQ ID NO: 2) LNA-AntimiR-21
In order to determine the biodistribution of a short fully LNA-modified LNA-antimiR (Compound 3205 (SEQ ID NO: 2), 8-mer) a whole body tissue distribution of radioactively labeled compound was done in mice. 35S-labelled Compound 3205 (SEQ ID NO: 2) was dosed to mice with a single intravenous administration and mice were sacrificed at different time-points, ranging from 5 min to 21 days.







TABLE 6(i)







Individual tissue concentrations


(μg Compound 3205/g tissue) after a single intravenous administration


of 35S-labelled Compound 3205 (SEQ ID NO: 2) in


female pigmented mice.











Max. Conc.





of oligo μg





Compound 3205/g
Time of max



Tissue
tissue
conc. hours
T1⁄2 hours













Adrenal gl.
13.6
0.083
374


Bile
4
1



Bone marrow
7.2
0.083
411


Brain
0.4
0.083



Brown fat
8.8
0.083



Gastric muc.
10.1
0.083



Heart blood
26.2
0.083
10.3


Kidney ctx.
58.7
24
104


Liver
11.8
0.083
588



10.7
24



Lung
13.2
0.083
289


Lymph node
5
0.083
262



2.4
48



Lymph
18.8
4




20.8
168



Myocardium
8.1
0.083
662


Ovary
13
0.083
198


Pancreas
5
0.083



Pituitary gl.
6.7
0.083



Salivary gl.
8.6
0.083
405



5.5
168



skel. Muscle
4.8
0.083



Skin pig.
5.4
0.25



Spleen
9.8
0.083
564


Thymus
3.8
0.083
185


Thyroid gl.
10.9
0.083
592


Urine
328.9
0.083



Uterus
9.6
0.25
177


Uvea of the eye
13.6
0.083



LOQ
0.045
0.083




0.033
24




0.03
168





The figures are mean values of three measurements for each tissue and ratio. The coefficient of variation (CV) is generally about 10%.













TABLE 6(ii)







Tissue to liver ratios after single intravenous administration of


35S-labelled Compound 3205 (SEQ ID NO: 2) in female pigmented mice.


35S-Compound 3205 (SEQ ID NO: 2)
















Animal no
10
11
12
13
14
15
16
17
18



















Surv. Time (h)
0.083
0.25
1 h
4 h
24 h
48 h
96 h
168
504


Organ











Adrenal gl
liver
liver
liver
liver
liver
liver
liver
liver
liver


Bile
1.15
1.08
0.52
0.27
0.24
0.26
0.23
0.18
0.17


Bone marrow
0.03
0.11
0.55
0.10
0.03
0.07
0.04
0.03
0.04


Brain
0.61
0.81
0.55
0.45
0.40
0.48
0.43
0.42
0.34


Brown fat
0.03
0.03
0.01
0.00
0.00
0.00
0.00
0.00
0.00


Gastric muc
0.75
0.57
0.29
0.12
0.07
0.12
0.08
0.10
0.07


Heart blood
0.86
0.71
0.31
0.22
0.10
0.21
0.15
0.16
0.12


Kidney ctx
2.23
1.91
0.74
0.11
0.01
0.00
0.00
0.00
0.00


Liver
2.87
3.94
6.45
6.95
5.51
6.68
3.92
2.24
0.40


Lung
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00


Lymph node
1.12
0.97
0.63
0.09
0.04
0.04
0.03
0.02
0.02


Lymph
0.43
0.30
0.25
0.19
0.11
0.32
0.20
0.17
0.12


Myocardium
0.82
1.09
1.78
2.78
1.03
2.05
1.62
3.17
1.89


Ovary
0.69
0.63
0.30
0.13
0.10
0.15
0.09
0.11
0.12


Pancreas
1.10
1.40
0.61
0.31
0.27
0.28
0.21
0.21
0.08


Pituitary gland
0.42
0.37
0.22
0.18
0.12
0.17
0.12
0.15
0.11


Salivary gland
0.57
0.54
0.28
0.11
0.15
0.16
0.12
0.10
0.08


Skel. muscle
0.73
0.81
0.38
0.25
0.25
0.42
0.23
0.85
0.24


Skin. pigm.
0.40
0.28
0.14
0.04
0.02
0.04
0.03
0.03
0.03


Spleen
0.34
0.69
0.65
0.36
0.20
0.26
0.20
0.19
0.13


Thymus
0.83
0.86
0.44
0.32
0.24
0.34
0.35
0.29
0.31


Thyroid gland
0.32
0.31
0.14
0.07
0.09
0.08
0.05
0.04
0.02


Urine
0.9
1.2
0.43
0.28
0.25
0.34
0.19
0.26
0.25


Uterus
27.96
39.48
9.90
5.44
0.24
0.39
0.12
0.15
0.03


Uvea of the eye
0.56
1.23
0.65
0.30
0.30
0.07
0.27
0.16
0.08









Conclusions: Compound 3205 (SEQ ID NO: 2) shows blood clearance of radioactivity with elimination half-lives of 8-10 hours. High levels of radioactivity were registered in the kidney cortex, lymph, liver, bone marrow, spleen, ovary and uterus. The highest level of radioactivity was registered in the kidney cortex showing five times higher levels than that of the liver for Compound 3205 (SEQ ID NO: 2). A strong retention of radioactivity was noticed in the kidney cortex, lymph, liver, bone marrow and spleen for Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21.
Materials and Methods:
Dose administration: All mice were weighed before administration. Nine female mice were given 10 mg/kg of 35S-Compound 3205 (SEQ ID NO: 2) intravenously in a tail vein. The volume given to each animal was 10 mL/kg of the test formulation. The specific activity 75.7 μCi/mg. Individual mice were killed 5 min, 15 min, 1 hour, 4 hours, 24 hours, 2 days, 4 days, 7 days and 21 days after administration of Compound 3205 (SEQ ID NO: 2). Whole body autoradiography: The mice were anaesthetized by sevoflurane, and then immediately immersed in heptane, cooled with dry ice to −80° C., ABR-SOP-0130. The frozen carcasses were embedded in a gel of aqueous carboxymethyl cellulose (CMC), frozen in ethanol, cooled with dry ice (−80° C.) and sectioned sagittaly for whole body autoradiography, according to the standard method, ABR-SOP-0131. From each animal 20 μm sections were cut at different levels with a cryomicrotome (Leica CM 3600) at a temperature of about −20° C. The obtained sections were caught on tape (Minnesota Mining and Manufacturing Co., No. 810) and numbered consecutively with radioactive ink. After being freeze-dried at −20° C. for about 24 hours, selected sections were covered with a thin layer of mylar foil, and put on imaging plates (Fuji, Japan). Exposure took place in light tight cassettes in a lead shielding box at −20° C., to protect the image plates from environmental radiation. After exposure the imaging plates were scanned at a pixel size of 50 μm and analyzed by radioluminography using a bioimaging analysis system (Bas 2500, Fuji, Japan), and described in ABR-SOP-0214. A water-soluble standard test solution of 35S radioactivity was mixed with whole blood and used for production of a calibration scale, ABR-SOP-0251. However, the different blood standards were dissolved in 500 uL Soluene-35. 4.5 mL Ultima Gold was then added to the dissolved samples. As 35S and 14C have very similar energy spectra, a standard 14C-programme (Packard 2200CA) was used when the radioactivity for the different blood samples was settled.
Pharmacokinetic calculations: The 35S radioactivity measured in whole blood and tissues was expressed as nCi/g tissue and recalculated to nmol equiv/g tissue for the pharmacokinetic evaluation. The pharmacokinetic parameters Cmax, t1/2 and AUC were determined for the whole blood and tissues by non-compartmental analysis using WinNonlin Professional (Pharsight Corporation, Mountain View, Calif., USA). After intravenous administration, the concentration was extrapolated back to zero and expressed as (C0). The elimination rate constant λ was estimated by linear regression analysis of the terminal slope of the logarithmic plasma concentration-time curve. The elimination half-life, t1/2, was calculated using the equation, t1/2=ln 2/λ. The last three time-points above LOQ were used in the elimination half-life calculations, if not stated otherwise.
Example 38
Assessment of let-7 Inhibition In Vivo by an 8-mer LNA-AntimiR, as Determined Through Ras Protein Quantification in Mouse Lung and Kidney
In order to investigate the possibility to antagonize the abundantly expressed let-7 family in vivo, mice were intravenously (i.v.) injected with an 8-mer LNA-antimiR antagonist or with saline. To measure treatment effect, proteins were isolated from lungs and kidneys. Because the Ras family of proteins (N-Ras, K-Ras, and H-Ras), in particular N-Ras and K-Ras, has previously been shown to be regulated (repressed) by the let-7 family by Johnson et al. (Cell, 2005), the aim was to analyze whether these let-7 targets could be de-repressed in vivo.
Results: As seen in FIG. 37, the 8-mer LNA-antimiR potently de-repressed Ras protein levels in the kidneys of treated mice, normalized against saline controls. The up-regulation in this organ was more than 3-fold, showing a clear in vivo effect. In the lungs, however, only a minimal (1.2-fold) Ras de-repression was observed (FIG. 1B), suggesting that insufficient amounts of LNA-antimiR has entered this organ in order to inhibit its massive amounts of let-7, as previously described by Johnson et al. (Cancer Research, 2007).
Conclusion: The 8-mer LNA-antimiR shows a clear effect in regulating target let-7 miRNA in vivo, as evaluated based on Ras protein levels in treated vs. control mice. Whereas the effect seems to be smaller in lungs, Ras levels in the kidney show a substantial up-regulation upon antimiRs-treatment.
Materials and Methods: Animals and dosing: C57BL/6 female mice were treated with 10 mg/kg LNA-antimiR or saline for three consecutive days (0, 1, and 2) and sacrificed on day 4. Tissue samples from lungs and kidneys were snapfrozen and stored at −80° C. until further processing.
Western blot analysis: Lung and kidney proteins from saline and LNA-antimiR-treated mice were separated on NuPAGE Bis Tris 4-12% (Invitrogen), using 100 μg per sample. The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to the manufacturer's instructions. Blocking, antibody dilution and detection was performed according to the manufacturer's specifications. For Ras detection, a primary rabbit-anti Ras antibody (SC-3339, Santa Cruz Biotechnology) and a secondary HRP-conjugated swine-anti-rabbit antibody (P0399, Dako) was used, and for tubulin detection, a primary tubulin alpha (MS-581-P1, Neomarkers) and a secondary HRP-conjugated goat-anti-mouse antibody (P0447, Dako) was used.
Example 40
In Vivo Efficacy Assessment of the 8-mer LNA-AntimiR (Compound 3205, SEQ ID NO: 2) in Targeting miR-21, as Determined by Pdcd4 Protein Up-Regulation in Mouse Kidney
We have shown that an 8-mer LNA-antimiR that is fully LNA-modified antagonizes miR-21 and has the ability to regulate the protein levels of the miR-21 target Pdcd4 in vitro. We therefore injected the LNA-antimiR into mice to determine the effects of the LNA-antimiR in vivo. The mice received 25 mg/kg of Compound 3205 (SEQ ID NO: 2) by i.v. injection every other day for 14 days (a total of 5 doses). The mice were sacrificed on day 14, the kidney was removed, and protein was isolated. In order to determine target regulation, Western blot analysis was performed.
Results: As shown in FIG. 38, treating mice with Compound 3205 (SEQ ID NO: 2) showed significantly increased Pdcd4 protein levels as compared to the saline control. While the normalized Pdcd4 versus Gapdh ratio was consistent in both saline samples, the protein up-regulation in the two LNA-antimiR-treated with Compound 3205 (SEQ ID NO: 2) mice were measured to 3.3- and 6.3-fold, respectively, demonstrating an in vivo pharmacological effect of the Compound 3205 (SEQ ID NO: 2) 8-mer LNA-antimiR.
Conclusion: The fully LNA-modified 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) antagonizes miR-21 in vivo, as demonstrated through its ability to de-repress (up-regulate) mouse kidney levels of Pdcd4, a validated miR-21 target.
Materials and Methods:
Animals and dosing: C57BL/6 female mice with average of 20 g body weight at first dosing were used in all experiments and received regular chow diet (Altromin no 1324, Brogaarden, Gentofte, Denmark). Substances were formulated in physiological saline (0.9% NaCl). The animals were dozed with LNA-antimiR or saline (0.9% NaCl), receiving an injection of 25 mg/kg every other day for 14 days, a total of 5 doses. Animals were sacrificed on day 14.
Western blot analysis: 80 μg kidney tissue from saline or LNA-treated mice was separated on NuPAGE Bis Tris 4-12% (Invitrogen). The proteins were transferred to a nitrocellulose membrane using iBlot (Invitrogen) according to the manufacturer's instructions. The membrane was incubated with Pdcd4 antibody (Bethyl Laboratories), followed by HRP-conjugated swine-anti-rabbit antibody (Dako). As equal loading control, GAPDH (Abcam) was used, followed by HRP-conjugated swine-anti-mouse antibody. The membranes were visualized by chemiluminiscence (ECL, Amersham).




















SEQ










ID
9-mer

8-mer

7-mer



microRNA
MicroRNASequence
NO
Compound

Compound

Compound























ebv-miR-BART1-3p
UAGCACCGCUAUCCACUAUGUC
 40
AGCGGTGCT

GCGGTGCT

CGGTGCT



 


ebv-miR-BART1-5p
UCUUAGUGGAAGUGACGUGCUGUG
 41
TCCACTAAG

CCACTAAG

CACTAAG



 


ebv-miR-BART10
UACAUAACCAUGGAGUUGGCUGU
 42
TGGTTATGT

GGTTATGT

GTTATGT



 


ebv-miR-BART10*
GCCACCUCUUUGGUUCUGUACA
 43
AAGAGGTGG

AGAGGTGG

GAGGTGG



 


ebv-miR-BART11-3p
ACGCACACCAGGCUGACUGCC
 44
TGGTGTGCG

GGTGTGCG

GTGTGCG



 


ebv-miR-BART11-5p
UCAGACAGUUUGGUGCGCUAGUUG
 45
AACTGTCTG

ACTGTCTG

CTGTCTG



 


ebv-miR-BART12
UCCUGUGGUGUUUGGUGUGGUU
 46
CACCACAGG

ACCACAGG

CCACAGG



 


ebv-miR-BART13
UGUAACUUGCCAGGGACGGCUGA
 47
GCAAGTTAC

CAAGTTAC

AAGTTAC



 


ebv-miR-BART13*
AACCGGCUCGUGGCUCGUACAG
 48
CGAGCCGGT

GAGCCGGT

AGCCGGT



 


ebv-miR-BART14
UAAAUGCUGCAGUAGUAGGGAU
 49
GCAGCATTT

CAGCATTT

AGCATTT



 


ebv-miR-BART14*
UACCCUACGCUGCCGAUUUACA
 50
GCGTAGGGT

CGTAGGGT

GTAGGGT



 


ebv-miR-BART15
GUCAGUGGUUUUGUUUCCUUGA
 51
AACCACTGA

ACCACTGA

CCACTGA



 


ebv-miR-BART16
UUAGAUAGAGUGGGUGUGUGCUCU
 52
CTCTATCTA

TCTATCTA

CTATCTA



 


ebv-miR-BART17-3p
UGUAUGCCUGGUGUCCCCUUAGU
 53
CAGGCATAC

AGGCATAC

GGCATAC



 


ebv-miR-BART17-5p
UAAGAGGACGCAGGCAUACAAG
 54
CGTCCTCTT

GTCCTCTT

TCCTCTT



 


ebv-miR-BART18-3p
UAUCGGAAGUUUGGGCUUCGUC
 55
ACTTCCGAT

CTTCCGAT

TTCCGAT



 


ebv-miR-BART18-5p
UCAAGUUCGCACUUCCUAUACA
 56
GCGAACTTG

CGAACTTG

GAACTTG



 


ebv-miR-BART19-3p
UUUUGUUUGCUUGGGAAUGCU
 57
GCAAACAAA

CAAACAAA

AAACAAA



 


ebv-miR-BART19-5p
ACAUUCCCCGCAAACAUGACAUG
 58
CGGGGAATG

GGGGAATG

GGGAATG



 


ebv-miR-BART2-3p
AAGGAGCGAUUUGGAGAAAAUAAA
 59
ATCGCTCCT

TCGCTCCT

CGCTCCT



 


ebv-miR-BART2-5p
UAUUUUCUGCAUUCGCCCUUGC
 60
GCAGAAAAT

CAGAAAAT

AGAAAAT



 


ebv-miR-BART20-3p
CAUGAAGGCACAGCCUGUUACC
 61
TGCCTTCAT

GCCTTCAT

CCTTCAT



 


ebv-miR-BART20-5p
UAGCAGGCAUGUCUUCAUUCC
 62
ATGCCTGCT

TGCCTGCT

GCCTGCT



 


ebv-miR-BART3
CGCACCACUAGUCACCAGGUGU
 63
TAGTGGTGC

AGTGGTGC

GTGGTGC



 


ebv-miR-BART3*
ACCUAGUGUUAGUGUUGUGCU
 64
AACACTAGG

ACACTAGG

CACTAGG



 


ebv-miR-BART4
GACCUGAUGCUGCUGGUGUGCU
 65
GCATCAGGT

CATCAGGT

ATCAGGT



 


ebv-miR-BART5
CAAGGUGAAUAUAGCUGCCCAUCG
 66
ATTCACCTT

TTCACCTT

TCACCTT



 


ebv-miR-BART6-3p
CGGGGAUCGGACUAGCCUUAGA
 67
CCGATCCCC

CGATCCCC

GATCCCC



 


ebv-miR-BART6-5p
UAAGGUUGGUCCAAUCCAUAGG
 68
ACCAACCTT

CCAACCTT

CAACCTT



 


ebv-miR-BART7
CAUCAUAGUCCAGUGUCCAGGG
 69
GACTATGAT

ACTATGAT

CTATGAT



 


ebv-miR-BART7*
CCUGGACCUUGACUAUGAAACA
 70
AAGGTCCAG

AGGTCCAG

GGTCCAG



 


ebv-miR-BART8
UACGGUUUCCUAGAUUGUACAG
 71
GGAAACCGT

GAAACCGT

AAACCGT



 


ebv-miR-BART8*
GUCACAAUCUAUGGGGUCGUAGA
 72
AGATTGTGA

GATTGTGA

ATTGTGA



 


ebv-miR-BART9
UAACACUUCAUGGGUCCCGUAGU
 73
TGAAGTGTT

GAAGTGTT

AAGTGTT



 


ebv-miR-BART9*
UACUGGACCCUGAAUUGGAAAC
 74
GGGTCCAGT

GGTCCAGT

GTCCAGT



 


ebv-miR-BHRF1-1
UAACCUGAUCAGCCCCGGAGUU
 75
GATCAGGTT

ATCAGGTT

TCAGGTT



 


ebv-miR-BHRF1-2
UAUCUUUUGCGGCAGAAAUUGA
 76
GCAAAAGAT

CAAAAGAT

AAAAGAT



 


ebv-miR-BHRF1-2*
AAAUUCUGUUGCAGCAGAUAGC
 77
AACAGAATT

ACAGAATT

CAGAATT



 


ebv-miR-BHRF1-3
UAACGGGAAGUGUGUAAGCACA
 78
CTTCCCGTT

TTCCCGTT

TCCCGTT



 


hcmv-miR-UL112
AAGUGACGGUGAGAUCCAGGCU
 79
ACCGTCACT

CCGTCACT

CGTCACT



 


hcmv-miR-UL148D
UCGUCCUCCCCUUCUUCACCG
 80
GGGAGGACG

GGAGGACG

GAGGACG



 


hcmv-miR-UL22A
UAACUAGCCUUCCCGUGAGA
 81
AGGCTAGTT

GGCTAGTT

GCTAGTT



 


hcmv-miR-UL22A*
UCACCAGAAUGCUAGUUUGUAG
 82
ATTCTGGTG

TTCTGGTG

TCTGGTG



 


hcmv-miR-UL36
UCGUUGAAGACACCUGGAAAGA
 83
TCTTCAACG

CTTCAACG

TTCAACG



 


hcmv-miR-UL36*
UUUCCAGGUGUUUUCAACGUGC
 84
CACCTGGAA

ACCTGGAA

CCTGGAA



 


hcmv-miR-UL70-3p
GGGGAUGGGCUGGCGCGCGG
 85
GCCCATCCC

CCCATCCC

CCATCCC



 


hcmv-miR-UL70-5p
UGCGUCUCGGCCUCGUCCAGA
 86
CCGAGACGC

CGAGACGC

GAGACGC



 


hcmv-miR-US25-1
AACCGCUCAGUGGCUCGGACC
 87
CTGAGCGGT

TGAGCGGT

GAGCGGT



 


hcmv-miR-US25-1*
UCCGAACGCUAGGUCGGUUCUC
 88
AGCGTTCGG

GCGTTCGG

CGTTCGG



 


hcmv-miR-US25-2-
AUCCACUUGGAGAGCUCCCGCGG
 89
CCAAGTGGA

CAAGTGGA

AAGTGGA



3p










 


hcmv-miR-US25-2-
AGCGGUCUGUUCAGGUGGAUGA
 90
ACAGACCGC

CAGACCGC

AGACCGC



5p










 


hcmv-miR-US33-3p
UCACGGUCCGAGCACAUCCA
 91
CGGACCGTG

GGACCGTG

GACCGTG



 


hcmv-miR-US33-5p
GAUUGUGCCCGGACCGUGGGCG
 92
GGGCACAAT

GGCACAAT

GCACAAT



 


hcmv-miR-US4
CGACAUGGACGUGCAGGGGGAU
 93
GTCCATGTC

TCCATGTC

CCATGTC



 


hcmv-miR-US5-1
UGACAAGCCUGACGAGAGCGU
 94
AGGCTTGTC

GGCTTGTC

GCTTGTC



 


hcmv-miR-US5-2
UUAUGAUAGGUGUGACGAUGUC
 95
CCTATCATA

CTATCATA

TATCATA



 


hsa-let-7a
UGAGGUAGUAGGUUGUAUAGUU
 96
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7a*
CUAUACAAUCUACUGUCUUUC
 97
GATTGTATA

ATTGTATA

TTGTATA



 


hsa-let-7b
UGAGGUAGUAGGUUGUGUGGUU
 98
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7b*
CUAUACAACCUACUGCCUUCCC
 99
GGTTGTATA

GTTGTATA

TTGTATA



 


hsa-let-7c
UGAGGUAGUAGGUUGUAUGGUU
100
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7c*
UAGAGUUACACCCUGGGAGUUA
101
TGTAACTCT

GTAACTCT

TAACTCT



 


hsa-let-7d
AGAGGUAGUAGGUUGCAUAGUU
102
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7d*
CUAUACGACCUGCUGCCUUUCU
103
GGTCGTATA

GTCGTATA

TCGTATA



 


hsa-let-7e
UGAGGUAGGAGGUUGUAUAGUU
104
TCCTACCTC

CCTACCTC

CTACCTC



 


hsa-let-7e*
CUAUACGGCCUCCUAGCUUUCC
105
GGCCGTATA

GCCGTATA

CCGTATA



 


hsa-let-7f
UGAGGUAGUAGAUUGUAUAGUU
106
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7f-1*
CUAUACAAUCUAUUGCCUUCCC
107
GATTGTATA

ATTGTATA

TTGTATA



 


hsa-let-7f-2*
CUAUACAGUCUACUGUCUUUCC
108
GACTGTATA

ACTGTATA

CTGTATA



 


hsa-let-7g
UGAGGUAGUAGUUUGUACAGUU
109
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7g*
CUGUACAGGCCACUGCCUUGC
110
GCCTGTACA

CCTGTACA

CTGTACA



 


hsa-let-7i
UGAGGUAGUAGUUUGUGCUGUU
111
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-let-7i*
CUGCGCAAGCUACUGCCUUGCU
112
GCTTGCGCA

CTTGCGCA

TTGCGCA



 


hsa-miR-1
UGGAAUGUAAAGAAGUAUGUAU
113
TTACATTCC

TACATTCC

ACATTCC



 


hsa-miR-100
AACCCGUAGAUCCGAACUUGUG
114
TCTACGGGT

CTACGGGT

TACGGGT



 


hsa-miR-100*
CAAGCUUGUAUCUAUAGGUAUG
115
TACAAGCTT

ACAAGCTT

CAAGCTT



 


hsa-miR-101
UACAGUACUGUGAUAACUGAA
116
CAGTACTGT

AGTACTGT

GTACTGT



 


hsa-miR-101*
CAGUUAUCACAGUGCUGAUGCU
117
GTGATAACT

TGATAACT

GATAACT



 


hsa-miR-103
AGCAGCAUUGUACAGGGCUAUGA
118
CAATGCTGC

AATGCTGC

ATGCTGC



 


hsa-miR-103-as
UCAUAGCCCUGUACAAUGCUGCU
119
AGGGCTATG

GGGCTATG

GGCTATG



 


hsa-miR-105
UCAAAUGCUCAGACUCCUGUGGU
120
GAGCATTTG

AGCATTTG

GCATTTG



 


hsa-miR-105*
ACGGAUGUUUGAGCAUGUGCUA
121
AAACATCCG

AACATCCG

ACATCCG



 


hsa-miR-106a
AAAAGUGCUUACAGUGCAGGUAG
122
AAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-106a*
CUGCAAUGUAAGCACUUCUUAC
123
TACATTGCA

ACATTGCA

CATTGCA



 


hsa-miR-106b
UAAAGUGCUGACAGUGCAGAU
124
CAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-106b*
CCGCACUGUGGGUACUUGCUGC
125
CACAGTGCG

ACAGTGCG

CAGTGCG



 


hsa-miR-107
AGCAGCAUUGUACAGGGCUAUCA
126
CAATGCTGC

AATGCTGC

ATGCTGC



 


hsa-miR-10a
UACCCUGUAGAUCCGAAUUUGUG
127
CTACAGGGT

TACAGGGT

ACAGGGT



 


hsa-miR-10a*
CAAAUUCGUAUCUAGGGGAAUA
128
TACGAATTT

ACGAATTT

CGAATTT



 


hsa-miR-10b
UACCCUGUAGAACCGAAUUUGUG
129
CTACAGGGT

TACAGGGT

ACAGGGT



 


hsa-miR-10b*
ACAGAUUCGAUUCUAGGGGAAU
130
TCGAATCTG

CGAATCTG

GAATCTG



 


hsa-miR-1178
UUGCUCACUGUUCUUCCCUAG
131
CAGTGAGCA

AGTGAGCA

GTGAGCA



 


hsa-miR-1179
AAGCAUUCUUUCAUUGGUUGG
132
AAGAATGCT

AGAATGCT

GAATGCT



 


hsa-miR-1180
UUUCCGGCUCGCGUGGGUGUGU
133
GAGCCGGAA

AGCCGGAA

GCCGGAA



 


hsa-miR-1181
CCGUCGCCGCCACCCGAGCCG
134
GCGGCGACG

CGGCGACG

GGCGACG



 


hsa-miR-1182
GAGGGUCUUGGGAGGGAUGUGAC
135
CAAGACCCT

AAGACCCT

AGACCCT



 


hsa-miR-1183
CACUGUAGGUGAUGGUGAGAGUGGGCA
136
ACCTACAGT

CCTACAGT

CTACAGT



 


hsa-miR-1184
CCUGCAGCGACUUGAUGGCUUCC
137
TCGCTGCAG

CGCTGCAG

GCTGCAG



 


hsa-miR-1185
AGAGGAUACCCUUUGUAUGUU
138
GGTATCCTC

GTATCCTC

TATCCTC



 


hsa-miR-1197
UAGGACACAUGGUCUACUUCU
139
ATGTGTCCT

TGTGTCCT

GTGTCCT



 


hsa-miR-1200
CUCCUGAGCCAUUCUGAGCCUC
140
GGCTCAGGA

GCTCAGGA

CTCAGGA



 


hsa-miR-1201
AGCCUGAUUAAACACAUGCUCUGA
141
TAATCAGGC

AATCAGGC

ATCAGGC



 


hsa-miR-1202
GUGCCAGCUGCAGUGGGGGAG
142
CAGCTGGCA

AGCTGGCA

GCTGGCA



 


hsa-miR-1203
CCCGGAGCCAGGAUGCAGCUC
143
TGGCTCCGG

GGCTCCGG

GCTCCGG



 


hsa-miR-1204
UCGUGGCCUGGUCUCCAUUAU
144
CAGGCCACG

AGGCCACG

GGCCACG



 


hsa-miR-1205
UCUGCAGGGUUUGCUUUGAG
145
ACCCTGCAG

CCCTGCAG

CCTGCAG



 


hsa-miR-1206
UGUUCAUGUAGAUGUUUAAGC
146
TACATGAAC

ACATGAAC

CATGAAC



 


hsa-miR-1207-3p
UCAGCUGGCCCUCAUUUC
147
GGCCAGCTG

GCCAGCTG

CCAGCTG



 


hsa-miR-1207-5p
UGGCAGGGAGGCUGGGAGGGG
148
CTCCCTGCC

TCCCTGCC

CCCTGCC



 


hsa-miR-1208
UCACUGUUCAGACAGGCGGA
149
TGAACAGTG

GAACAGTG

AACAGTG



 


hsa-miR-122
UGGAGUGUGACAAUGGUGUUUG
150
TCACACTCC

CACACTCC

ACACTCC



 


hsa-miR-122*
AACGCCAUUAUCACACUAAAUA
151
TAATGGCGT

AATGGCGT

ATGGCGT



 


hsa-miR-1224-3p
CCCCACCUCCUCUCUCCUCAG
152
GGAGGTGGG

GAGGTGGG

AGGTGGG



 


hsa-miR-1224-5p
GUGAGGACUCGGGAGGUGG
153
GAGTCCTCA

AGTCCTCA

GTCCTCA



 


hsa-miR-1225-3p
UGAGCCCCUGUGCCGCCCCCAG
154
CAGGGGCTC

AGGGGCTC

GGGGCTC



 


hsa-miR-1225-5p
GUGGGUACGGCCCAGUGGGGGG
155
CCGTACCCA

CGTACCCA

GTACCCA



 


hsa-miR-1226
UCACCAGCCCUGUGUUCCCUAG
156
GGGCTGGTG

GGCTGGTG

GCTGGTG



 


hsa-miR-1226*
GUGAGGGCAUGCAGGCCUGGAUGGGG
157
ATGCCCTCA

TGCCCTCA

GCCCTCA



 


hsa-miR-1227
CGUGCCACCCUUUUCCCCAG
158
GGGTGGCAC

GGTGGCAC

GTGGCAC



 


hsa-miR-1228
UCACACCUGCCUCGCCCCCC
159
GCAGGTGTG

CAGGTGTG

AGGTGTG



 


hsa-miR-1228*
GUGGGCGGGGGCAGGUGUGUG
160
CCCCGCCCA

CCCGCCCA

CCGCCCA



 


hsa-miR-1229
CUCUCACCACUGCCCUCCCACAG
161
GTGGTGAGA

TGGTGAGA

GGTGAGA



 


hsa-miR-1231
GUGUCUGGGCGGACAGCUGC
162
GCCCAGACA

CCCAGACA

CCAGACA



 


hsa-miR-1233
UGAGCCCUGUCCUCCCGCAG
163
ACAGGGCTC

CAGGGCTC

AGGGCTC



 


hsa-miR-1234
UCGGCCUGACCACCCACCCCAC
164
GTCAGGCCG

TCAGGCCG

CAGGCCG



 


hsa-miR-1236
CCUCUUCCCCUUGUCUCUCCAG
165
GGGGAAGAG

GGGAAGAG

GGAAGAG



 


hsa-miR-1237
UCCUUCUGCUCCGUCCCCCAG
166
AGCAGAAGG

GCAGAAGG

CAGAAGG



 


hsa-miR-1238
CUUCCUCGUCUGUCUGCCCC
167
GACGAGGAA

ACGAGGAA

CGAGGAA



 


hsa-miR-124
UAAGGCACGCGGUGAAUGCC
168
GCGTGCCTT

CGTGCCTT

GTGCCTT



 


hsa-miR-124*
CGUGUUCACAGCGGACCUUGAU
169
TGTGAACAC

GTGAACAC

TGAACAC



 


hsa-miR-1243
AACUGGAUCAAUUAUAGGAGUG
170
TGATCCAGT

GATCCAGT

ATCCAGT



 


hsa-miR-1244
AAGUAGUUGGUUUGUAUGAGAUGGUU
171
CCAACTACT

CAACTACT

AACTACT



 


hsa-miR-1245
AAGUGAUCUAAAGGCCUACAU
172
TAGATCACT

AGATCACT

GATCACT



 


hsa-miR-1246
AAUGGAUUUUUGGAGCAGG
173
AAAATCCAT

AAATCCAT

AATCCAT



 


hsa-miR-1247
ACCCGUCCCGUUCGUCCCCGGA
174
CGGGACGGG

GGGACGGG

GGACGGG



 


hsa-miR-1248
ACCUUCUUGUAUAAGCACUGUGCUAAA
175
ACAAGAAGG

CAAGAAGG

AAGAAGG



 


hsa-miR-1249
ACGCCCUUCCCCCCCUUCUUCA
176
GGAAGGGCG

GAAGGGCG

AAGGGCG



 


hsa-miR-1250
ACGGUGCUGGAUGUGGCCUUU
177
CCAGCACCG

CAGCACCG

AGCACCG



 


hsa-miR-1251
ACUCUAGCUGCCAAAGGCGCU
178
CAGCTAGAG

AGCTAGAG

GCTAGAG



 


hsa-miR-1252
AGAAGGAAAUUGAAUUCAUUUA
179
ATTTCCTTC

TTTCCTTC

TTCCTTC



 


hsa-miR-1253
AGAGAAGAAGAUCAGCCUGCA
180
CTTCTTCTC

TTCTTCTC

TCTTCTC



 


hsa-miR-1254
AGCCUGGAAGCUGGAGCCUGCAGU
181
CTTCCAGGC

TTCCAGGC

TCCAGGC



 


hsa-miR-1255a
AGGAUGAGCAAAGAAAGUAGAUU
182
TGCTCATCC

GCTCATCC

CTCATCC



 


hsa-miR-1255b
CGGAUGAGCAAAGAAAGUGGUU
183
TGCTCATCC

GCTCATCC

CTCATCC



 


hsa-miR-1256
AGGCAUUGACUUCUCACUAGCU
184
GTCAATGCC

TCAATGCC

CAATGCC



 


hsa-miR-1257
AGUGAAUGAUGGGUUCUGACC
185
ATCATTCAC

TCATTCAC

CATTCAC



 


hsa-miR-1258
AGUUAGGAUUAGGUCGUGGAA
186
AATCCTAAC

ATCCTAAC

TCCTAAC



 


hsa-miR-1259
AUAUAUGAUGACUUAGCUUUU
187
CATCATATA

ATCATATA

TCATATA



 


hsa-miR-125a-3p
ACAGGUGAGGUUCUUGGGAGCC
188
CCTCACCTG

CTCACCTG

TCACCTG



 


hsa-miR-125a-5p
UCCCUGAGACCCUUUAACCUGUGA
189
GTCTCAGGG

TCTCAGGG

CTCAGGG



 


hsa-miR-125b
UCCCUGAGACCCUAACUUGUGA
190
GTCTCAGGG

TCTCAGGG

CTCAGGG



 


hsa-miR-125b-1*
ACGGGUUAGGCUCUUGGGAGCU
191
CCTAACCCG

CTAACCCG

TAACCCG



 


hsa-miR-125b-2*
UCACAAGUCAGGCUCUUGGGAC
192
TGACTTGTG

GACTTGTG

ACTTGTG



 


hsa-miR-126
UCGUACCGUGAGUAAUAAUGCG
193
CACGGTACG

ACGGTACG

CGGTACG



 


hsa-miR-126*
CAUUAUUACUUUUGGUACGCG
194
AGTAATAAT

GTAATAAT

TAATAAT



 


hsa-miR-1260
AUCCCACCUCUGCCACCA
195
GAGGTGGGA

AGGTGGGA

GGTGGGA



 


hsa-miR-1261
AUGGAUAAGGCUUUGGCUU
196
CCTTATCCA

CTTATCCA

TTATCCA



 


hsa-miR-1262
AUGGGUGAAUUUGUAGAAGGAU
197
ATTCACCCA

TTCACCCA

TCACCCA



 


hsa-miR-1263
AUGGUACCCUGGCAUACUGAGU
198
AGGGTACCA

GGGTACCA

GGTACCA



 


hsa-miR-1264
CAAGUCUUAUUUGAGCACCUGUU
199
ATAAGACTT

TAAGACTT

AAGACTT



 


hsa-miR-1265
CAGGAUGUGGUCAAGUGUUGUU
200
CCACATCCT

CACATCCT

ACATCCT



 


hsa-miR-1266
CCUCAGGGCUGUAGAACAGGGCU
201
AGCCCTGAG

GCCCTGAG

CCCTGAG



 


hsa-miR-1267
CCUGUUGAAGUGUAAUCCCCA
202
CTTCAACAG

TTCAACAG

TCAACAG



 


hsa-miR-1268
CGGGCGUGGUGGUGGGGG
203
ACCACGCCC

CCACGCCC

CACGCCC



 


hsa-miR-1269
CUGGACUGAGCCGUGCUACUGG
204
CTCAGTCCA

TCAGTCCA

CAGTCCA



 


hsa-miR-127-3p
UCGGAUCCGUCUGAGCUUGGCU
205
ACGGATCCG

CGGATCCG

GGATCCG



 


hsa-miR-127-5p
CUGAAGCUCAGAGGGCUCUGAU
206
TGAGCTTCA

GAGCTTCA

AGCTTCA



 


hsa-miR-1270
CUGGAGAUAUGGAAGAGCUGUGU
207
ATATCTCCA

TATCTCCA

ATCTCCA



 


hsa-miR-1271
CUUGGCACCUAGCAAGCACUCA
208
AGGTGCCAA

GGTGCCAA

GTGCCAA



 


hsa-miR-1272
GAUGAUGAUGGCAGCAAAUUCUGAAA
209
CATCATCAT

ATCATCAT

TCATCAT



 


hsa-miR-1273
GGGCGACAAAGCAAGACUCUUUCUU
210
TTTGTCGCC

TTGTCGCC

TGTCGCC



 


hsa-miR-1274a
GUCCCUGUUCAGGCGCCA
211
GAACAGGGA

AACAGGGA

ACAGGGA



 


hsa-miR-1274b
UCCCUGUUCGGGCGCCA
212
CGAACAGGG

GAACAGGG

AACAGGG



 


hsa-miR-1275
GUGGGGGAGAGGCUGUC
213
TCTCCCCCA

CTCCCCCA

TCCCCCA



 


hsa-miR-1276
UAAAGAGCCCUGUGGAGACA
214
GGGCTCTTT

GGCTCTTT

GCTCTTT



 


hsa-miR-1277
UACGUAGAUAUAUAUGUAUUUU
215
TATCTACGT

ATCTACGT

TCTACGT



 


hsa-miR-1278
UAGUACUGUGCAUAUCAUCUAU
216
CACAGTACT

ACAGTACT

CAGTACT



 


hsa-miR-1279
UCAUAUUGCUUCUUUCU
217
AGCAATATG

GCAATATG

CAATATG



 


hsa-miR-128
UCACAGUGAACCGGUCUCUUU
218
TTCACTGTG

TCACTGTG

CACTGTG



 


hsa-miR-1280
UCCCACCGCUGCCACCC
219
AGCGGTGGG

GCGGTGGG

CGGTGGG



 


hsa-miR-1281
UCGCCUCCUCCUCUCCC
220
GAGGAGGCG

AGGAGGCG

GGAGGCG



 


hsa-miR-1282
UCGUUUGCCUUUUUCUGCUU
221
AGGCAAACG

GGCAAACG

GCAAACG



 


hsa-miR-1283
UCUACAAAGGAAAGCGCUUUCU
222
CCTTTGTAG

CTTTGTAG

TTTGTAG



 


hsa-miR-1284
UCUAUACAGACCCUGGCUUUUC
223
TCTGTATAG

CTGTATAG

TGTATAG



 


hsa-miR-1285
UCUGGGCAACAAAGUGAGACCU
224
GTTGCCCAG

TTGCCCAG

TGCCCAG



 


hsa-miR-1286
UGCAGGACCAAGAUGAGCCCU
225
TGGTCCTGC

GGTCCTGC

GTCCTGC



 


hsa-miR-1287
UGCUGGAUCAGUGGUUCGAGUC
226
TGATCCAGC

GATCCAGC

ATCCAGC



 


hsa-miR-1288
UGGACUGCCCUGAUCUGGAGA
227
GGGCAGTCC

GGCAGTCC

GCAGTCC



 


hsa-miR-1289
UGGAGUCCAGGAAUCUGCAUUUU
228
CTGGACTCC

TGGACTCC

GGACTCC



 


hsa-miR-129*
AAGCCCUUACCCCAAAAAGUAU
229
GTAAGGGCT

TAAGGGCT

AAGGGCT



 


hsa-miR-129-3p
AAGCCCUUACCCCAAAAAGCAU
230
GTAAGGGCT

TAAGGGCT

AAGGGCT



 


hsa-miR-129-5p
CUUUUUGCGGUCUGGGCUUGC
231
CCGCAAAAA

CGCAAAAA

GCAAAAA



 


hsa-miR-1290
UGGAUUUUUGGAUCAGGGA
232
CAAAAATCC

AAAAATCC

AAAATCC



 


hsa-miR-1291
UGGCCCUGACUGAAGACCAGCAGU
233
GTCAGGGCC

TCAGGGCC

CAGGGCC



 


hsa-miR-1292
UGGGAACGGGUUCCGGCAGACGCUG
234
CCCGTTCCC

CCGTTCCC

CGTTCCC



 


hsa-miR-1293
UGGGUGGUCUGGAGAUUUGUGC
235
AGACCACCC

GACCACCC

ACCACCC



 


hsa-miR-1294
UGUGAGGUUGGCAUUGUUGUCU
236
CAACCTCAC

AACCTCAC

ACCTCAC



 


hsa-miR-1295
UUAGGCCGCAGAUCUGGGUGA
237
TGCGGCCTA

GCGGCCTA

CGGCCTA



 


hsa-miR-1296
UUAGGGCCCUGGCUCCAUCUCC
238
AGGGCCCTA

GGGCCCTA

GGCCCTA



 


hsa-miR-1297
UUCAAGUAAUUCAGGUG
239
ATTACTTGA

TTACTTGA

TACTTGA



 


hsa-miR-1298
UUCAUUCGGCUGUCCAGAUGUA
240
GCCGAATGA

CCGAATGA

CGAATGA



 


hsa-miR-1299
UUCUGGAAUUCUGUGUGAGGGA
241
AATTCCAGA

ATTCCAGA

TTCCAGA



 


hsa-miR-1300
UUGAGAAGGAGGCUGCUG
242
TCCTTCTCA

CCTTCTCA

CTTCTCA



 


hsa-miR-1301
UUGCAGCUGCCUGGGAGUGACUUC
243
GCAGCTGCA

CAGCTGCA

AGCTGCA



 


hsa-miR-1302
UUGGGACAUACUUAUGCUAAA
244
TATGTCCCA

ATGTCCCA

TGTCCCA



 


hsa-miR-1303
UUUAGAGACGGGGUCUUGCUCU
245
CGTCTCTAA

GTCTCTAA

TCTCTAA



 


hsa-miR-1304
UUUGAGGCUACAGUGAGAUGUG
246
TAGCCTCAA

AGCCTCAA

GCCTCAA



 


hsa-miR-1305
UUUUCAACUCUAAUGGGAGAGA
247
GAGTTGAAA

AGTTGAAA

GTTGAAA



 


hsa-miR-1306
ACGUUGGCUCUGGUGGUG
248
GAGCCAACG

AGCCAACG

GCCAACG



 


hsa-miR-1307
ACUCGGCGUGGCGUCGGUCGUG
249
CACGCCGAG

ACGCCGAG

CGCCGAG



 


hsa-miR-1308
GCAUGGGUGGUUCAGUGG
250
CCACCCATG

CACCCATG

ACCCATG



 


hsa-miR-130a
CAGUGCAAUGUUAAAAGGGCAU
251
CATTGCACT

ATTGCACT

TTGCACT



 


hsa-miR-130a*
UUCACAUUGUGCUACUGUCUGC
252
ACAATGTGA

CAATGTGA

AATGTGA



 


hsa-miR-130b
CAGUGCAAUGAUGAAAGGGCAU
253
CATTGCACT

ATTGCACT

TTGCACT



 


hsa-miR-130b*
ACUCUUUCCCUGUUGCACUAC
254
GGGAAAGAG

GGAAAGAG

GAAAGAG



 


hsa-miR-132
UAACAGUCUACAGCCAUGGUCG
255
TAGACTGTT

AGACTGTT

GACTGTT



 


hsa-miR-132*
ACCGUGGCUUUCGAUUGUUACU
256
AAGCCACGG

AGCCACGG

GCCACGG



 


hsa-miR-1321
CAGGGAGGUGAAUGUGAU
257
CACCTCCCT

ACCTCCCT

CCTCCCT



 


hsa-miR-1322
GAUGAUGCUGCUGAUGCUG
258
CAGCATCAT

AGCATCAT

GCATCAT



 


hsa-miR-1323
UCAAAACUGAGGGGCAUUUUCU
259
TCAGTTTTG

CAGTTTTG

AGTTTTG



 


hsa-miR-1324
CCAGACAGAAUUCUAUGCACUUUC
260
TTCTGTCTG

TCTGTCTG

CTGTCTG



 


hsa-miR-133a
UUUGGUCCCCUUCAACCAGCUG
261
GGGGACCAA

GGGACCAA

GGACCAA



 


hsa-miR-133b
UUUGGUCCCCUUCAACCAGCUA
262
GGGGACCAA

GGGACCAA

GGACCAA



 


hsa-miR-134
UGUGACUGGUUGACCAGAGGGG
263
ACCAGTCAC

CCAGTCAC

CAGTCAC



 


hsa-miR-135a
UAUGGCUUUUUAUUCCUAUGUGA
264
AAAAGCCAT

AAAGCCAT

AAGCCAT



 


hsa-miR-135a*
UAUAGGGAUUGGAGCCGUGGCG
265
AATCCCTAT

ATCCCTAT

TCCCTAT



 


hsa-miR-135b
UAUGGCUUUUCAUUCCUAUGUGA
266
AAAAGCCAT

AAAGCCAT

AAGCCAT



 


hsa-miR-135b*
AUGUAGGGCUAAAAGCCAUGGG
267
AGCCCTACA

GCCCTACA

CCCTACA



 


hsa-miR-136
ACUCCAUUUGUUUUGAUGAUGGA
268
CAAATGGAG

AAATGGAG

AATGGAG



 


hsa-miR-136*
CAUCAUCGUCUCAAAUGAGUCU
269
GACGATGAT

ACGATGAT

CGATGAT



 


hsa-miR-137
UUAUUGCUUAAGAAUACGCGUAG
270
TAAGCAATA

AAGCAATA

AGCAATA



 


hsa-miR-138
AGCUGGUGUUGUGAAUCAGGCCG
271
AACACCAGC

ACACCAGC

CACCAGC



 


hsa-miR-138-1*
GCUACUUCACAACACCAGGGCC
272
GTGAAGTAG

TGAAGTAG

GAAGTAG



 


hsa-miR-138-2*
GCUAUUUCACGACACCAGGGUU
273
GTGAAATAG

TGAAATAG

GAAATAG



 


hsa-miR-139-3p
GGAGACGCGGCCCUGUUGGAGU
274
CCGCGTCTC

CGCGTCTC

GCGTCTC



 


hsa-miR-139-5p
UCUACAGUGCACGUGUCUCCAG
275
GCACTGTAG

CACTGTAG

ACTGTAG



 


hsa-miR-140-3p
UACCACAGGGUAGAACCACGG
276
CCCTGTGGT

CCTGTGGT

CTGTGGT



 


hsa-miR-140-5p
CAGUGGUUUUACCCUAUGGUAG
277
AAAACCACT

AAACCACT

AACCACT



 


hsa-miR-141
UAACACUGUCUGGUAAAGAUGG
278
GACAGTGTT

ACAGTGTT

CAGTGTT



 


hsa-miR-141*
CAUCUUCCAGUACAGUGUUGGA
279
CTGGAAGAT

TGGAAGAT

GGAAGAT



 


hsa-miR-142-3p
UGUAGUGUUUCCUACUUUAUGGA
280
AAACACTAC

AACACTAC

ACACTAC



 


hsa-miR-142-5p
CAUAAAGUAGAAAGCACUACU
281
CTACTTTAT

TACTTTAT

ACTTTAT



 


hsa-miR-143
UGAGAUGAAGCACUGUAGCUC
282
CTTCATCTC

TTCATCTC

TCATCTC



 


hsa-miR-143*
GGUGCAGUGCUGCAUCUCUGGU
283
GCACTGCAC

CACTGCAC

ACTGCAC



 


hsa-miR-144
UACAGUAUAGAUGAUGUACU
284
CTATACTGT

TATACTGT

ATACTGT



 


hsa-miR-144*
GGAUAUCAUCAUAUACUGUAAG
285
GATGATATC

ATGATATC

TGATATC



 


hsa-miR-145
GUCCAGUUUUCCCAGGAAUCCCU
286
AAAACTGGA

AAACTGGA

AACTGGA



 


hsa-miR-145*
GGAUUCCUGGAAAUACUGUUCU
287
CCAGGAATC

CAGGAATC

AGGAATC



 


hsa-miR-1468
CUCCGUUUGCCUGUUUCGCUG
288
GCAAACGGA

CAAACGGA

AAACGGA



 


hsa-miR-1469
CUCGGCGCGGGGCGCGGGCUCC
289
CCGCGCCGA

CGCGCCGA

GCGCCGA



 


hsa-miR-146a
UGAGAACUGAAUUCCAUGGGUU
290
TCAGTTCTC

CAGTTCTC

AGTTCTC



 


hsa-miR-146a*
CCUCUGAAAUUCAGUUCUUCAG
291
ATTTCAGAG

TTTCAGAG

TTCAGAG



 


hsa-miR-146b-3p
UGCCCUGUGGACUCAGUUCUGG
292
CCACAGGGC

CACAGGGC

ACAGGGC



 


hsa-miR-146b-5p
UGAGAACUGAAUUCCAUAGGCU
293
TCAGTTCTC

CAGTTCTC

AGTTCTC



 


hsa-miR-147
GUGUGUGGAAAUGCUUCUGC
294
TTCCACACA

TCCACACA

CCACACA



 


hsa-miR-1470
GCCCUCCGCCCGUGCACCCCG
295
GGCGGAGGG

GCGGAGGG

CGGAGGG



 


hsa-miR-1471
GCCCGCGUGUGGAGCCAGGUGU
296
ACACGCGGG

CACGCGGG

ACGCGGG



 


hsa-miR-147b
GUGUGCGGAAAUGCUUCUGCUA
297
TTCCGCACA

TCCGCACA

CCGCACA



 


hsa-miR-148a
UCAGUGCACUACAGAACUUUGU
298
AGTGCACTG

GTGCACTG

TGCACTG



 


hsa-miR-148a*
AAAGUUCUGAGACACUCCGACU
299
TCAGAACTT

CAGAACTT

AGAACTT



 


hsa-miR-148b
UCAGUGCAUCACAGAACUUUGU
300
GATGCACTG

ATGCACTG

TGCACTG



 


hsa-miR-148b*
AAGUUCUGUUAUACACUCAGGC
301
AACAGAACT

ACAGAACT

CAGAACT



 


hsa-miR-149
UCUGGCUCCGUGUCUUCACUCCC
302
CGGAGCCAG

GGAGCCAG

GAGCCAG



 


hsa-miR-149*
AGGGAGGGACGGGGGCUGUGC
303
GTCCCTCCC

TCCCTCCC

CCCTCCC



 


hsa-miR-150
UCUCCCAACCCUUGUACCAGUG
304
GGTTGGGAG

GTTGGGAG

TTGGGAG



 


hsa-miR-150*
CUGGUACAGGCCUGGGGGACAG
305
CCTGTACCA

CTGTACCA

TGTACCA



 


hsa-miR-151-3p
CUAGACUGAAGCUCCUUGAGG
306
TTCAGTCTA

TCAGTCTA

CAGTCTA



 


hsa-miR-151-5p
UCGAGGAGCUCACAGUCUAGU
307
AGCTCCTCG

GCTCCTCG

CTCCTCG



 


hsa-miR-152
UCAGUGCAUGACAGAACUUGG
308
CATGCACTG

ATGCACTG

TGCACTG



 


hsa-miR-153
UUGCAUAGUCACAAAAGUGAUC
309
GACTATGCA

ACTATGCA

CTATGCA



 


hsa-miR-1537
AAAACCGUCUAGUUACAGUUGU
310
AGACGGTTT

GACGGTTT

ACGGTTT



 


hsa-miR-1538
CGGCCCGGGCUGCUGCUGUUCCU
311
GCCCGGGCC

CCCGGGCC

CCGGGCC



 


hsa-miR-1539
UCCUGCGCGUCCCAGAUGCCC
312
ACGCGCAGG

CGCGCAGG

GCGCAGG



 


hsa-miR-154
UAGGUUAUCCGUGUUGCCUUCG
313
GGATAACCT

GATAACCT

ATAACCT



 


hsa-miR-154*
AAUCAUACACGGUUGACCUAUU
314
GTGTATGAT

TGTATGAT

GTATGAT



 


hsa-miR-155
UUAAUGCUAAUCGUGAUAGGGGU
315
TTAGCATTA

TAGCATTA

AGCATTA



 


hsa-miR-155*
CUCCUACAUAUUAGCAUUAACA
316
TATGTAGGA

ATGTAGGA

TGTAGGA



 


hsa-miR-15a
UAGCAGCACAUAAUGGUUUGUG
317
TGTGCTGCT

GTGCTGCT

TGCTGCT



 


hsa-miR-15a*
CAGGCCAUAUUGUGCUGCCUCA
318
ATATGGCCT

TATGGCCT

ATGGCCT



 


hsa-miR-15b
UAGCAGCACAUCAUGGUUUACA
319
TGTGCTGCT

GTGCTGCT

TGCTGCT



 


hsa-miR-15b*
CGAAUCAUUAUUUGCUGCUCUA
320
TAATGATTC

AATGATTC

ATGATTC



 


hsa-miR-16
UAGCAGCACGUAAAUAUUGGCG
321
CGTGCTGCT

GTGCTGCT

TGCTGCT



 


hsa-miR-16-1*
CCAGUAUUAACUGUGCUGCUGA
322
TTAATACTG

TAATACTG

AATACTG



 


hsa-miR-16-2*
CCAAUAUUACUGUGCUGCUUUA
323
GTAATATTG

TAATATTG

AATATTG



 


hsa-miR-17
CAAAGUGCUUACAGUGCAGGUAG
324
AAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-17*
ACUGCAGUGAAGGCACUUGUAG
325
TCACTGCAG

CACTGCAG

ACTGCAG



 


hsa-miR-181a
AACAUUCAACGCUGUCGGUGAGU
326
GTTGAATGT

TTGAATGT

TGAATGT



 


hsa-miR-181a*
ACCAUCGACCGUUGAUUGUACC
327
GGTCGATGG

GTCGATGG

TCGATGG



 


hsa-miR-181a-2*
ACCACUGACCGUUGACUGUACC
328
GGTCAGTGG

GTCAGTGG

TCAGTGG



 


hsa-miR-181b
AACAUUCAUUGCUGUCGGUGGGU
329
AATGAATGT

ATGAATGT

TGAATGT



 


hsa-miR-181c
AACAUUCAACCUGUCGGUGAGU
330
GTTGAATGT

TTGAATGT

TGAATGT



 


hsa-miR-181c*
AACCAUCGACCGUUGAGUGGAC
331
GTCGATGGT

TCGATGGT

CGATGGT



 


hsa-miR-181d
AACAUUCAUUGUUGUCGGUGGGU
332
AATGAATGT

ATGAATGT

TGAATGT



 


hsa-miR-182
UUUGGCAAUGGUAGAACUCACACU
333
CATTGCCAA

ATTGCCAA

TTGCCAA



 


hsa-miR-182*
UGGUUCUAGACUUGCCAACUA
334
TCTAGAACC

CTAGAACC

TAGAACC



 


hsa-miR-1825
UCCAGUGCCCUCCUCUCC
335
GGGCACTGG

GGCACTGG

GCACTGG



 


hsa-miR-1826
AUUGAUCAUCGACACUUCGAACGCAAU
336
GATGATCAA

ATGATCAA

TGATCAA



 


hsa-miR-1827
UGAGGCAGUAGAUUGAAU
337
TACTGCCTC

ACTGCCTC

CTGCCTC



 


hsa-miR-183
UAUGGCACUGGUAGAAUUCACU
338
CAGTGCCAT

AGTGCCAT

GTGCCAT



 


hsa-miR-183*
GUGAAUUACCGAAGGGCCAUAA
339
GGTAATTCA

GTAATTCA

TAATTCA



 


hsa-miR-184
UGGACGGAGAACUGAUAAGGGU
340
TCTCCGTCC

CTCCGTCC

TCCGTCC



 


hsa-miR-185
UGGAGAGAAAGGCAGUUCCUGA
341
TTTCTCTCC

TTCTCTCC

TCTCTCC



 


hsa-miR-185*
AGGGGCUGGCUUUCCUCUGGUC
342
GCCAGCCCC

CCAGCCCC

CAGCCCC



 


hsa-miR-186
CAAAGAAUUCUCCUUUUGGGCU
343
GAATTCTTT

AATTCTTT

ATTCTTT



 


hsa-miR-186*
GCCCAAAGGUGAAUUUUUUGGG
344
ACCTTTGGG

CCTTTGGG

CTTTGGG



 


hsa-miR-187
UCGUGUCUUGUGUUGCAGCCGG
345
CAAGACACG

AAGACACG

AGACACG



 


hsa-miR-187*
GGCUACAACACAGGACCCGGGC
346
TGTTGTAGC

GTTGTAGC

TTGTAGC



 


hsa-miR-188-3p
CUCCCACAUGCAGGGUUUGCA
347
CATGTGGGA

ATGTGGGA

TGTGGGA



 


hsa-miR-188-5p
CAUCCCUUGCAUGGUGGAGGG
348
GCAAGGGAT

CAAGGGAT

AAGGGAT



 


hsa-miR-18a
UAAGGUGCAUCUAGUGCAGAUAG
349
ATGCACCTT

TGCACCTT

GCACCTT



 


hsa-miR-18a*
ACUGCCCUAAGUGCUCCUUCUGG
350
TTAGGGCAG

TAGGGCAG

AGGGCAG



 


hsa-miR-18b
UAAGGUGCAUCUAGUGCAGUUAG
351
ATGCACCTT

TGCACCTT

GCACCTT



 


hsa-miR-18b*
UGCCCUAAAUGCCCCUUCUGGC
352
ATTTAGGGC

TTTAGGGC

TTAGGGC



 


hsa-miR-190
UGAUAUGUUUGAUAUAUUAGGU
353
AAACATATC

AACATATC

ACATATC



 


hsa-miR-1908
CGGCGGGGACGGCGAUUGGUC
354
GTCCCCGCC

TCCCCGCC

CCCCGCC



 


hsa-miR-1909
CGCAGGGGCCGGGUGCUCACCG
355
GGCCCCTGC

GCCCCTGC

CCCCTGC



 


hsa-miR-1909*
UGAGUGCCGGUGCCUGCCCUG
356
CCGGCACTC

CGGCACTC

GGCACTC



 


hsa-miR-190b
UGAUAUGUUUGAUAUUGGGUU
357
AAACATATC

AACATATC

ACATATC



 


hsa-miR-191
CAACGGAAUCCCAAAAGCAGCUG
358
GATTCCGTT

ATTCCGTT

TTCCGTT



 


hsa-miR-191*
GCUGCGCUUGGAUUUCGUCCCC
359
CAAGCGCAG

AAGCGCAG

AGCGCAG



 


hsa-miR-1910
CCAGUCCUGUGCCUGCCGCCU
360
ACAGGACTG

CAGGACTG

AGGACTG



 


hsa-miR-1911
UGAGUACCGCCAUGUCUGUUGGG
361
GCGGTACTC

CGGTACTC

GGTACTC



 


hsa-miR-1911*
CACCAGGCAUUGUGGUCUCC
362
ATGCCTGGT

TGCCTGGT

GCCTGGT



 


hsa-miR-1912
UACCCAGAGCAUGCAGUGUGAA
363
GCTCTGGGT

CTCTGGGT

TCTGGGT



 


hsa-miR-1913
UCUGCCCCCUCCGCUGCUGCCA
364
AGGGGGCAG

GGGGGCAG

GGGGCAG



 


hsa-miR-1914
CCCUGUGCCCGGCCCACUUCUG
365
GGGCACAGG

GGCACAGG

GCACAGG



 


hsa-miR-1914*
GGAGGGGUCCCGCACUGGGAGG
366
GGACCCCTC

GACCCCTC

ACCCCTC



 


hsa-miR-1915
CCCCAGGGCGACGCGGCGGG
367
CGCCCTGGG

GCCCTGGG

CCCTGGG



 


hsa-miR-1915*
ACCUUGCCUUGCUGCCCGGGCC
368
AAGGCAAGG

AGGCAAGG

GGCAAGG



 


hsa-miR-192
CUGACCUAUGAAUUGACAGCC
369
CATAGGTCA

ATAGGTCA

TAGGTCA



 


hsa-miR-192*
CUGCCAAUUCCAUAGGUCACAG
370
GAATTGGCA

AATTGGCA

ATTGGCA



 


hsa-miR-193a-3p
AACUGGCCUACAAAGUCCCAGU
371
TAGGCCAGT

AGGCCAGT

GGCCAGT



 


hsa-miR-193a-5p
UGGGUCUUUGCGGGCGAGAUGA
372
CAAAGACCC

AAAGACCC

AAGACCC



 


hsa-miR-193b
AACUGGCCCUCAAAGUCCCGCU
373
AGGGCCAGT

GGGCCAGT

GGCCAGT



 


hsa-miR-193b*
CGGGGUUUUGAGGGCGAGAUGA
374
CAAAACCCC

AAAACCCC

AAACCCC



 


hsa-miR-194
UGUAACAGCAACUCCAUGUGGA
375
TGCTGTTAC

GCTGTTAC

CTGTTAC



 


hsa-miR-194*
CCAGUGGGGCUGCUGUUAUCUG
376
GCCCCACTG

CCCCACTG

CCCACTG



 


hsa-miR-195
UAGCAGCACAGAAAUAUUGGC
377
TGTGCTGCT

GTGCTGCT

TGCTGCT



 


hsa-miR-195*
CCAAUAUUGGCUGUGCUGCUCC
378
CCAATATTG

CAATATTG

AATATTG



 


hsa-miR-196a
UAGGUAGUUUCAUGUUGUUGGG
379
AAACTACCT

AACTACCT

ACTACCT



 


hsa-miR-196a*
CGGCAACAAGAAACUGCCUGAG
380
CTTGTTGCC

TTGTTGCC

TGTTGCC



 


hsa-miR-196b
UAGGUAGUUUCCUGUUGUUGGG
381
AAACTACCT

AACTACCT

ACTACCT



 


hsa-miR-197
UUCACCACCUUCUCCACCCAGC
382
AGGTGGTGA

GGTGGTGA

GTGGTGA



 


hsa-miR-198
GGUCCAGAGGGGAGAUAGGUUC
383
CCTCTGGAC

CTCTGGAC

TCTGGAC



 


hsa-miR-199a-5p
CCCAGUGUUCAGACUACCUGUUC
384
GAACACTGG

AACACTGG

ACACTGG



 


hsa-miR-199b-3p
ACAGUAGUCUGCACAUUGGUUA
385
AGACTACTG

GACTACTG

ACTACTG



 


hsa-miR-199b-5p
CCCAGUGUUUAGACUAUCUGUUC
386
AAACACTGG

AACACTGG

ACACTGG



 


hsa-miR-19a
UGUGCAAAUCUAUGCAAAACUGA
387
GATTTGCAC

ATTTGCAC

TTTGCAC



 


hsa-miR-19a*
AGUUUUGCAUAGUUGCACUACA
388
ATGCAAAAC

TGCAAAAC

GCAAAAC



 


hsa-miR-19b
UGUGCAAAUCCAUGCAAAACUGA
389
GATTTGCAC

ATTTGCAC

TTTGCAC



 


hsa-miR-19b-1*
AGUUUUGCAGGUUUGCAUCCAGC
390
CTGCAAAAC

TGCAAAAC

GCAAAAC



 


hsa-miR-19b-2*
AGUUUUGCAGGUUUGCAUUUCA
391
CTGCAAAAC

TGCAAAAC

GCAAAAC



 


hsa-miR-200a
UAACACUGUCUGGUAACGAUGU
392
GACAGTGTT

ACAGTGTT

CAGTGTT



 


hsa-miR-200a*
CAUCUUACCGGACAGUGCUGGA
393
CGGTAAGAT

GGTAAGAT

GTAAGAT



 


hsa-miR-200b
UAAUACUGCCUGGUAAUGAUGA
394
GGCAGTATT

GCAGTATT

CAGTATT



 


hsa-miR-200b*
CAUCUUACUGGGCAGCAUUGGA
395
CAGTAAGAT

AGTAAGAT

GTAAGAT



 


hsa-miR-200c
UAAUACUGCCGGGUAAUGAUGGA
396
GGCAGTATT

GCAGTATT

CAGTATT



 


hsa-miR-200c*
CGUCUUACCCAGCAGUGUUUGG
397
GGGTAAGAC

GGTAAGAC

GTAAGAC



 


hsa-miR-202
AGAGGUAUAGGGCAUGGGAA
398
CTATACCTC

TATACCTC

ATACCTC



 


hsa-miR-202*
UUCCUAUGCAUAUACUUCUUUG
399
TGCATAGGA

GCATAGGA

CATAGGA



 


hsa-miR-203
GUGAAAUGUUUAGGACCACUAG
400
AACATTTCA

ACATTTCA

CATTTCA



 


hsa-miR-204
UUCCCUUUGUCAUCCUAUGCCU
401
ACAAAGGGA

CAAAGGGA

AAAGGGA



 


hsa-miR-205
UCCUUCAUUCCACCGGAGUCUG
402
GAATGAAGG

AATGAAGG

ATGAAGG



 


hsa-miR-206
UGGAAUGUAAGGAAGUGUGUGG
403
TTACATTCC

TACATTCC

ACATTCC



 


hsa-miR-208a
AUAAGACGAGCAAAAAGCUUGU
404
CTCGTCTTA

TCGTCTTA

CGTCTTA



 


hsa-miR-208b
AUAAGACGAACAAAAGGUUUGU
405
TTCGTCTTA

TCGTCTTA

CGTCTTA



 


hsa-miR-20a
UAAAGUGCUUAUAGUGCAGGUAG
406
AAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-20a*
ACUGCAUUAUGAGCACUUAAAG
407
ATAATGCAG

TAATGCAG

AATGCAG



 


hsa-miR-20b
CAAAGUGCUCAUAGUGCAGGUAG
408
GAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-20b*
ACUGUAGUAUGGGCACUUCCAG
409
ATACTACAG

TACTACAG

ACTACAG



 


hsa-miR-21
UAGCUUAUCAGACUGAUGUUGA
410
TGATAAGCT

GATAAGCT

ATAAGCT



 


hsa-miR-21*
CAACACCAGUCGAUGGGCUGU
411
ACTGGTGTT

CTGGTGTT

TGGTGTT



 


hsa-miR-210
CUGUGCGUGUGACAGCGGCUGA
412
ACACGCACA

CACGCACA

ACGCACA



 


hsa-miR-211
UUCCCUUUGUCAUCCUUCGCCU
413
ACAAAGGGA

CAAAGGGA

AAAGGGA



 


hsa-miR-212
UAACAGUCUCCAGUCACGGCC
414
GAGACTGTT

AGACTGTT

GACTGTT



 


hsa-miR-214
ACAGCAGGCACAGACAGGCAGU
415
TGCCTGCTG

GCCTGCTG

CCTGCTG



 


hsa-miR-214*
UGCCUGUCUACACUUGCUGUGC
416
TAGACAGGC

AGACAGGC

GACAGGC



 


hsa-miR-215
AUGACCUAUGAAUUGACAGAC
417
CATAGGTCA

ATAGGTCA

TAGGTCA



 


hsa-miR-216a
UAAUCUCAGCUGGCAACUGUGA
418
GCTGAGATT

CTGAGATT

TGAGATT



 


hsa-miR-216b
AAAUCUCUGCAGGCAAAUGUGA
419
GCAGAGATT

CAGAGATT

AGAGATT



 


hsa-miR-217
UACUGCAUCAGGAACUGAUUGGA
420
TGATGCAGT

GATGCAGT

ATGCAGT



 


hsa-miR-218
UUGUGCUUGAUCUAACCAUGU
421
TCAAGCACA

CAAGCACA

AAGCACA



 


hsa-miR-218-1*
AUGGUUCCGUCAAGCACCAUGG
422
ACGGAACCA

CGGAACCA

GGAACCA



 


hsa-miR-218-2*
CAUGGUUCUGUCAAGCACCGCG
423
CAGAACCAT

AGAACCAT

GAACCAT



 


hsa-miR-219-1-3p
AGAGUUGAGUCUGGACGUCCCG
424
ACTCAACTC

CTCAACTC

TCAACTC



 


hsa-miR-219-2-3p
AGAAUUGUGGCUGGACAUCUGU
425
CCACAATTC

CACAATTC

ACAATTC



 


hsa-miR-219-5p
UGAUUGUCCAAACGCAAUUCU
426
TGGACAATC

GGACAATC

GACAATC



 


hsa-miR-22
AAGCUGCCAGUUGAAGAACUGU
427
CTGGCAGCT

TGGCAGCT

GGCAGCT



 


hsa-miR-22*
AGUUCUUCAGUGGCAAGCUUUA
428
CTGAAGAAC

TGAAGAAC

GAAGAAC



 


hsa-miR-220a
CCACACCGUAUCUGACACUUU
429
TACGGTGTG

ACGGTGTG

CGGTGTG



 


hsa-miR-220b
CCACCACCGUGUCUGACACUU
430
ACGGTGGTG

CGGTGGTG

GGTGGTG



 


hsa-miR-220c
ACACAGGGCUGUUGUGAAGACU
431
AGCCCTGTG

GCCCTGTG

CCCTGTG



 


hsa-miR-221
AGCUACAUUGUCUGCUGGGUUUC
432
CAATGTAGC

AATGTAGC

ATGTAGC



 


hsa-miR-221*
ACCUGGCAUACAAUGUAGAUUU
433
TATGCCAGG

ATGCCAGG

TGCCAGG



 


hsa-miR-222
AGCUACAUCUGGCUACUGGGU
434
AGATGTAGC

GATGTAGC

ATGTAGC



 


hsa-miR-222*
CUCAGUAGCCAGUGUAGAUCCU
435
GGCTACTGA

GCTACTGA

CTACTGA



 


hsa-miR-223
UGUCAGUUUGUCAAAUACCCCA
436
CAAACTGAC

AAACTGAC

AACTGAC



 


hsa-miR-223*
CGUGUAUUUGACAAGCUGAGUU
437
CAAATACAC

AAATACAC

AATACAC



 


hsa-miR-224
CAAGUCACUAGUGGUUCCGUU
438
TAGTGACTT

AGTGACTT

GTGACTT



 


hsa-miR-23a
AUCACAUUGCCAGGGAUUUCC
439
GCAATGTGA

CAATGTGA

AATGTGA



 


hsa-miR-23a*
GGGGUUCCUGGGGAUGGGAUUU
440
CAGGAACCC

AGGAACCC

GGAACCC



 


hsa-miR-23b
AUCACAUUGCCAGGGAUUACC
441
GCAATGTGA

CAATGTGA

AATGTGA



 


hsa-miR-23b*
UGGGUUCCUGGCAUGCUGAUUU
442
CAGGAACCC

AGGAACCC

GGAACCC



 


hsa-miR-24
UGGCUCAGUUCAGCAGGAACAG
443
AACTGAGCC

ACTGAGCC

CTGAGCC



 


hsa-miR-24-1*
UGCCUACUGAGCUGAUAUCAGU
444
TCAGTAGGC

CAGTAGGC

AGTAGGC



 


hsa-miR-24-2*
UGCCUACUGAGCUGAAACACAG
445
TCAGTAGGC

CAGTAGGC

AGTAGGC



 


hsa-miR-25
CAUUGCACUUGUCUCGGUCUGA
446
AAGTGCAAT

AGTGCAAT

GTGCAAT



 


hsa-miR-25*
AGGCGGAGACUUGGGCAAUUG
447
GTCTCCGCC

TCTCCGCC

CTCCGCC



 


hsa-miR-26a
UUCAAGUAAUCCAGGAUAGGCU
448
ATTACTTGA

TTACTTGA

TACTTGA



 


hsa-miR-26a-1*
CCUAUUCUUGGUUACUUGCACG
449
CAAGAATAG

AAGAATAG

AGAATAG



 


hsa-miR-26a-2*
CCUAUUCUUGAUUACUUGUUUC
450
CAAGAATAG

AAGAATAG

AGAATAG



 


hsa-miR-26b
UUCAAGUAAUUCAGGAUAGGU
451
ATTACTTGA

TTACTTGA

TACTTGA



 


hsa-miR-26b*
CCUGUUCUCCAUUACUUGGCUC
452
GGAGAACAG

GAGAACAG

AGAACAG



 


hsa-miR-27a
UUCACAGUGGCUAAGUUCCGC
453
CCACTGTGA

CACTGTGA

ACTGTGA



 


hsa-miR-27a*
AGGGCUUAGCUGCUUGUGAGCA
454
GCTAAGCCC

CTAAGCCC

TAAGCCC



 


hsa-miR-27b
UUCACAGUGGCUAAGUUCUGC
455
CCACTGTGA

CACTGTGA

ACTGTGA



 


hsa-miR-27b*
AGAGCUUAGCUGAUUGGUGAAC
456
GCTAAGCTC

CTAAGCTC

TAAGCTC



 


hsa-miR-28-3p
CACUAGAUUGUGAGCUCCUGGA
457
CAATCTAGT

AATCTAGT

ATCTAGT



 


hsa-miR-28-5p
AAGGAGCUCACAGUCUAUUGAG
458
TGAGCTCCT

GAGCTCCT

AGCTCCT



 


hsa-miR-296-3p
GAGGGUUGGGUGGAGGCUCUCC
459
CCCAACCCT

CCAACCCT

CAACCCT



 


hsa-miR-296-5p
AGGGCCCCCCCUCAAUCCUGU
460
GGGGGGCCC

GGGGGCCC

GGGGCCC



 


hsa-miR-297
AUGUAUGUGUGCAUGUGCAUG
461
ACACATACA

CACATACA

ACATACA



 


hsa-miR-298
AGCAGAAGCAGGGAGGUUCUCCCA
462
TGCTTCTGC

GCTTCTGC

CTTCTGC



 


hsa-miR-299-3p
UAUGUGGGAUGGUAAACCGCUU
463
ATCCCACAT

TCCCACAT

CCCACAT



 


hsa-miR-299-5p
UGGUUUACCGUCCCACAUACAU
464
CGGTAAACC

GGTAAACC

GTAAACC



 


hsa-miR-29a
UAGCACCAUCUGAAAUCGGUUA
465
GATGGTGCT

ATGGTGCT

TGGTGCT



 


hsa-miR-29a*
ACUGAUUUCUUUUGGUGUUCAG
466
AGAAATCAG

GAAATCAG

AAATCAG



 


hsa-miR-29b
UAGCACCAUUUGAAAUCAGUGUU
467
AATGGTGCT

ATGGTGCT

TGGTGCT



 


hsa-miR-29b-1*
GCUGGUUUCAUAUGGUGGUUUAGA
468
TGAAACCAG

GAAACCAG

AAACCAG



 


hsa-miR-29b-2*
CUGGUUUCACAUGGUGGCUUAG
469
GTGAAACCA

TGAAACCA

GAAACCA



 


hsa-miR-29c
UAGCACCAUUUGAAAUCGGUUA
470
AATGGTGCT

ATGGTGCT

TGGTGCT



 


hsa-miR-29c*
UGACCGAUUUCUCCUGGUGUUC
471
AAATCGGTC

AATCGGTC

ATCGGTC



 


hsa-miR-300
UAUACAAGGGCAGACUCUCUCU
472
CCCTTGTAT

CCTTGTAT

CTTGTAT



 


hsa-miR-301a
CAGUGCAAUAGUAUUGUCAAAGC
473
TATTGCACT

ATTGCACT

TTGCACT



 


hsa-miR-301b
CAGUGCAAUGAUAUUGUCAAAGC
474
CATTGCACT

ATTGCACT

TTGCACT



 


hsa-miR-302a
UAAGUGCUUCCAUGUUUUGGUGA
475
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-302a*
ACUUAAACGUGGAUGUACUUGCU
476
ACGTTTAAG

CGTTTAAG

GTTTAAG



 


hsa-miR-302b
UAAGUGCUUCCAUGUUUUAGUAG
477
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-302b*
ACUUUAACAUGGAAGUGCUUUC
478
ATGTTAAAG

TGTTAAAG

GTTAAAG



 


hsa-miR-302c
UAAGUGCUUCCAUGUUUCAGUGG
479
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-302c*
UUUAACAUGGGGGUACCUGCUG
480
CCATGTTAA

CATGTTAA

ATGTTAA



 


hsa-miR-302d
UAAGUGCUUCCAUGUUUGAGUGU
481
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-302d*
ACUUUAACAUGGAGGCACUUGC
482
ATGTTAAAG

TGTTAAAG

GTTAAAG



 


hsa-miR-302e
UAAGUGCUUCCAUGCUU
483
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-302f
UAAUUGCUUCCAUGUUU
484
GAAGCAATT

AAGCAATT

AGCAATT



 


hsa-miR-30a
UGUAAACAUCCUCGACUGGAAG
485
GATGTTTAC

ATGTTTAC

TGTTTAC



 


hsa-miR-30a*
CUUUCAGUCGGAUGUUUGCAGC
486
CGACTGAAA

GACTGAAA

ACTGAAA



 


hsa-miR-30b
UGUAAACAUCCUACACUCAGCU
487
GATGTTTAC

ATGTTTAC

TGTTTAC



 


hsa-miR-30b*
CUGGGAGGUGGAUGUUUACUUC
488
CACCTCCCA

ACCTCCCA

CCTCCCA



 


hsa-miR-30c
UGUAAACAUCCUACACUCUCAGC
489
GATGTTTAC

ATGTTTAC

TGTTTAC



 


hsa-miR-30c-1*
CUGGGAGAGGGUUGUUUACUCC
490
CCTCTCCCA

CTCTCCCA

TCTCCCA



 


hsa-miR-30c-2*
CUGGGAGAAGGCUGUUUACUCU
491
CTTCTCCCA

TTCTCCCA

TCTCCCA



 


hsa-miR-30d
UGUAAACAUCCCCGACUGGAAG
492
GATGTTTAC

ATGTTTAC

TGTTTAC



 


hsa-miR-30d*
CUUUCAGUCAGAUGUUUGCUGC
493
TGACTGAAA

GACTGAAA

ACTGAAA



 


hsa-miR-30e
UGUAAACAUCCUUGACUGGAAG
494
GATGTTTAC

ATGTTTAC

TGTTTAC



 


hsa-miR-30e*
CUUUCAGUCGGAUGUUUACAGC
495
CGACTGAAA

GACTGAAA

ACTGAAA



 


hsa-miR-31
AGGCAAGAUGCUGGCAUAGCU
496
CATCTTGCC

ATCTTGCC

TCTTGCC



 


hsa-miR-31*
UGCUAUGCCAACAUAUUGCCAU
497
TGGCATAGC

GGCATAGC

GCATAGC



 


hsa-miR-32
UAUUGCACAUUACUAAGUUGCA
498
ATGTGCAAT

TGTGCAAT

GTGCAAT



 


hsa-miR-32*
CAAUUUAGUGUGUGUGAUAUUU
499
CACTAAATT

ACTAAATT

CTAAATT



 


hsa-miR-320a
AAAAGCUGGGUUGAGAGGGCGA
500
CCCAGCTTT

CCAGCTTT

CAGCTTT



 


hsa-miR-320b
AAAAGCUGGGUUGAGAGGGCAA
501
CCCAGCTTT

CCAGCTTT

CAGCTTT



 


hsa-miR-320c
AAAAGCUGGGUUGAGAGGGU
502
CCCAGCTTT

CCAGCTTT

CAGCTTT



 


hsa-miR-320d
AAAAGCUGGGUUGAGAGGA
503
CCCAGCTTT

CCAGCTTT

CAGCTTT



 


hsa-miR-323-3p
CACAUUACACGGUCGACCUCU
504
GTGTAATGT

TGTAATGT

GTAATGT



 


hsa-miR-323-5p
AGGUGGUCCGUGGCGCGUUCGC
505
CGGACCACC

GGACCACC

GACCACC



 


hsa-miR-324-3p
ACUGCCCCAGGUGCUGCUGG
506
CTGGGGCAG

TGGGGCAG

GGGGCAG



 


hsa-miR-324-5p
CGCAUCCCCUAGGGCAUUGGUGU
507
AGGGGATGC

GGGGATGC

GGGATGC



 


hsa-miR-325
CCUAGUAGGUGUCCAGUAAGUGU
508
ACCTACTAG

CCTACTAG

CTACTAG



 


hsa-miR-326
CCUCUGGGCCCUUCCUCCAG
509
GGCCCAGAG

GCCCAGAG

CCCAGAG



 


hsa-miR-328
CUGGCCCUCUCUGCCCUUCCGU
510
AGAGGGCCA

GAGGGCCA

AGGGCCA



 


hsa-miR-329
AACACACCUGGUUAACCUCUUU
511
CAGGTGTGT

AGGTGTGT

GGTGTGT



 


hsa-miR-330-3p
GCAAAGCACACGGCCUGCAGAGA
512
TGTGCTTTG

GTGCTTTG

TGCTTTG



 


hsa-miR-330-5p
UCUCUGGGCCUGUGUCUUAGGC
513
GGCCCAGAG

GCCCAGAG

CCCAGAG



 


hsa-miR-331-3p
GCCCCUGGGCCUAUCCUAGAA
514
GCCCAGGGG

CCCAGGGG

CCAGGGG



 


hsa-miR-331-5p
CUAGGUAUGGUCCCAGGGAUCC
515
CCATACCTA

CATACCTA

ATACCTA



 


hsa-miR-335
UCAAGAGCAAUAACGAAAAAUGU
516
TTGCTCTTG

TGCTCTTG

GCTCTTG



 


hsa-miR-335*
UUUUUCAUUAUUGCUCCUGACC
517
TAATGAAAA

AATGAAAA

ATGAAAA



 


hsa-miR-337-3p
CUCCUAUAUGAUGCCUUUCUUC
518
CATATAGGA

ATATAGGA

TATAGGA



 


hsa-miR-337-5p
GAACGGCUUCAUACAGGAGUU
519
GAAGCCGTT

AAGCCGTT

AGCCGTT



 


hsa-miR-338-3p
UCCAGCAUCAGUGAUUUUGUUG
520
TGATGCTGG

GATGCTGG

ATGCTGG



 


hsa-miR-338-5p
AACAAUAUCCUGGUGCUGAGUG
521
GGATATTGT

GATATTGT

ATATTGT



 


hsa-miR-339-3p
UGAGCGCCUCGACGACAGAGCCG
522
GAGGCGCTC

AGGCGCTC

GGCGCTC



 


hsa-miR-339-5p
UCCCUGUCCUCCAGGAGCUCACG
523
AGGACAGGG

GGACAGGG

GACAGGG



 


hsa-miR-33a
GUGCAUUGUAGUUGCAUUGCA
524
TACAATGCA

ACAATGCA

CAATGCA



 


hsa-miR-33a*
CAAUGUUUCCACAGUGCAUCAC
525
GGAAACATT

GAAACATT

AAACATT



 


hsa-miR-33b
GUGCAUUGCUGUUGCAUUGC
526
AGCAATGCA

GCAATGCA

CAATGCA



 


hsa-miR-33b*
CAGUGCCUCGGCAGUGCAGCCC
527
CGAGGCACT

GAGGCACT

AGGCACT



 


hsa-miR-340
UUAUAAAGCAAUGAGACUGAUU
528
TGCTTTATA

GCTTTATA

CTTTATA



 


hsa-miR-340*
UCCGUCUCAGUUACUUUAUAGC
529
CTGAGACGG

TGAGACGG

GAGACGG



 


hsa-miR-342-3p
UCUCACACAGAAAUCGCACCCGU
530
CTGTGTGAG

TGTGTGAG

GTGTGAG



 


hsa-miR-342-5p
AGGGGUGCUAUCUGUGAUUGA
531
TAGCACCCC

AGCACCCC

GCACCCC



 


hsa-miR-345
GCUGACUCCUAGUCCAGGGCUC
532
AGGAGTCAG

GGAGTCAG

GAGTCAG



 


hsa-miR-346
UGUCUGCCCGCAUGCCUGCCUCU
533
CGGGCAGAC

GGGCAGAC

GGCAGAC



 


hsa-miR-34a
UGGCAGUGUCUUAGCUGGUUGU
534
GACACTGCC

ACACTGCC

CACTGCC



 


hsa-miR-34a*
CAAUCAGCAAGUAUACUGCCCU
535
TTGCTGATT

TGCTGATT

GCTGATT



 


hsa-miR-34b
CAAUCACUAACUCCACUGCCAU
536
TTAGTGATT

TAGTGATT

AGTGATT



 


hsa-miR-34b*
UAGGCAGUGUCAUUAGCUGAUUG
537
ACACTGCCT

CACTGCCT

ACTGCCT



 


hsa-miR-34c-3p
AAUCACUAACCACACGGCCAGG
538
GTTAGTGAT

TTAGTGAT

TAGTGAT



 


hsa-miR-34c-5p
AGGCAGUGUAGUUAGCUGAUUGC
539
TACACTGCC

ACACTGCC

CACTGCC



 


hsa-miR-361-3p
UCCCCCAGGUGUGAUUCUGAUUU
540
ACCTGGGGG

CCTGGGGG

CTGGGGG



 


hsa-miR-361-5p
UUAUCAGAAUCUCCAGGGGUAC
541
ATTCTGATA

TTCTGATA

TCTGATA



 


hsa-miR-362-3p
AACACACCUAUUCAAGGAUUCA
542
TAGGTGTGT

AGGTGTGT

GGTGTGT



 


hsa-miR-362-5p
AAUCCUUGGAACCUAGGUGUGAGU
543
TCCAAGGAT

CCAAGGAT

CAAGGAT



 


hsa-miR-363
AAUUGCACGGUAUCCAUCUGUA
544
CCGTGCAAT

CGTGCAAT

GTGCAAT



 


hsa-miR-363*
CGGGUGGAUCACGAUGCAAUUU
545
GATCCACCC

ATCCACCC

TCCACCC



 


hsa-miR-365
UAAUGCCCCUAAAAAUCCUUAU
546
AGGGGCATT

GGGGCATT

GGGCATT



 


hsa-miR-367
AAUUGCACUUUAGCAAUGGUGA
547
AAGTGCAAT

AGTGCAAT

GTGCAAT



 


hsa-miR-367*
ACUGUUGCUAAUAUGCAACUCU
548
TAGCAACAG

AGCAACAG

GCAACAG



 


hsa-miR-369-3p
AAUAAUACAUGGUUGAUCUUU
549
ATGTATTAT

TGTATTAT

GTATTAT



 


hsa-miR-369-5p
AGAUCGACCGUGUUAUAUUCGC
550
CGGTCGATC

GGTCGATC

GTCGATC



 


hsa-miR-370
GCCUGCUGGGGUGGAACCUGGU
551
CCCAGCAGG

CCAGCAGG

CAGCAGG



 


hsa-miR-371-3p
AAGUGCCGCCAUCUUUUGAGUGU
552
GGCGGCACT

GCGGCACT

CGGCACT



 


hsa-miR-371-5p
ACUCAAACUGUGGGGGCACU
553
CAGTTTGAG

AGTTTGAG

GTTTGAG



 


hsa-miR-372
AAAGUGCUGCGACAUUUGAGCGU
554
GCAGCACTT

CAGCACTT

AGCACTT



 


hsa-miR-373
GAAGUGCUUCGAUUUUGGGGUGU
555
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-373*
ACUCAAAAUGGGGGCGCUUUCC
556
CATTTTGAG

ATTTTGAG

TTTTGAG



 


hsa-miR-374a
UUAUAAUACAACCUGAUAAGUG
557
TGTATTATA

GTATTATA

TATTATA



 


hsa-miR-374a*
CUUAUCAGAUUGUAUUGUAAUU
558
ATCTGATAA

TCTGATAA

CTGATAA



 


hsa-miR-374b
AUAUAAUACAACCUGCUAAGUG
559
TGTATTATA

GTATTATA

TATTATA



 


hsa-miR-374b*
CUUAGCAGGUUGUAUUAUCAUU
560
ACCTGCTAA

CCTGCTAA

CTGCTAA



 


hsa-miR-375
UUUGUUCGUUCGGCUCGCGUGA
561
AACGAACAA

ACGAACAA

CGAACAA



 


hsa-miR-376a
AUCAUAGAGGAAAAUCCACGU
562
CCTCTATGA

CTCTATGA

TCTATGA



 


hsa-miR-376a*
GUAGAUUCUCCUUCUAUGAGUA
563
GAGAATCTA

AGAATCTA

GAATCTA



 


hsa-miR-376b
AUCAUAGAGGAAAAUCCAUGUU
564
CCTCTATGA

CTCTATGA

TCTATGA



 


hsa-miR-376c
AACAUAGAGGAAAUUCCACGU
565
CCTCTATGT

CTCTATGT

TCTATGT



 


hsa-miR-377
AUCACACAAAGGCAACUUUUGU
566
TTTGTGTGA

TTGTGTGA

TGTGTGA



 


hsa-miR-377*
AGAGGUUGCCCUUGGUGAAUUC
567
GGCAACCTC

GCAACCTC

CAACCTC



 


hsa-miR-378
ACUGGACUUGGAGUCAGAAGG
568
CAAGTCCAG

AAGTCCAG

AGTCCAG



 


hsa-miR-378*
CUCCUGACUCCAGGUCCUGUGU
569
GAGTCAGGA

AGTCAGGA

GTCAGGA



 


hsa-miR-379
UGGUAGACUAUGGAACGUAGG
570
TAGTCTACC

AGTCTACC

GTCTACC



 


hsa-miR-379*
UAUGUAACAUGGUCCACUAACU
571
ATGTTACAT

TGTTACAT

GTTACAT



 


hsa-miR-380
UAUGUAAUAUGGUCCACAUCUU
572
ATATTACAT

TATTACAT

ATTACAT



 


hsa-miR-380*
UGGUUGACCAUAGAACAUGCGC
573
TGGTCAACC

GGTCAACC

GTCAACC



 


hsa-miR-381
UAUACAAGGGCAAGCUCUCUGU
574
CCCTTGTAT

CCTTGTAT

CTTGTAT



 


hsa-miR-382
GAAGUUGUUCGUGGUGGAUUCG
575
GAACAACTT

AACAACTT

ACAACTT



 


hsa-miR-383
AGAUCAGAAGGUGAUUGUGGCU
576
CTTCTGATC

TTCTGATC

TCTGATC



 


hsa-miR-384
AUUCCUAGAAAUUGUUCAUA
577
TTCTAGGAA

TCTAGGAA

CTAGGAA



 


hsa-miR-409-3p
GAAUGUUGCUCGGUGAACCCCU
578
AGCAACATT

GCAACATT

CAACATT



 


hsa-miR-409-5p
AGGUUACCCGAGCAACUUUGCAU
579
CGGGTAACC

GGGTAACC

GGTAACC



 


hsa-miR-410
AAUAUAACACAGAUGGCCUGU
580
GTGTTATAT

TGTTATAT

GTTATAT



 


hsa-miR-411
UAGUAGACCGUAUAGCGUACG
581
CGGTCTACT

GGTCTACT

GTCTACT



 


hsa-miR-411*
UAUGUAACACGGUCCACUAACC
582
GTGTTACAT

TGTTACAT

GTTACAT



 


hsa-miR-412
ACUUCACCUGGUCCACUAGCCGU
583
CAGGTGAAG

AGGTGAAG

GGTGAAG



 


hsa-miR-421
AUCAACAGACAUUAAUUGGGCGC
584
GTCTGTTGA

TCTGTTGA

CTGTTGA



 


hsa-miR-422a
ACUGGACUUAGGGUCAGAAGGC
585
TAAGTCCAG

AAGTCCAG

AGTCCAG



 


hsa-miR-423-3p
AGCUCGGUCUGAGGCCCCUCAGU
586
AGACCGAGC

GACCGAGC

ACCGAGC



 


hsa-miR-423-5p
UGAGGGGCAGAGAGCGAGACUUU
587
CTGCCCCTC

TGCCCCTC

GCCCCTC



 


hsa-miR-424
CAGCAGCAAUUCAUGUUUUGAA
588
ATTGCTGCT

TTGCTGCT

TGCTGCT



 


hsa-miR-424*
CAAAACGUGAGGCGCUGCUAU
589
TCACGTTTT

CACGTTTT

ACGTTTT



 


hsa-miR-425
AAUGACACGAUCACUCCCGUUGA
590
TCGTGTCAT

CGTGTCAT

GTGTCAT



 


hsa-miR-425*
AUCGGGAAUGUCGUGUCCGCCC
591
CATTCCCGA

ATTCCCGA

TTCCCGA



 


hsa-miR-429
UAAUACUGUCUGGUAAAACCGU
592
GACAGTATT

ACAGTATT

CAGTATT



 


hsa-miR-431
UGUCUUGCAGGCCGUCAUGCA
593
CTGCAAGAC

TGCAAGAC

GCAAGAC



 


hsa-miR-431*
CAGGUCGUCUUGCAGGGCUUCU
594
AGACGACCT

GACGACCT

ACGACCT



 


hsa-miR-432
UCUUGGAGUAGGUCAUUGGGUGG
595
TACTCCAAG

ACTCCAAG

CTCCAAG



 


hsa-miR-432*
CUGGAUGGCUCCUCCAUGUCU
596
AGCCATCCA

GCCATCCA

CCATCCA



 


hsa-miR-433
AUCAUGAUGGGCUCCUCGGUGU
597
CCATCATGA

CATCATGA

ATCATGA



 


hsa-miR-448
UUGCAUAUGUAGGAUGUCCCAU
598
ACATATGCA

CATATGCA

ATATGCA



 


hsa-miR-449a
UGGCAGUGUAUUGUUAGCUGGU
599
TACACTGCC

ACACTGCC

CACTGCC



 


hsa-miR-449b
AGGCAGUGUAUUGUUAGCUGGC
600
TACACTGCC

ACACTGCC

CACTGCC



 


hsa-miR-450a
UUUUGCGAUGUGUUCCUAAUAU
601
CATCGCAAA

ATCGCAAA

TCGCAAA



 


hsa-miR-450b-3p
UUGGGAUCAUUUUGCAUCCAUA
602
ATGATCCCA

TGATCCCA

GATCCCA



 


hsa-miR-450b-5p
UUUUGCAAUAUGUUCCUGAAUA
603
TATTGCAAA

ATTGCAAA

TTGCAAA



 


hsa-miR-451
AAACCGUUACCAUUACUGAGUU
604
GTAACGGTT

TAACGGTT

AACGGTT



 


hsa-miR-452
AACUGUUUGCAGAGGAAACUGA
605
GCAAACAGT

CAAACAGT

AAACAGT



 


hsa-miR-452*
CUCAUCUGCAAAGAAGUAAGUG
606
TGCAGATGA

GCAGATGA

CAGATGA



 


hsa-miR-453
AGGUUGUCCGUGGUGAGUUCGCA
607
CGGACAACC

GGACAACC

GACAACC



 


hsa-miR-454
UAGUGCAAUAUUGCUUAUAGGGU
608
TATTGCACT

ATTGCACT

TTGCACT



 


hsa-miR-454*
ACCCUAUCAAUAUUGUCUCUGC
609
TTGATAGGG

TGATAGGG

GATAGGG



 


hsa-miR-455-3p
GCAGUCCAUGGGCAUAUACAC
610
CATGGACTG

ATGGACTG

TGGACTG



 


hsa-miR-455-5p
UAUGUGCCUUUGGACUACAUCG
611
AAGGCACAT

AGGCACAT

GGCACAT



 


hsa-miR-483-3p
UCACUCCUCUCCUCCCGUCUU
612
AGAGGAGTG

GAGGAGTG

AGGAGTG



 


hsa-miR-483-5p
AAGACGGGAGGAAAGAAGGGAG
613
CTCCCGTCT

TCCCGTCT

CCCGTCT



 


hsa-miR-484
UCAGGCUCAGUCCCCUCCCGAU
614
CTGAGCCTG

TGAGCCTG

GAGCCTG



 


hsa-miR-485-3p
GUCAUACACGGCUCUCCUCUCU
615
CGTGTATGA

GTGTATGA

TGTATGA



 


hsa-miR-485-5p
AGAGGCUGGCCGUGAUGAAUUC
616
GCCAGCCTC

CCAGCCTC

CAGCCTC



 


hsa-miR-486-3p
CGGGGCAGCUCAGUACAGGAU
617
AGCTGCCCC

GCTGCCCC

CTGCCCC



 


hsa-miR-486-5p
UCCUGUACUGAGCUGCCCCGAG
618
CAGTACAGG

AGTACAGG

GTACAGG



 


hsa miR-487a
AAUCAUACAGGGACAUCCAGUU
619
CTGTATGAT

TGTATGAT

GTATGAT



 


hsa-miR-487b
AAUCGUACAGGGUCAUCCACUU
620
CTGTACGAT

TGTACGAT

GTACGAT



 


hsa-miR-488
UUGAAAGGCUAUUUCUUGGUC
621
AGCCTTTCA

GCCTTTCA

CCTTTCA



 


hsa-miR-488*
CCCAGAUAAUGGCACUCUCAA
622
ATTATCTGG

TTATCTGG

TATCTGG



 


hsa-miR-489
GUGACAUCACAUAUACGGCAGC
623
GTGATGTCA

TGATGTCA

GATGTCA



 


hsa-miR-490-3p
CAACCUGGAGGACUCCAUGCUG
624
CTCCAGGTT

TCCAGGTT

CCAGGTT



 


hsa-miR-490-5p
CCAUGGAUCUCCAGGUGGGU
625
AGATCCATG

GATCCATG

ATCCATG



 


hsa-miR-491-3p
CUUAUGCAAGAUUCCCUUCUAC
626
CTTGCATAA

TTGCATAA

TGCATAA



 


hsa-miR-491-5p
AGUGGGGAACCCUUCCAUGAGG
627
GTTCCCCAC

TTCCCCAC

TCCCCAC



 


hsa-miR-492
AGGACCUGCGGGACAAGAUUCUU
628
CGCAGGTCC

GCAGGTCC

CAGGTCC



 


nsa-miR-493
UGAAGGUCUACUGUGUGCCAGG
629
TAGACCTTC

AGACCTTC

GACCTTC



 


hsa-miR-493*
UUGUACAUGGUAGGCUUUCAUU
630
CCATGTACA

CATGTACA

ATGTACA



 


hsa-miR-494
UGAAACAUACACGGGAAACCUC
631
GTATGTTTC

TATGTTTC

ATGTTTC



 


hsa-miR-495
AAACAAACAUGGUGCACUUCUU
632
ATGTTTGTT

TGTTTGTT

GTTTGTT



 


hsa-miR-496
UGAGUAUUACAUGGCCAAUCUC
633
GTAATACTC

TAATACTC

AATACTC



 


hsa-miR-497
CAGCAGCACACUGUGGUUUGU
634
TGTGCTGCT

GTGCTGCT

TGCTGCT



 


hsa-miR-497*
CAAACCACACUGUGGUGUUAGA
635
GTGTGGTTT

TGTGGTTT

GTGGTTT



 


hsa-miR-498
UUUCAAGCCAGGGGGCGUUUUUC
636
TGGCTTGAA

GGCTTGAA

GCTTGAA



 


hsa-miR-499-3p
AACAUCACAGCAAGUCUGUGCU
637
CTGTGATGT

TGTGATGT

GTGATGT



 


hsa-miR-499-5p
UUAAGACUUGCAGUGAUGUUU
638
CAAGTCTTA

AAGTCTTA

AGTCTTA



 


hsa-miR-500
UAAUCCUUGCUACCUGGGUGAGA
639
GCAAGGATT

CAAGGATT

AAGGATT



 


hsa-miR-500*
AUGCACCUGGGCAAGGAUUCUG
640
CCAGGTGCA

CAGGTGCA

AGGTGCA



 


hsa-miR-501-3p
AAUGCACCCGGGCAAGGAUUCU
641
CGGGTGCAT

GGGTGCAT

GGTGCAT



 


hsa-miR-501-5p
AAUCCUUUGUCCCUGGGUGAGA
642
ACAAAGGAT

CAAAGGAT

AAAGGAT



 


hsa-miR-502-3p
AAUGCACCUGGGCAAGGAUUCA
643
CAGGTGCAT

AGGTGCAT

GGTGCAT



 


hsa-miR-502-5p
AUCCUUGCUAUCUGGGUGCUA
644
TAGCAAGGA

AGCAAGGA

GCAAGGA



 


hsa-miR-503
UAGCAGCGGGAACAGUUCUGCAG
645
CCCGCTGCT

CCGCTGCT

CGCTGCT



 


hsa-miR-504
AGACCCUGGUCUGCACUCUAUC
646
ACCAGGGTC

CCAGGGTC

CAGGGTC



 


hsa-miR-505
CGUCAACACUUGCUGGUUUCCU
647
AGTGTTGAC

GTGTTGAC

TGTTGAC



 


hsa-miR-505*
GGGAGCCAGGAAGUAUUGAUGU
648
CCTGGCTCC

CTGGCTCC

TGGCTCC



 


hsa-miR-506
UAAGGCACCCUUCUGAGUAGA
649
GGGTGCCTT

GGTGCCTT

GTGCCTT



 


hsa-miR-507
UUUUGCACCUUUUGGAGUGAA
650
AGGTGCAAA

GGTGCAAA

GTGCAAA



 


hsa-miR-508-3p
UGAUUGUAGCCUUUUGGAGUAGA
651
GCTACAATC

CTACAATC

TACAATC



 


hsa-miR-508-5p
UACUCCAGAGGGCGUCACUCAUG
652
CTCTGGAGT

TCTGGAGT

CTGGAGT



 


hsa-miR-509-3-5p
UACUGCAGACGUGGCAAUCAUG
653
GTCTGCAGT

TCTGCAGT

CTGCAGT



 


hsa-miR-509-3p
UGAUUGGUACGUCUGUGGGUAG
654
GTACCAATC

TACCAATC

ACCAATC



 


hsa-miR-509-5p
UACUGCAGACAGUGGCAAUCA
655
GTCTGCAGT

TCTGCAGT

CTGCAGT



 


hsa-miR-510
UACUCAGGAGAGUGGCAAUCAC
656
CTCCTGAGT

TCCTGAGT

CCTGAGT



 


hsa-miR-511
GUGUCUUUUGCUCUGCAGUCA
657
CAAAAGACA

AAAAGACA

AAAGACA



 


hsa-miR-512-3p
AAGUGCUGUCAUAGCUGAGGUC
658
GACAGCACT

ACAGCACT

CAGCACT



 


hsa-miR-512-5p
CACUCAGCCUUGAGGGCACUUUC
659
AGGCTGAGT

GGCTGAGT

GCTGAGT



 


hsa-miR-513a-3p
UAAAUUUCACCUUUCUGAGAAGG
660
GTGAAATTT

TGAAATTT

GAAATTT



 


hsa-miR-513a-5p
UUCACAGGGAGGUGUCAU
661
TCCCTGTGA

CCCTGTGA

CCTGTGA



 


hsa-miR-513b
UUCACAAGGAGGUGUCAUUUAU
662
TCCTTGTGA

CCTTGTGA

CTTGTGA



 


hsa-miR-513c
UUCUCAAGGAGGUGUCGUUUAU
663
TCCTTGAGA

CCTTGAGA

CTTGAGA



 


hsa-miR-514
AUUGACACUUCUGUGAGUAGA
664
AAGTGTCAA

AGTGTCAA

GTGTCAA



 


hsa-miR-515-3p
GAGUGCCUUCUUUUGGAGCGUU
665
GAAGGCACT

AAGGCACT

AGGCACT



 


hsa-miR-515-5p
UUCUCCAAAAGAAAGCACUUUCUG
666
TTTTGGAGA

TTTGGAGA

TTGGAGA



 


hsa-miR-516a-3p
UGCUUCCUUUCAGAGGGU
667
AAAGGAAGC

AAGGAAGC

AGGAAGC



 


hsa-miR-516a-5p
UUCUCGAGGAAAGAAGCACUUUC
668
TCCTCGAGA

CCTCGAGA

CTCGAGA



 


hsa-miR-516b
AUCUGGAGGUAAGAAGCACUUU
669
ACCTCCAGA

CCTCCAGA

CTCCAGA



 


hsa-miR-517*
CCUCUAGAUGGAAGCACUGUCU
670
CATCTAGAG

ATCTAGAG

TCTAGAG



 


hsa-miR-517a
AUCGUGCAUCCCUUUAGAGUGU
671
GATGCACGA

ATGCACGA

TGCACGA



 


hsa-miR-517b
UCGUGCAUCCCUUUAGAGUGUU
672
GGATGCACG

GATGCACG

ATGCACG



 


hsa-miR-517c
AUCGUGCAUCCUUUUAGAGUGU
673
GATGCACGA

ATGCACGA

TGCACGA



 


hsa miR-518A-3p
GAAAGCGCUUCCCUUUGCUGGA
674
AAGCGCTTT

AGCGCTTT

GCGCTTT



 


hsa-miR-518b
CAAAGCGCUCCCCUUUAGAGGU
675
GAGCGCTTT

AGCGCTTT

GCGCTTT



 


hsa-miR-518c
CAAAGCGCUUCUCUUUAGAGUGU
676
AAGCGCTTT

AGCGCTTT

GCGCTTT



 


hsa-miR-518c*
UCUCUGGAGGGAAGCACUUUCUG
677
CCTCCAGAG

CTCCAGAG

TCCAGAG



 


hsa-miR-518d-3p
CAAAGCGCUUCCCUUUGGAGC
678
AAGCGCTTT

AGCGCTTT

GCGCTTT



 


hsa-miR-518d-5p
CUCUAGAGGGAAGCACUUUCUG
679
CCCTCTAGA

CCTCTAGA

CTCTAGA



 


hsa-miR-518e
AAAGCGCUUCCCUUCAGAGUG
680
GAAGCGCTT

AAGCGCTT

AGCGCTT



 


hsa-miR-518f
GAAAGCGCUUCUCUUUAGAGG
681
AAGCGCTTT

AGCGCTTT

GCGCTTT



 


hsa-miR-518f*
CUCUAGAGGGAAGCACUUUCUC
682
CCCTCTAGA

CCTCTAGA

CTCTAGA



 


hsa-miR-519a
AAAGUGCAUCCUUUUAGAGUGU
683
GATGCACTT

ATGCACTT

TGCACTT



 


nsa-miR-519a*
CUCUAGAGGGAAGCGCUUUCUG
684
CCCTCTAGA

CCTCTAGA

CTCTAGA



 


hsa-miR-519b-3p
AAAGUGCAUCCUUUUAGAGGUU
685
GATGCACTT

ATGCACTT

TGCACTT



 


hsa-miR-519c-3p
AAAGUGCAUCUUUUUAGAGGAU
686
GATGCACTT

ATGCACTT

TGCACTT



 


hsa-miR-519d
CAAAGUGCCUCCCUUUAGAGUG
687
AGGCACTTT

GGCACTTT

GCACTTT



 


hsa-miR-519e
AAGUGCCUCCUUUUAGAGUGUU
688
GGAGGCACT

GAGGCACT

AGGCACT



 


hsa-miR-519e*
UUCUCCAAAAGGGAGCACUUUC
689
TTTTGGAGA

TTTGGAGA

TTGGAGA



 


hsa-miR-520a-3p
AAAGUGCUUCCCUUUGGACUGU
690
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-520a-5p
CUCCAGAGGGAAGUACUUUCU
691
CCCTCTGGA

CCTCTGGA

CTCTGGA



 


hsa-miR-520b
AAAGUGCUUCCUUUUAGAGGG
692
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-520c-3p
AAAGUGCUUCCUUUUAGAGGGU
693
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-520d-3p
AAAGUGCUUCUCUUUGGUGGGU
694
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-520d-5p
CUACAAAGGGAAGCCCUUUC
695
CCCTTTGTA

CCTTTGTA

CTTTGTA



 


hsa-miR-520e
AAAGUGCUUCCUUUUUGAGGG
696
GAAGCACTT

AAGCACTT

AGCACTT



 


hsa-miR-520f
AAGUGCUUCCUUUUAGAGGGUU
697
GGAAGCACT

GAAGCACT

AAGCACT



 


hsa-miR-520g
ACAAAGUGCUUCCCUUUAGAGUGU
698
AGCACTTTG

GCACTTTG

CACTTTG



 


hsa-miR-520h
ACAAAGUGCUUCCCUUUAGAGU
699
AGCACTTTG

GCACTTTG

CACTTTG



 


hsa-miR-521
AACGCACUUCCCUUUAGAGUGU
700
GAAGTGCGT

AAGTGCGT

AGTGCGT



 


hsa-miR-522
AAAAUGGUUCCCUUUAGAGUGU
701
GAACCATTT

AACCATTT

ACCATTT



 


hsa-miR-523
GAACGCGCUUCCCUAUAGAGGGU
702
AAGCGCGTT

AGCGCGTT

GCGCGTT



 


hsa-miR-524-3p
GAAGGCGCUUCCCUUUGGAGU
703
AAGCGCCTT

AGCGCCTT

GCGCCTT



 


hsa-miR-524-5p
CUACAAAGGGAAGCACUUUCUC
704
CCCTTTGTA

CCTTTGTA

CTTTGTA



 


hsa-miR-525-3p
GAAGGCGCUUCCCUUUAGAGCG
705
AAGCGCCTT

AGCGCCTT

GCGCCTT



 


hsa-miR-525-5p
CUCCAGAGGGAUGCACUUUCU
706
CCCTCTGGA

CCTCTGGA

CTCTGGA



 


hsa-miR-526b
CUCUUGAGGGAAGCACUUUCUGU
707
CCCTCAAGA

CCTCAAGA

CTCAAGA



 


hsa-miR-526b*
GAAAGUGCUUCCUUUUAGAGGC
708
AAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-527
CUGCAAAGGGAAGCCCUUUC
709
CCCTTTGCA

CCTTTGCA

CTTTGCA



 


hsa-miR-532-3p
CCUCCCACACCCAAGGCUUGCA
710
GTGTGGGAG

TGTGGGAG

GTGGGAG



 


hsa-miR-532-5p
CAUGCCUUGAGUGUAGGACCGU
711
TCAAGGCAT

CAAGGCAT

AAGGCAT



 


hsa-miR-539
GGAGAAAUUAUCCUUGGUGUGU
712
TAATTTCTC

AATTTCTC

ATTTCTC



 


hsa-miR-541
UGGUGGGCACAGAAUCUGGACU
713
GTGCCCACC

TGCCCACC

GCCCACC



 


hsa-miR-541*
AAAGGAUUCUGCUGUCGGUCCCACU
714
AGAATCCTT

GAATCCTT

AATCCTT



 


hsa-miR-542-3p
UGUGACAGAUUGAUAACUGAAA
715
ATCTGTCAC

TCTGTCAC

CTGTCAC



 


hsa-miR-542-5p
UCGGGGAUCAUCAUGUCACGAGA
716
TGATCCCCG

GATCCCCG

ATCCCCG



 


hsa-miR-543
AAACAUUCGCGGUGCACUUCUU
717
GCGAATGTT

CGAATGTT

GAATGTT



 


hsa-miR-544
AUUCUGCAUUUUUAGCAAGUUC
718
AATGCAGAA

ATGCAGAA

TGCAGAA



 


hsa-miR-545
UCAGCAAACAUUUAUUGUGUGC
719
TGTTTGCTG

GTTTGCTG

TTTGCTG



 


hsa-miR-545*
UCAGUAAAUGUUUAUUAGAUGA
720
CATTTACTG

ATTTACTG

TTTACTG



 


hsa-miR-548a-3p
CAAAACUGGCAAUUACUUUUGC
721
GCCAGTTTT

CCAGTTTT

CAGTTTT



 


hsa-miR-548a-5p
AAAAGUAAUUGCGAGUUUUACC
722
AATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548b-3p
CAAGAACCUCAGUUGCUUUUGU
723
GAGGTTCTT

AGGTTCTT

GGTTCTT



 


hsa-miR-548b-5p
AAAAGUAAUUGUGGUUUUGGCC
724
AATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548c-3p
CAAAAAUCUCAAUUACUUUUGC
725
GAGATTTTT

AGATTTTT

GATTTTT



 


hsa-miR-548c-5p
AAAAGUAAUUGCGGUUUUUGCC
726
AATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548d-3p
CAAAAACCACAGUUUCUUUUGC
727
GTGGTTTTT

TGGTTTTT

GGTTTTT



 


hsa-miR-548d-5p
AAAAGUAAUUGUGGUUUUUGCC
728
AATTACTTT

ATTACTTT

TTACTTT



 


hsa miR-548e
AAAAACUGAGACUACUUUUGCA
729
CTCAGTTTT

TCAGTTTT

CAGTTTT



 


hsa-miR-548f
AAAAACUGUAAUUACUUUU
730
TACAGTTTT

ACAGTTTT

CAGTTTT



 


hsa-miR-548g
AAAACUGUAAUUACUUUUGUAC
731
TTACAGTTT

TACAGTTT

ACAGTTT



 


hsa-miR-548h
AAAAGUAAUCGCGGUUUUUGUC
732
GATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548i
AAAAGUAAUUGCGGAUUUUGCC
733
AATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548j
AAAAGUAAUUGCGGUCUUUGGU
734
AATTACTTT

ATTACTTT

TTACTTT



 


hsa-miR-548k
AAAAGUACUUGCGGAUUUUGCU
735
AAGTACTTT

AGTACTTT

GTACTTT



 


hsa-miR-548l
AAAAGUAUUUGCGGGUUUUGUC
736
AAATACTTT

AATACTTT

ATACTTT



 


hsa-miR-548m
CAAAGGUAUUUGUGGUUUUUG
737
AATACCTTT

ATACCTTT

TACCTTT



 


hsa-miR-548n
CAAAAGUAAUUGUGGAUUUUGU
738
ATTACTTTT

TTACTTTT

TACTTTT



 


nsa-miR-548o
CCAAAACUGCAGUUACUUUUGC
739
GCAGTTTTG

CAGTTTTG

AGTTTTG



 


hsa-miR-548p
UAGCAAAAACUGCAGUUACUUU
740
GTTTTTGCT

TTTTTGCT

TTTTGCT



 


hsa-miR-549
UGACAACUAUGGAUGAGCUCU
741
ATAGTTGTC

TAGTTGTC

AGTTGTC



 


hsa-miR-550
AGUGCCUGAGGGAGUAAGAGCCC
742
CTCAGGCAC

TCAGGCAC

CAGGCAC



 


hsa-miR-550*
UGUCUUACUCCCUCAGGCACAU
743
GAGTAAGAC

AGTAAGAC

GTAAGAC



 


hsa-miR-551a
GCGACCCACUCUUGGUUUCCA
744
AGTGGGTCG

GTGGGTCG

TGGGTCG



 


hsa-miR-551b
GCGACCCAUACUUGGUUUCAG
745
TATGGGTCG

ATGGGTCG

TGGGTCG



 


hsa-miR-551b*
GAAAUCAAGCGUGGGUGAGACC
746
GCTTGATTT

CTTGATTT

TTGATTT



 


hsa-miR-552
AACAGGUGACUGGUUAGACAA
747
GTCACCTGT

TCACCTGT

CACCTGT



 


hsa-miR-553
AAAACGGUGAGAUUUUGUUUU
748
TCACCGTTT

CACCGTTT

ACCGTTT



 


hsa-miR-554
GCUAGUCCUGACUCAGCCAGU
749
CAGGACTAG

AGGACTAG

GGACTAG



 


hsa-miR-555
AGGGUAAGCUGAACCUCUGAU
750
AGCTTACCC

GCTTACCC

CTTACCC



 


hsa-miR-556-3p
AUAUUACCAUUAGCUCAUCUUU
751
ATGGTAATA

TGGTAATA

GGTAATA



 


hsa-miR-556-5p
GAUGAGCUCAUUGUAAUAUGAG
752
TGAGCTCAT

GAGCTCAT

AGCTCAT



 


hsa-miR-557
GUUUGCACGGGUGGGCCUUGUCU
753
CCGTGCAAA

CGTGCAAA

GTGCAAA



 


hsa-miR-558
UGAGCUGCUGUACCAAAAU
754
CAGCAGCTC

AGCAGCTC

GCAGCTC



 


hsa-miR-559
UAAAGUAAAUAUGCACCAAAA
755
ATTTACTTT

TTTACTTT

TTACTTT



 


hsa-miR-561
CAAAGUUUAAGAUCCUUGAAGU
756
TTAAACTTT

TAAACTTT

AAACTTT



 


hsa-miR-562
AAAGUAGCUGUACCAUUUGC
757
CAGCTACTT

AGCTACTT

GCTACTT



 


hsa-miR-563
AGGUUGACAUACGUUUCCC
758
ATGTCAACC

TGTCAACC

GTCAACC



 


hsa-miR-564
AGGCACGGUGUCAGCAGGC
759
CACCGTGCC

ACCGTGCC

CCGTGCC



 


hsa-miR-566
GGGCGCCUGUGAUCCCAAC
760
ACAGGCGCC

CAGGCGCC

AGGCGCC



 


hsa-miR-567
AGUAUGUUCUUCCAGGACAGAAC
761
AGAACATAC

GAACATAC

AACATAC



 


hsa-miR-568
AUGUAUAAAUGUAUACACAC
762
ATTTATACA

TTTATACA

TTATACA



 


hsa-miR-569
AGUUAAUGAAUCCUGGAAAGU
763
TTCATTAAC

TCATTAAC

CATTAAC



 


hsa-miR-570
CGAAAACAGCAAUUACCUUUGC
764
GCTGTTTTC

CTGTTTTC

TGTTTTC



 


hsa-miR-571
UGAGUUGGCCAUCUGAGUGAG
765
GGCCAACTC

GCCAACTC

CCAACTC



 


hsa-miR-572
GUCCGCUCGGCGGUGGCCCA
766
CCGAGCGGA

CGAGCGGA

GAGCGGA



 


hsa-miR-573
CUGAAGUGAUGUGUAACUGAUCAG
767
ATCACTTCA

TCACTTCA

CACTTCA



 


hsa-miR-574-3p
CACGCUCAUGCACACACCCACA
768
CATGAGCGT

ATGAGCGT

TGAGCGT



 


hsa-miR-474-5p
UGAGUGUGUGUGUGUGAGUGUGU
769
CACACACTC

ACACACTC

CACACTC



 


hsa-miR-575
GAGCCAGUUGGACAGGAGC
770
CAACTGGCT

AACTGGCT

ACTGGCT



 


hsa-miR-576-3p
AAGAUGUGGAAAAAUUGGAAUC
771
TCCACATCT

CCACATCT

CACATCT



 


hsa-miR-576-5p
AUUCUAAUUUCUCCACGUCUUU
772
AAATTAGAA

AATTAGAA

ATTAGAA



 


hsa-miR-577
UAGAUAAAAUAUUGGUACCUG
773
ATTTTATCT

TTTTATCT

TTTATCT



 


hsa-miR-578
CUUCUUGUGCUCUAGGAUUGU
774
GCACAAGAA

CACAAGAA

ACAAGAA



 


hsa-miR-579
UUCAUUUGGUAUAAACCGCGAUU
775
ACCAAATGA

CCAAATGA

CAAATGA



 


hsa-miR-580
UUGAGAAUGAUGAAUCAUUAGG
776
TCATTCTCA

CATTCTCA

ATTCTCA



 


hsa-miR-581
UCUUGUGUUCUCUAGAUCAGU
777
GAACACAAG

AACACAAG

ACACAAG



 


hsa-miR-582-3p
UAACUGGUUGAACAACUGAACC
778
CAACCAGTT

AACCAGTT

ACCAGTT



 


hsa-miR-582-5p
UUACAGUUGUUCAACCAGUUACU
779
ACAACTGTA

CAACTGTA

AACTGTA



 


hsa-miR-583
CAAAGAGGAAGGUCCCAUUAC
780
TTCCTCTTT

TCCTCTTT

CCTCTTT



 


hsa-miR-584
UUAUGGUUUGCCUGGGACUGAG
781
CAAACCATA

AAACCATA

AACCATA



 


hsa-miR-585
UGGGCGUAUCUGUAUGCUA
782
GATACGCCC

ATACGCCC

TACGCCC



 


hsa-miR-586
UAUGCAUUGUAUUUUUAGGUCC
783
ACAATGCAT

CAATGCAT

AATGCAT



 


hsa miR-587
UUUCCAUAGGUGAUGAGUCAC
784
CCTATGGAA

CTATGGAA

TATGGAA



 


hsa-miR-588
UUGGCCACAAUGGGUUAGAAC
785
TTGTGGCCA

TGTGGCCA

GTGGCCA



 


hsa-miR-589
UGAGAACCACGUCUGCUCUGAG
786
GTGGTTCTC

TGGTTCTC

GGTTCTC



 


hsa-miR-589*
UCAGAACAAAUGCCGGUUCCCAGA
787
TTTGTTCTG

TTGTTCTG

TGTTCTG



 


hsa-miR-590-3p
UAAUUUUAUGUAUAAGCUAGU
788
CATAAAATT

ATAAAATT

TAAAATT



 


hsa-miR-590-5p
GAGCUUAUUCAUAAAAGUGCAG
789
GAATAAGCT

AATAAGCT

ATAAGCT



 


hsa-miR-591
AGACCAUGGGUUCUCAUUGU
790
CCCATGGTC

CCATGGTC

CATGGTC



 


hsa-miR-592
UUGUGUCAAUAUGCGAUGAUGU
791
ATTGACACA

TTGACACA

TGACACA



 


hsa-miR-593
UGUCUCUGCUGGGGUUUCU
792
AGCAGAGAC

GCAGAGAC

CAGAGAC



 


hsa-miR-593*
AGGCACCAGCCAGGCAUUGCUCAGC
793
GCTGGTGCC

CTGGTGCC

TGGTGCC



 


hsa-miR-595
GAAGUGUGCCGUGGUGUGUCU
794
GGCACACTT

GCACACTT

CACACTT



 


hsa-miR-596
AAGCCUGCCCGGCUCCUCGGG
795
GGGCAGGCT

GGCAGGCT

GCAGGCT



 


hsa-miR-597
UGUGUCACUCGAUGACCACUGU
796
GAGTGACAC

AGTGACAC

GTGACAC



 


hsa-miR-598
UACGUCAUCGUUGUCAUCGUCA
797
CGATGACGT

GATGACGT

ATGACGT



 


hsa-miR-599
GUUGUGUCAGUUUAUCAAAC
798
CTGACACAA

TGACACAA

GACACAA



 


hsa-miR-600
ACUUACAGACAAGAGCCUUGCUC
799
GTCTGTAAG

TCTGTAAG

CTGTAAG



 


hsa-miR-601
UGGUCUAGGAUUGUUGGAGGAG
800
TCCTAGACC

CCTAGACC

CTAGACC



 


hsa-miR-602
GACACGGGCGACAGCUGCGGCCC
801
CGCCCGTGT

GCCCGTGT

CCCGTGT



 


hsa-miR-603
CACACACUGCAAUUACUUUUGC
802
GCAGTGTGT

CAGTGTGT

AGTGTGT



 


hsa-miR-604
AGGCUGCGGAAUUCAGGAC
803
TCCGCAGCC

CCGCAGCC

CGCAGCC



 


hsa-miR-605
UAAAUCCCAUGGUGCCUUCUCCU
804
ATGGGATTT

TGGGATTT

GGGATTT



 


hsa-miR-606
AAACUACUGAAAAUCAAAGAU
805
TCAGTAGTT

CAGTAGTT

AGTAGTT



 


hsa-miR-607
GUUCAAAUCCAGAUCUAUAAC
806
GGATTTGAA

GATTTGAA

ATTTGAA



 


hsa-miR-608
AGGGGUGGUGUUGGGACAGCUCCGU
807
CACCACCCC

ACCACCCC

CCACCCC



 


hsa-miR-609
AGGGUGUUUCUCUCAUCUCU
808
GAAACACCC

AAACACCC

AACACCC



 


hsa-miR-610
UGAGCUAAAUGUGUGCUGGGA
809
ATTTAGCTC

TTTAGCTC

TTAGCTC



 


hsa-miR-611
GCGAGGACCCCUCGGGGUCUGAC
810
GGGTCCTCG

GGTCCTCG

GTCCTCG



 


hsa-miR-612
GCUGGGCAGGGCUUCUGAGCUCCUU
811
CCTGCCCAG

CTGCCCAG

TGCCCAG



 


hsa-miR-613
AGGAAUGUUCCUUCUUUGCC
812
GAACATTCC

AACATTCC

ACATTCC



 


hsa-miR-614
GAACGCCUGUUCUUGCCAGGUGG
813
ACAGGCGTT

CAGGCGTT

AGGCGTT



 


hsa-miR-615-3p
UCCGAGCCUGGGUCUCCCUCUU
814
CAGGCTCGG

AGGCTCGG

GGCTCGG



 


hsa-miR-615-5p
GGGGGUCCCCGGUGCUCGGAUC
815
GGGGACCCC

GGGACCCC

GGACCCC



 


hsa-miR-616
AGUCAUUGGAGGGUUUGAGCAG
816
TCCAATGAC

CCAATGAC

CAATGAC



 


hsa-miR-616*
ACUCAAAACCCUUCAGUGACUU
817
GGTTTTGAG

GTTTTGAG

TTTTGAG



 


hsa-miR-617
AGACUUCCCAUUUGAAGGUGGC
818
TGGGAAGTC

GGGAAGTC

GGAAGTC



 


hsa-miR-618
AAACUCUACUUGUCCUUCUGAGU
819
AGTAGAGTT

GTAGAGTT

TAGAGTT



 


hsa-miR-619
GACCUGGACAUGUUUGUGCCCAGU
820
TGTCCAGGT

GTCCAGGT

TCCAGGT



 


hsa-miR-620
AUGGAGAUAGAUAUAGAAAU
821
CTATCTCCA

TATCTCCA

ATCTCCA



 


hsa-miR-621
GGCUAGCAACAGCGCUUACCU
822
GTTGCTAGC

TTGCTAGC

TGCTAGC



 


hsa-miR-622
ACAGUCUGCUGAGGUUGGAGC
823
AGCAGACTG

GCAGACTG

CAGACTG



 


hsa-miR-623
AUCCCUUGCAGGGGCUGUUGGGU
824
TGCAAGGGA

GCAAGGGA

CAAGGGA



 


hsa-miR-624
CACAAGGUAUUGGUAUUACCU
825
ATACCTTGT

TACCTTGT

ACCTTGT



 


hsa-miR-624*
UAGUACCAGUACCUUGUGUUCA
826
ACTGGTACT

CTGGTACT

TGGTACT



 


hsa-miR-625
AGGGGGAAAGUUCUAUAGUCC
827
CTTTCCCCC

TTTCCCCC

TTCCCCC



 


hsa-miR-625*
GACUAUAGAACUUUCCCCCUCA
828
TTCTATAGT

TCTATAGT

CTATAGT



 


hsa-miR-626
AGCUGUCUGAAAAUGUCUU
829
TCAGACAGC

CAGACAGC

AGACAGC



 


hsa-miR-627
GUGAGUCUCUAAGAAAAGAGGA
830
AGAGACTCA

GAGACTCA

AGACTCA



 


hsa-miR-628-3p
UCUAGUAAGAGUGGCAGUCGA
831
TCTTACTAG

CTTACTAG

TTACTAG



 


hsa-miR-628-5p
AUGCUGACAUAUUUACUAGAGG
832
ATGTCAGCA

TGTCAGCA

GTCAGCA



 


hsa-miR-629
UGGGUUUACGUUGGGAGAACU
833
CGTAAACCC

GTAAACCC

TAAACCC



 


hsa-miR-629*
GUUCUCCCAACGUAAGCCCAGC
834
TTGGGAGAA

TGGGAGAA

GGGAGAA



 


hsa-miR-630
AGUAUUCUGUACCAGGGAAGGU
835
ACAGAATAC

CAGAATAC

AGAATAC



 


hsa-miR-631
AGACCUGGCCCAGACCUCAGC
836
GGCCAGGTC

GCCAGGTC

CCAGGTC



 


hsa-miR-632
GUGUCUGCUUCCUGUGGGA
837
AAGCAGACA

AGCAGACA

GCAGACA



 


hsa-miR-633
CUAAUAGUAUCUACCACAAUAAA
838
ATACTATTA

TACTATTA

ACTATTA



 


hsa-miR-634
AACCAGCACCCCAACUUUGGAC
839
GGTGCTGGT

GTGCTGGT

TGCTGGT



 


hsa-miR-635
ACUUGGGCACUGAAACAAUGUCC
840
GTGCCCAAG

TGCCCAAG

GCCCAAG



 


hsa-miR-636
UGUGCUUGCUCGUCCCGCCCGCA
841
AGCAAGCAC

GCAAGCAC

CAAGCAC



 


hsa-miR-637
ACUGGGGGCUUUCGGGCUCUGCGU
842
AGCCCCCAG

GCCCCCAG

CCCCCAG



 


hsa-miR-638
AGGGAUCGCGGGCGGGUGGCGGCCU
843
CGCGATCCC

GCGATCCC

CGATCCC



 


hsa-miR-639
AUCGCUGCGGUUGCGAGCGCUGU
844
CCGCAGCGA

CGCAGCGA

GCAGCGA



 


hsa-miR-640
AUGAUCCAGGAACCUGCCUCU
845
CCTGGATCA

CTGGATCA

TGGATCA



 


hsa-miR-641
AAAGACAUAGGAUAGAGUCACCUC
846
CTATGTCTT

TATGTCTT

ATGTCTT



 


hsa-miR-642
GUCCCUCUCCAAAUGUGUCUUG
847
GGAGAGGGA

GAGAGGGA

AGAGGGA



 


hsa-miR-643
ACUUGUAUGCUAGCUCAGGUAG
848
GCATACAAG

CATACAAG

ATACAAG



 


hsa-miR-644
AGUGUGGCUUUCUUAGAGC
849
AAGCCACAC

AGCCACAC

GCCACAC



 


hsa-miR-645
UCUAGGCUGGUACUGCUGA
850
CCAGCCTAG

CAGCCTAG

AGCCTAG



 


hsa-miR-646
AAGCAGCUGCCUCUGAGGC
851
GCAGCTGCT

CAGCTGCT

AGCTGCT



 


hsa-miR-647
GUGGCUGCACUCACUUCCUUC
852
GTGCAGCCA

TGCAGCCA

GCAGCCA



 


hsa-miR-648
AAGUGUGCAGGGCACUGGU
853
CTGCACACT

TGCACACT

GCACACT



 


hsa-miR-649
AAACCUGUGUUGUUCAAGAGUC
854
ACACAGGTT

CACAGGTT

ACAGGTT



 


hsa-miR-650
AGGAGGCAGCGCUCUCAGGAC
855
GCTGCCTCC

CTGCCTCC

TGCCTCC



 


hsa-miR-651
UUUAGGAUAAGCUUGACUUUUG
856
TTATCCTAA

TATCCTAA

ATCCTAA



 


hsa-miR-652
AAUGGCGCCACUAGGGUUGUG
857
TGGCGCCAT

GGCGCCAT

GCGCCAT



 


hsa-miR-653
GUGUUGAAACAAUCUCUACUG
858
GTTTCAACA

TTTCAACA

TTCAACA



 


hsa-miR-654-3p
UAUGUCUGCUGACCAUCACCUU
859
AGCAGACAT

GCAGACAT

CAGACAT



 


hsa-miR-654-5p
UGGUGGGCCGCAGAACAUGUGC
860
CGGCCCACC

GGCCCACC

GCCCACC



 


hsa-miR-655
AUAAUACAUGGUUAACCUCUUU
861
CATGTATTA

ATGTATTA

TGTATTA



 


hsa-miR-656
AAUAUUAUACAGUCAACCUCU
862
GTATAATAT

TATAATAT

ATAATAT



 


hsa-miR-657
GGCAGGUUCUCACCCUCUCUAGG
863
AGAACCTGC

GAACCTGC

AACCTGC



 


hsa-miR-658
GGCGGAGGGAAGUAGGUCCGUUGGU
864
TCCCTCCGC

CCCTCCGC

CCTCCGC



 


hsa-miR-659
CUUGGUUCAGGGAGGGUCCCCA
865
CTGAACCAA

TGAACCAA

GAACCAA



 


hsa-miR-660
UACCCAUUGCAUAUCGGAGUUG
866
GCAATGGGT

CAATGGGT

AATGGGT



 


hsa-miR-661
UGCCUGGGUCUCUGGCCUGCGCGU
867
GACCCAGGC

ACCCAGGC

CCCAGGC



 


hsa-miR-662
UCCCACGUUGUGGCCCAGCAG
868
CAACGTGGG

AACGTGGG

ACGTGGG



 


hsa-miR-663
AGGCGGGGCGCCGCGGGACCGC
869
CGCCCCGCC

GCCCCGCC

CCCCGCC



 


hsa-miR-663b
GGUGGCCCGGCCGUGCCUGAGG
870
CCGGGCCAC

CGGGCCAC

GGGCCAC



 


hsa-miR-664
UAUUCAUUUAUCCCCAGCCUACA
871
TAAATGAAT

AAATGAAT

AATGAAT



 


hsa-miR-664*
ACUGGCUAGGGAAAAUGAUUGGAU
872
CCTAGCCAG

CTAGCCAG

TAGCCAG



 


hsa-miR-665
ACCAGGAGGCUGAGGCCCCU
873
GCCTCCTGG

CCTCCTGG

CTCCTGG



 


hsa-miR-668
UGUCACUCGGCUCGGCCCACUAC
874
CCGAGTGAC

CGAGTGAC

GAGTGAC



 


hsa-miR-671-3p
UCCGGUUCUCAGGGCUCCACC
875
GAGAACCGG

AGAACCGG

GAACCGG



 


hsa-miR-671-5p
AGGAAGCCCUGGAGGGGCUGGAG
876
AGGGCTTCC

GGGCTTCC

GGCTTCC



 


hsa-miR-675
UGGUGCGGAGAGGGCCCACAGUG
877
CTCCGCACC

TCCGCACC

CCGCACC



 


hsa-miR-675b
CUGUAUGCCCUCACCGCUCA
878
GGGCATACA

GGCATACA

GCATACA



 


hsa-miR-7
UGGAAGACUAGUGAUUUUGUUGU
879
TAGTCTTCC

AGTCTTCC

GTCTTCC



 


hsa-miR-7-1*
CAACAAAUCACAGUCUGCCAUA
880
TGATTTGTT

GATTTGTT

ATTTGTT



 


hsa-miR-7-2*
CAACAAAUCCCAGUCUACCUAA
881
GGATTTGTT

GATTTGTT

ATTTGTT



 


hsa-miR-708
AAGGAGCUUACAAUCUAGCUGGG
882
TAAGCTCCT

AAGCTCCT

AGCTCCT



 


hsa-miR-708*
CAACUAGACUGUGAGCUUCUAG
883
AGTCTAGTT

GTCTAGTT

TCTAGTT



 


hsa-miR-720
UCUCGCUGGGGCCUCCA
884
CCCAGCGAG

CCAGCGAG

CAGCGAG



 


hsa-miR-744
UGCGGGGCUAGGGCUAACAGCA
885
TAGCCCCGC

AGCCCCGC

GCCCCGC



 


hsa-miR-744*
CUGUUGCCACUAACCUCAACCU
886
GTGGCAACA

TGGCAACA

GGCAACA



 


hsa-miR-758
UUUGUGACCUGGUCCACUAACC
887
AGGTCACAA

GGTCACAA

GTCACAA



 


hsa-miR-760
CGGCUCUGGGUCUGUGGGGA
888
CCCAGAGCC

CCAGAGCC

CAGAGCC



 


hsa-miR-765
UGGAGGAGAAGGAAGGUGAUG
889
TTCTCCTCC

TCTCCTCC

CTCCTCC



 


hsa-miR-766
ACUCCAGCCCCACAGCCUCAGC
890
GGGCTGGAG

GGCTGGAG

GCTGGAG



 


hsa-miR-767-3p
UCUGCUCAUACCCCAUGGUUUCU
891
TATGAGCAG

ATGAGCAG

TGAGCAG



 


hsa-miR-767-5p
UGCACCAUGGUUGUCUGAGCAUG
892
CCATGGTGC

CATGGTGC

ATGGTGC



 


hsa-miR-769-3p
CUGGGAUCUCCGGGGUCUUGGUU
893
GAGATCCCA

AGATCCCA

GATCCCA



 


hsa-miR-769-5p
UGAGACCUCUGGGUUCUGAGCU
894
AGAGGTCTC

GAGGTCTC

AGGTCTC



 


hsa-miR-770-5p
UCCAGUACCACGUGUCAGGGCCA
895
TGGTACTGG

GGTACTGG

GTACTGG



 


hsa-miR-802
CAGUAACAAAGAUUCAUCCUUGU
896
TTTGTTACT

TTGTTACT

TGTTACT



 


hsa-miR-873
GCAGGAACUUGUGAGUCUCCU
897
AAGTTCCTG

AGTTCCTG

GTTCCTG



 


hsa-miR-874
CUGCCCUGGCCCGAGGGACCGA
898
GCCAGGGCA

CCAGGGCA

CAGGGCA



 


hsa-miR-875-3p
CCUGGAAACACUGAGGUUGUG
899
TGTTTCCAG

GTTTCCAG

TTTCCAG



 


hsa-miR-875-5p
UAUACCUCAGUUUUAUCAGGUG
900
CTGAGGTAT

TGAGGTAT

GAGGTAT



 


hsa-miR-876-3p
UGGUGGUUUACAAAGUAAUUCA
901
TAAACCACC

AAACCACC

AACCACC



 


hsa-miR-876-5p
UGGAUUUCUUUGUGAAUCACCA
902
AAGAAATCC

AGAAATCC

GAAATCC



 


hsa-miR-877
GUAGAGGAGAUGGCGCAGGG
903
TCTCCTCTA

CTCCTCTA

TCCTCTA



 


hsa-miR-877*
UCCUCUUCUCCCUCCUCCCAG
904
GAGAAGAGG

AGAAGAGG

GAAGAGG



 


hsa-miR-885-3p
AGGCAGCGGGGUGUAGUGGAUA
905
CCCGCTGCC

CCGCTGCC

CGCTGCC



 


hsa-miR-885-5p
UCCAUUACACUACCCUGCCUCU
906
GTGTAATGG

TGTAATGG

GTAATGG



 


hsa-miR-886-3p
CGCGGGUGCUUACUGACCCUU
907
AGCACCCGC

GCACCCGC

CACCCGC



 


hsa-miR-886-5p
CGGGUCGGAGUUAGCUCAAGCGG
908
CTCCGACCC

TCCGACCC

CCGACCC



 


hsa-miR-887
GUGAACGGGCGCCAUCCCGAGG
909
GCCCGTTCA

CCCGTTCA

CCGTTCA



 


hsa-miR-888
UACUCAAAAAGCUGUCAGUCA
910
TTTTTGAGT

TTTTGAGT

TTTGAGT



 


hsa-miR-888*
GACUGACACCUCUUUGGGUGAA
911
GGTGTCAGT

GTGTCAGT

TGTCAGT



 


hsa-miR-889
UUAAUAUCGGACAACCAUUGU
912
CCGATATTA

CGATATTA

GATATTA



 


hsa-miR-890
UACUUGGAAAGGCAUCAGUUG
913
TTTCCAAGT

TTCCAAGT

TCCAAGT



 


hsa-miR-891a
UGCAACGAACCUGAGCCACUGA
914
GTTCGTTGC

TTCGTTGC

TCGTTGC



 


hsa-miR-891b
UGCAACUUACCUGAGUCAUUGA
915
GTAAGTTGC

TAAGTTGC

AAGTTGC



 


hsa-miR-892a
CACUGUGUCCUUUCUGCGUAG
916
GGACACAGT

GACACAGT

ACACAGT



 


hsa-miR-892b
CACUGGCUCCUUUCUGGGUAGA
917
GGAGCCAGT

GAGCCAGT

AGCCAGT



 


hsa-miR-9
UCUUUGGUUAUCUAGCUGUAUGA
918
TAACCAAAG

AACCAAAG

ACCAAAG



 


hsa-miR-9*
AUAAAGCUAGAUAACCGAAAGU
919
CTAGCTTTA

TAGCTTTA

AGCTTTA



 


hsa-miR-920
GGGGAGCUGUGGAAGCAGUA
920
ACAGCTCCC

CAGCTCCC

AGCTCCC



 


hsa-miR-921
CUAGUGAGGGACAGAACCAGGAUUC
921
CCCTCACTA

CCTCACTA

CTCACTA



 


hsa-miR-922
GCAGCAGAGAAUAGGACUACGUC
922
TCTCTGCTG

CTCTGCTG

TCTGCTG



 


hsa-miR-923
GUCAGCGGAGGAAAAGAAACU
923
CTCCGCTGA

TCCGCTGA

CCGCTGA



 


hsa-miR-924
AGAGUCUUGUGAUGUCUUGC
924
ACAAGACTC

CAAGACTC

AAGACTC



 


hsa-miR-92a
UAUUGCACUUGUCCCGGCCUGU
925
AAGTGCAAT

AGTGCAAT

GTGCAAT



 


hsa-miR-92a-1*
AGGUUGGGAUCGGUUGCAAUGCU
926
ATCCCAACC

TCCCAACC

CCCAACC



 


hsa-miR-92a-2*
GGGUGGGGAUUUGUUGCAUUAC
927
ATCCCCACC

TCCCCACC

CCCCACC



 


hsa-miR-92b
UAUUGCACUCGUCCCGGCCUCC
928
GAGTGCAAT

AGTGCAAT

GTGCAAT



 


hsa-miR-92b*
AGGGACGGGACGCGGUGCAGUG
929
TCCCGTCCC

CCCGTCCC

CCGTCCC



 


hsa-miR-93
CAAAGUGCUGUUCGUGCAGGUAG
930
CAGCACTTT

AGCACTTT

GCACTTT



 


hsa-miR-93*
ACUGCUGAGCUAGCACUUCCCG
931
GCTCAGCAG

CTCAGCAG

TCAGCAG



 


hsa-miR-933
UGUGCGCAGGGAGACCUCUCCC
932
CCTGCGCAC

CTGCGCAC

TGCGCAC



 


hsa-miR-934
UGUCUACUACUGGAGACACUGG
933
GTAGTAGAC

TAGTAGAC

AGTAGAC



 


hsa-miR-935
CCAGUUACCGCUUCCGCUACCGC
934
CGGTAACTG

GGTAACTG

GTAACTG



 


hsa-miR-936
ACAGUAGAGGGAGGAAUCGCAG
935
CCTCTACTG

CTCTACTG

TCTACTG



 


hsa-miR-937
AUCCGCGCUCUGACUCUCUGCC
936
GAGCGCGGA

AGCGCGGA

GCGCGGA



 


hsa-miR-938
UGCCCUUAAAGGUGAACCCAGU
937
TTTAAGGGC

TTAAGGGC

TAAGGGC



 


hsa-miR-939
UGGGGAGCUGAGGCUCUGGGGGUG
938
CAGCTCCCC

AGCTCCCC

GCTCCCC



 


hsa-miR-940
AAGGCAGGGCCCCCGCUCCCC
939
GCCCTGCCT

CCCTGCCT

CCTGCCT



 


hsa-miR-941
CACCCGGCUGUGUGCACAUGUGC
940
CAGCCGGGT

AGCCGGGT

GCCGGGT



 


hsa-miR-942
UCUUCUCUGUUUUGGCCAUGUG
941
ACAGAGAAG

CAGAGAAG

AGAGAAG



 


hsa-miR-943
CUGACUGUUGCCGUCCUCCAG
942
CAACAGTCA

AACAGTCA

ACAGTCA



 


hsa-miR-944
AAAUUAUUGUACAUCGGAUGAG
943
ACAATAATT

CAATAATT

AATAATT



 


hsa-miR-95
UUCAACGGGUAUUUAUUGAGCA
944
ACCCGTTGA

CCCGTTGA

CCGTTGA



 


hsa-miR-96
UUUGGCACUAGCACAUUUUUGCU
945
TAGTGCCAA

AGTGCCAA

GTGCCAA



 


hsa-miR-96*
AAUCAUGUGCAGUGCCAAUAUG
946
GCACATGAT

CACATGAT

ACATGAT



 


hsa-miR-98
UGAGGUAGUAAGUUGUAUUGUU
947
TACTACCTC

ACTACCTC

CTACCTC



 


hsa-miR-99a
AACCCGUAGAUCCGAUCUUGUG
948
TCTACGGGT

CTACGGGT

TACGGGT



 


hsa-miR-99a*
CAAGCUCGCUUCUAUGGGUCUG
949
AGCGAGCTT

GCGAGCTT

CGAGCTT



 


hsa-miR-99b
CACCCGUAGAACCGACCUUGCG
950
TCTACGGGT

CTACGGGT

TACGGGT



 


hsa-miR-99b*
CAAGCUCGUGUCUGUGGGUCCG
951
CACGAGCTT

ACGAGCTT

CGAGCTT



 


hsvl-miR-H1
UGGAAGGACGGGAAGUGGAAG
952
CGTCCTTCC

GTCCTTCC

TCCTTCC



 


hsvl-miR-H2-3p
CCUGAGCCAGGGACGAGUGCGACU
953
CTGGCTCAG

TGGCTCAG

GGCTCAG



 


hsvl-miR-H2-5p
UCGCACGCGCCCGGCACAGACU
954
GCGCGTGCG

CGCGTGCG

GCGTGCG



 


hsvl-miR-H3
CUGGGACUGUGCGGUUGGGA
955
ACAGTCCCA

CAGTCCCA

AGTCCCA



 


hsvl-miR-H4-3p
CUUGCCUGUCUAACUCGCUAGU
956
GACAGGCAA

ACAGGCAA

CAGGCAA



 


hsvl-miR-H4-5p
GGUAGAGUUUGACAGGCAAGCA
957
AAACTCTAC

AACTCTAC

ACTCTAC



 


hsvl-miR-H5
GUCAGAGAUCCAAACCCUCCGG
958
GATCTCTGA

ATCTCTGA

TCTCTGA



 


hsvl-miR-H6
CACUUCCCGUCCUUCCAUCCC
959
ACGGGAAGT

CGGGAAGT

GGGAAGT



 


kshv-miR-K12-1
AUUACAGGAAACUGGGUGUAAGC
960
TTCCTGTAA

TCCTGTAA

CCTGTAA



 


kshv-miR-K12-10a
UAGUGUUGUCCCCCCGAGUGGC
961
GACAACACT

ACAACACT

CAACACT



 


kshv-miR-K12-10b
UGGUGUUGUCCCCCCGAGUGGC
962
GACAACACC

ACAACACC

CAACACC



 


kshv-miR-K12-11
UUAAUGCUUAGCCUGUGUCCGA
963
TAAGCATTA

AAGCATTA

AGCATTA



 


kshv-miR-K12-12
ACCAGGCCACCAUUCCUCUCCG
964
GTGGCCTGG

TGGCCTGG

GGCCTGG



 


kshv-miR-K12-2
AACUGUAGUCCGGGUCGAUCUG
965
GACTACAGT

ACTACAGT

CTACAGT



 


kshv-miR-K12-3
UCACAUUCUGAGGACGGCAGCGA
966
CAGAATGTG

AGAATGTG

GAATGTG



 


kshv-miR-K12-3*
UCGCGGUCACAGAAUGUGACA
967
GTGACCGCG

TGACCGCG

GACCGCG



 


kshv-miR-K12-4-3p
UAGAAUACUGAGGCCUAGCUGA
968
CAGTATTCT

AGTATTCT

GTATTCT



 


kshv-miR-K12-4-5p
AGCUAAACCGCAGUACUCUAGG
969
CGGTTTAGC

GGTTTAGC

GTTTAGC



 


kshv-miR-K12-5
UAGGAUGCCUGGAACUUGCCGG
970
AGGCATCCT

GGCATCCT

GCATCCT



 


kshv-miR-K12-6-3p
UGAUGGUUUUCGGGCUGUUGAG
971
AAAACCATC

AAACCATC

AACCATC



 


kshv-miR-K12-6-5p
CCAGCAGCACCUAAUCCAUCGG
972
GTGCTGCTG

TGCTGCTG

GCTGCTG



 


kshv-miR-K12-7
UGAUCCCAUGUUGCUGGCGCU
973
CATGGGATC

ATGGGATC

TGGGATC



 


kshv-miR-K12-8
UAGGCGCGACUGAGAGAGCACG
974
GTCGCGCCT

TCGCGCCT

CGCGCCT



 


kshv-miR-K12-9
CUGGGUAUACGCAGCUGCGUAA
975
GTATACCCA

TATACCCA

ATACCCA



 


kshv-miR-K12-9*
ACCCAGCUGCGUAAACCCCGCU
976
GCAGCTGGG

CAGCTGGG

AGCTGGG

## Claims

1. An oligomer of a contiguous sequence of 7, 8, 9 or 10 nucleotide units in length, for use in reducing the effective amount of a microRNA target in a cell or an organism, wherein at least 70% of the nucleotide units of the oligomer are selected from the group consisting of LNA (Locked Nucleic Acid) units and 2′ substituted nucleotide analogues, and wherein at least 50% of the nucleotide units of the oligomer are LNA units, and wherein at least one of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence is a phosphorothioate internucleoside linkage, and wherein the contiguous nucleotide sequence is complementary to a corresponding renion of the microRNA sequence of miR-122 (SEQ ID NO: 150).

2. The oligomer according to claim 1, for use in the treatment of a medical disorder or disease selected from the group consisting of: hepatitis C virus infection and hypercholesterolemia and related disorders.

3. A pharmaceutical composition comprising an oligomer according to claim 1, and a pharmaceutically acceptable diluent, carrier, salt of adjuvant.

4. The pharmaceutical composition according to claim 3, wherein the composition further comprises a second independent active ingredient that is an inhibitor of the VLDL (very-low density lipoprotein) assembly pathway or an MTP (microsomal transfer protein) inhibitor.

5. A method for the treatment of a disease or medical disorder associated with the presence or over-expression of a microRNA, comprising the step of administering a the pharmaceutical composition according to claim 3 to a patient who is suffering from, or is likely to suffer from said disease or medical disorder.

6. The oligomer according to claim 1, wherein at least 75% of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

7. The oligomer according to claim 1, wherein all the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

8. The oligomer according claim 6, wherein the nucleotide analogue units are selected from the group consisting of DNA, 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, T-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′-MOE RNA unit.

9. The oligomer according to claim 6 which comprises a 3′ terminal LNA unit and a 5′ terminal LNA unit.

10. The oligomer according to claim 6, wherein the length of the oligomer is 7, 8 or 9 contiguous nucleotides, wherein the contiguous nucleotide units are independently selected from the group consisting of LNA units and 2′ substituted nucleotide analogue units.

11. The oligomer according to claim 7, wherein all of the nucleotide units of the contiguous nucleotide sequence are LNA units.

12. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 7 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.

13. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 8 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.

14. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 9 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.

15. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is 10 nucleotide units in length, all the nucleotide units are LNA units, and all the internucleoside linkages are phosphorothioate.

16. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

17. The oligomer according to claim 6, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

18. The oligomer according to claim 7, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

19. The oligomer according to claim 12, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

20. The oligomer according to claim 13, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

21. The oligomer according to claim 14, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence which is complementary to the seed sequence of miR-122 (SEQ ID NO: 150).

22. The oligomer according to claim 11, wherein said contiguous nucleotide sequence of the oligomer does not comprise a nucleotide which corresponds to the first nucleotide present in the microRNA sequence of miR-122 (SEQ ID NO: 150) counted from the 5′ end.

23. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(c) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(d) 5′-ACACTCC-3′ (SEQ ID NO: 984).

24. The oligomer according to claim 6, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(c) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(d) 5′-ACACTCC-3′ (SEQ ID NO: 984).

25. The oligomer according to claim 7, wherein the contiguous nucleotide sequence of the oligomer comprises a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(b) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(c) 5′-ACACTCC-3′ (SEQ ID NO: 984).

26. The oligomer according to claim 1, wherein the contiguous nucleotide sequence of the oligomer is a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(c) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(d) 5′-ACACTCC-3′ (SEQ ID NO: 984).

27. The oligomer according to claim 6, wherein the contiguous nucleotide sequence of the oligomer is a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(c) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(d) 5′-ACACTCC-3′ (SEQ ID NO: 984).

28. The oligomer according to claim 7, wherein the contiguous nucleotide sequence of the oligomer is a sequence selected from:
(a) 5′-GTCACACTCC-3′ (SEQ ID NO: 981),
(b) 5′-TCACACTCC-3′ (SEQ ID NO: 982),
(b) 5′-CACACTCC-3′ (SEQ ID NO: 983), or
(c) 5′-ACACTCC-3′ (SEQ ID NO: 984).

29. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 7 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.

30. The oligomer according to claim 29, wherein the oligomer sequence comprises 5′-ACACTCC-3′ (SEQ ID NO: 984).

31. The oligomer according to claim 29, wherein the oligomer sequence is 5′-ACACTCC-3′ (SEQ ID NO: 984).

32. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 8 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.

33. The oligomer according to claim 32, wherein the oligomer sequence comprises 5′-CACACTCC-3′ (SEQ ID NO: 983).

34. The oligomer according to claim 32, wherein the oligomer sequence is 5′-CACACTCC-3′ (SEQ ID NO: 983).

35. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 9 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.

36. The oligomer according to claim 35, wherein the oligomer sequence comprises 5′-TCACACTCC-3′ (SEQ ID NO: 982).

37. The oligomer according to claim 35, wherein the oligomer sequence is 5′-TCACACTCC-3′ (SEQ ID NO: 982).

38. The oligomer according to claim 11, wherein the contiguous nucleotide sequence of the oligomer is 10 nucleotide units in length, all the nucleotide units are LNA units, all the internucleoside linkages are phosphorothioate, and all cytosine LNA units are 5-methylcytosines.

39. The oligomer according to claim 38, wherein the oligomer sequence comprises 5′-GTCACACTCC-3′ (SEQ ID NO: 981).

40. The oligomer according to claim 38, wherein the oligomer sequence is 5′-GTCACACTCC-3′ (SEQ ID NO: 981).

41. The method according to claim 5, wherein the disease or medical disorder is hepatitis C(HCV).

42. The oligomer according to claim 1, wherein the oligomer is conjugated with at least one non-nucleotide or non-polynucleotide moiety.

43. The oligomer according to claim 42, wherein the non-nucleotide or non-polynucleotide moiety is selected a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combination thereof.

44. The oligomer according to claim 43, wherein the protein is an antibody.

45. The oligomer according to claim 43, wherein the polymer is polyethylene glycol.

46. The oligomer according to claim 1, wherein at least one of the internucleoside linkages present between the nucleotide units of the contiguous nucleotide sequence is not a phosphorothioate or a phosphodiester internucleoside linkage.

47. The oligomer according to claim 1, wherein at least one cytosine LNA units is not 5-methylcytosine.

48. The oligomer according to claim 1, wherein at least one cytosine LNA unit is 5-methylcytosine.

49. The oligomer according to claim 1, wherein all cytosine LNA units are 5-methylcytosines.

50. The oligomer according to claim 6, wherein at least one cytosine LNA units is not 5-methylcytosine.

51. The oligomer according to claim 6, wherein at least one cytosine LNA unit is 5-methylcytosine.

52. The oligomer according to claim 6, wherein all cytosine LNA units are 5-methylcytosines.