OLIGONUCLEOTIDES FOR MODULATING TARGET RNA ACTIVITY

• US 20100021914A1
• Filed: 11/23/2007
• Published: 01/28/2010
• Est. Priority Date: 11/23/2006
• Status: Abandoned Application

• Current Assignee: MIRX Therapeutics APS
• Original Assignee: Querdenker APS
• Inventors: Møller, Thorleif
• Application Number:
  US12/516,205
• Publication Number:
  US 20100021914A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/12 in functional genomics, i.e. for the determination of gene function

Abstract

The present invention describes oligonucleotides that bind to microRNA target sites in target RNAs, such as mRNAs. The oligonucleotides of the invention may mediate RNase H degradation of the target RNA, mediate RNAi of the target RNA or prevent microRNA regulation of the target RNA. The oligonucleotides of the invention are useful e.g. as research tools for studying microRNA:mRNA interactions and for therapeutic development. The present invention also describes methods of identifying microRNA target sites, methods of validating microRNA target sites, methods of identifying oligonucleotides of the invention and methods of modulating the activity of a target RNA using the oligonucleotides of the invention.

Claims

1. An oligonucleotide comprising a antisense sequence that comprises a guide sequence corresponding to the seed sequence of a microRNA, with the proviso that the oligonucleotide is not a microRNA or does not comprise a sequence corresponding to the complete sequence of a microRNA.

2. The oligonucleotide of claim 1, wherein the microRNA is a human microRNA

3. The oligonucleotide of claim 1 comprising a sequence selected from the group consisting of sequences that are capable of base pairing to the complementary sequence of a sequence selected from the group consisting of position 1-20, position 1-19, position 1-18, position 1-17, position 1-16, position 1-15, position 1-14, position 1-13, position 1-12, position 1-11, position 1-10, position 1-9, position 1-8, position 1-7, position 1-6, position 2-20, position 2-19, position 2-18, position 2-17, position 2-16, position 2-15, position 2-14, position 2-13, position 2-12, position 2-11, position 2-10, position 2-9, position 2-8, position 2-7, position 2-6, position 3-20, position 3-19, position 3-18, position 3-17, position 3-16, position 3-15, position 3-14, position 3-13, position 3-12, position 3-11, position 3-10 and position 3-9 of any SEQ ID NOs:

1-723

4. The oligonucleotide of claim 1, wherein the antisense sequence comprises an sequence selected from the group consisting of position 1-20, position 1-19, position 1-18, position 1-17, position 1-16, position 1-15, position 1-14, position 1-13, position 1-12, position 1-11, position 1-10, position 1-9, position 1-8, position 1-7, position 1-6, position 2-20, position 2-19, position 2-18, position 2-17, position 2-16, position 2-15, position 2-14, position 2-13, position 2-12, position 2-11, position 2-10, position 2-9, position 2-8, position 2-7, position 2-6, position 3-20, position 3-19, position 3-18, position 3-17, position 3-16, position 3-15, position 3-14, position 3-13, position 3-12, position 3-11, position 3-10 and position 3-9 of any SEQ ID NOs:

1-723, wherein

a. A may be exchanged with only G, C, U, T or I

b. G may be exchanged with only A or I

c. C may be exchanged with only A, U or T

d. U may be exchanged with only C, A, T or I

and wherein 3 additional positions may be exchanged with any base.

5. The oligonucleotide of claim 3, wherein

a. A may be exchanged with only G, C, U, T or I

b. G may be exchanged with only A or I

c. C may be exchanged with only A or U

d. U may be exchanged with only C, A, T or I

and wherein 3 additional positions may be exchanged with any base.

6. The oligonucleotide of claim 3, wherein

a. A may be exchanged with only C, U, T or I

b. G may be exchanged with only I

c. C may be exchanged with only A, U or T

d. U may be exchanged with only C, A, T or I

and wherein 3 additional positions may be exchanged with any base.

7. The oligonucleotide of claim 3, wherein

a. A may be exchanged with only C, U, or I

b. G may be exchanged with only I

c. C may be exchanged with only A or U

d. U may be exchanged with only C, A, T or I

and wherein 3 additional positions may be exchanged with any base.

8. The oligonucleotide of claim 3, wherein

a. A may be exchanged with only G or I

b. G may be exchanged with only I or A

c. C may be exchanged with only A, U or T

d. U may be exchanged with only C or T

and wherein 3 additional positions may be exchanged with any base.

9. The oligonucleotide of claim 3, wherein

a. A may be exchanged with only G

b. G may be exchanged with only A or G

c. C may be exchanged with only T or U

d. U may be exchanged with only C or T

and wherein 3 additional positions may be exchanged with any base.

10. The oligonucleotide of claim 3, wherein U may be exchanged with only T

and wherein 3 additional positions may be exchanged with any base.

11. The oligonucleotide of claim 1, wherein 2 additional positions may be exchanged with any base.

12. The oligonucleotide of claim 1, wherein 1 additional position may be exchanged with any base.

13. The oligonucleotide of claim 1, wherein no additional positions may be exchanged with any base.

39. The oligonucleotide of claim 1, wherein the oligonucleotide do not comprise any DNA units.

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37. The oligonucleotide comprising a repeating pattern of one or more LNA units and one or more units that are substituted in the 2′

-position.

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57. A method comprising the steps of:

a. Providing a target sequence of a target RNA regulated by a microRNA, said target sequence being the sequence of the target RNA involved in microRNA regulation.

b. Designing an oligonucleotide sequence that comprises a stretch of bases of at least 6 bases that is complementary to the target sequence

c. Synthesizing the oligonucleotide sequence of step b, thereby providing the oligonucleotide of step b, said oligonucleotide being a candidate regulator of the activity of a target RNA.

58. The method of claim 57 further comprising testing the steps of:

a. Providing a reporter system for activity of the target RNA

b. Determining the activity of the target RNA in the presence of the oligonucleotide of claim 57 step c

c. Determining the activity of the target RNA in the absence of the oligonucleotide of claim 57 step c

d. Comparing the activity levels in b and c and thereby verifying whether the oligonucleotide is indeed a capable of regulating the activity of the RNA and/or whether the potential target sequence of the RNA is indeed a target sequence.

60. The method of claim 57, wherein the target sequence of the target RNA comprises a sequence complementary to the seed sequence of a microRNAs.

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72. A method comprising the steps of

a. Providing a reporter system for expression of a target mRNA

b. Providing a oligonucleotide that is complementary to a part of the target mRNA

c. Determining the expression of the target mRNA in the presence of the oligonucleotide of step b

d. Determining the expression of the target mRNA in the absence of the oligonucleotide of step b

e. Comparing the expression levels in c and d and thereby verifying whether the oligonucleotide affect the expression of the mRNA.

75. The method of claim 72, wherein a series of oligonucleotides are provided that each are complementary to a part of the target mRNA and where the series of oligonucleotides has an overall coverage of more than 50% for a particular target region of the target mRNA and wherein each oligonucleotide in the series are tested for activity.

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