Methods and compositions involving miRNA and miRNA inhibitor molecules

• US 8,563,708 B2
• Filed: 08/10/2007
• Issued: 10/22/2013
• Est. Priority Date: 11/12/2004
• Status: Expired due to Fees

• Current Assignee: Asuragen, Inc. (Bio-Techne Corporation)
• Original Assignee: Asuragen, Inc. (Bio-Techne Corporation)
• Inventors: Brown, David, Ford, Lance, Cheng, Angie, Jarvis, Rich, Byrom, Mike, Ovcharenko, Dmitriy, Devroe, Eric, Kelnar, Kevin
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US11/837,487
• Publication Number:
  US 20080176766A1
• Time in Patent Office: 2,265 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61N 5/10 X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy A61N5/01 takes precedence
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 15/111 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 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/141 : MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/312 : Phosphonates
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/344 : Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/35 : Nature of the modification
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3535 : Nitrogen
• CPC Class Codes: C12N 2310/533 : having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/12 : in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/50 : Methods for regulating/modulating their activity
• CPC Class Codes: C12N 2330/10 : naturally occurring
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/178 : miRNA, siRNA or ncRNA

Abstract

The present invention concerns methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. Moreover, the present invention concerns methods and compositions for identifying miRNAs with specific cellular functions that are relevant to therapeutic, diagnostic, and prognostic applications wherein synthetic miRNAs and/or miRNA inhibitors are used in library screening assays.

Claims

1. A synthetic RNA molecule comprising two polynucleotides each between 17 and 25 residues in length comprising

a) a first polynucleotide comprising an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and

b) a second polynucleotide comprising a complementary region whose sequence from 5′

to 3′

is 100% complementary to the miRNA sequence,

wherein i) the 5′

terminus of the second polynucleotide is replaced with a lower alkylamine group and/or ii) there are one or more sugar modifications with a 2′

oxygen-methyl (2′

O-Me) in the first or last 1 to 6 residues of the second polynucleotide.

2. The RNA molecule of claim 1, comprising one or more sugar modifications in the first 1 to 6 residues of the second polynucleotide.

3. The RNA molecule of claim 1, comprising one or more sugar modifications in the last 1 to 6 residues of the second polynucleotide.

4. The RNA molecule of claim 1, comprising one or more sugar modifications in the first or last 2 to 4 residues of the second polynucleotide.

5. The RNA molecule of claim 4, comprising more than one sugar modification in the first or last 2 to 4 residues of the second polynucleotide.

6. The RNA molecule of claim 1, comprising noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′

end of the complementary region and the corresponding nucleotides in the complementary region.

7. The RNA molecules of claim 6, wherein there is noncomplementarity with at least 2 nucleotides in the complementary region.

8. The RNA of claim 1, wherein the RNA molecule further comprises a flanking region of 1-5 nucleotides at either the 5′

or 3′

region of either or both polynucleotides.

9. A synthetic RNA molecule comprising two polynucleotides each between 17 and 25 residues in length comprising

a) a first polynucleotide comprising an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and

b) a second polynucleotide comprising a complementary region whose sequence from 5′

to 3′

is 100% complementary to the miRNA sequence,

wherein the 5′

terminus of the second polynucleotide is replaced with a lower alkylamine group.