Natural antisense and non-coding RNA transcripts as drug targets

• US 8,288,354 B2
• Filed: 12/28/2006
• Issued: 10/16/2012
• Est. Priority Date: 12/28/2005
• Status: Active Grant

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: The Scripps Research Institute
• Inventors: Wahlestedt, Claes
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US12/159,607
• Publication Number:
  US 20090258925A1
• Time in Patent Office: 2,119 Days
• Field of Search: 514/44, 536/23.1, 536/24.3, 536/24.33, 536/24.5, 435/6, 435/91.1, 435/325, 435/375
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• 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 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/22 Anxiolytics
• CPC Class Codes: A61P 25/24 Antidepressants
• 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 25/32 Alcohol-abuse
• CPC Class Codes: A61P 25/36 : Opioid-abuse
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 27/16 : Otologicals
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 39/02 : Antidotes
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/08 : Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock artificial tears A61P27/04
• 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 15/113 : 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 2310/111 : spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2330/10 : naturally occurring

Abstract

Small interfering RNA (siRNA) knock down antisense transcripts, and regulate the expression of their sense partners. This regulation can either be discordant (antisense knockdown results in sense transcript elevation) or concordant (antisense knockdown results in concomitant sense transcript reduction).

Claims

1. A method of treating a disease or condition in a subject in need of treatment thereof by modulating expression of a targeted gene encoding a drug target in a target cell, comprising:

targeting a nucleic acid molecule to a naturally-occurring anti-sense transcript of a sense strand of the targeted gene in the target cell, wherein the nucleic acid molecule targeting the anti-sense transcript is complementary to the anti-sense transcript; and

, wherein the anti-sense transcript is a coding RNA transcript or a noncoding RNA transcript lacking any extensive open reading frame, wherein the targeted gene is selected from the group consisting of CD97, TS-α

, C/EBP delta, CDC23, PINK1, HIF1α

, Gnbp3g, Adrenomedullin AM1 receptor, 3-oxoacid CoA transferase, Cathepsin W and BACE1, whereby expression of the sense strand in the target cell is modulated and expression of the gene encoding the drug target is up-regulated or down-regulated.

2. The method of claim 1, wherein the nucleic acid molecule is an interference RNA molecule.

3. The method of claim 1, wherein said nucleic acid molecule is about 5 to about 50 residues in length.

4. The method of claim 3, wherein said nucleic acid molecule is about 5 to about 40 residues in length, about 5 to about 30 residues in length, about 10 to about 30 residues in length, about 15 to about 25 residues in length, about 10 to about 25 residues in length, about 12 to about 20 residues in length, or about 20 to about 25 residues in length.

5. The method of claim 1, wherein said nucleic acid molecule:

comprises a modified backbone;

is a chimera;

is chemically linked to one or more moieties or conjugates;

is composed of linked regions;

comprises at least one modified or substituted nucleobase; and

/or combinations thereof.

6. The method of claim 5, wherein said at least one modified or substituted nucleobase comprises a modified sugar moiety or a modified base moiety.

7. The method of claim 5, wherein said at least one modified or substituted nucleobase comprises a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, or a 2′

-O-alkyl modified sugar moiety.

8. The method of claim 5, wherein said at least one modified or substituted nucleobase comprises:

an LNA;

hypoxanthine;

6-methyladenine;

5-Me pyrimidine;

5-methylcytosine;

5-hydroxymethylcytosine (HMC);

glycosyl HMC;

gentobiosyl HMC;

2-aminoadenine;

2-(methylamino)adenine;

2-(imidazolylalky)adenine;

2-(aminoalklyamino)adenine;

a heterosubstituted alkyladenine;

2-thiouracil;

2-thiothymine;

5-bromouracil;

5-hydroxymethyluracil;

8-azaguanine;

7deazaguanine;

N₆ (6-aminohexyl)adenine;

or 2,6-diaminopurine.

9. The method of claim 5, wherein said nucleic acid comprising a modified backbone comprises:

a PNA backbone linkage;

a phosphorothioate backbone linkage;

a morpholino backbone linkage;

a phosphotriester backbone linkage;

a methyl phosphonate backbone linkage;

a short chain alkyl or cycloalkyl intersugar linkage;

a short chain heteroatomic or heterocyclic intersugar linkage; and

/or combinations thereof.

10. The method of claim 5, wherein said one or more moiety or conjugate comprises:

a lipid;

a cholesteryl;

a cholic acid;

a thioether;

a thiocholesterol;

an aliphatic chain;

a phospholipid;

a polyamine;

a polyethylene glycol chain;

or adamantane acetic acid.

11. The method of claim 1, wherein the nucleic acid molecule is a ribozyme, small interfering RNA (sRNAi), double stranded RNA (dsRNA), inverted repeat, short hairpin RNA (shRNA), small temporally regulated RNA, clustered inhibitory RNA (cRNAi), radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, or a complex or compound clustered inhibitory RNA.

27. The method of claim 1 wherein modulating expression of the targeted gene is down-regulating expression of the targeted gene, the method further comprising targeting a nucleic acid molecule to a sense transcript of the target gene, wherein the sense transcript is complementary to the sense strand of the targeted gene, whereby expression of the sense strand in the target cell is down-regulated.

12. A method of treating a disease or condition in a subject in need of treatment thereof by modulating expression of a drug target protein in a target cell, comprising:

targeting a nucleic acid molecule to a naturally-occurring anti-sense transcript of a sense strand of a targeted gene selected from the group consisting of CD97, TS-α

, C/EBP delta, CDC23, PINK1, HIF1α

, Gnbp3g, Adrenomedullin AM1 receptor, 3-oxoacid CoA transferase, Cathepsin W and BACE1, wherein the nucleic acid molecule targeting the anti-sense transcript is complementary to the anti-sense transcript and the nucleic acid binds to the anti-sense transcript, whereby expression of the sense strand in the target cell is modulated and expression of the drug target protein is up-regulated or down-regulated by at least 10%.

13. The method of claim 12, wherein the nucleic acid molecule is an interference RNA molecule.

14. The method of claim 12, wherein said nucleic acid molecule is about 5 to about 50 residues in length.

15. The method of claim 14, wherein said nucleic acid molecule is about 5 to about 40 residues in length, about 5 to about 30 residues in length, about 10 to about 30 residues in length, about 15 to about 25 residues in length, about 10 to about 25 residues in length, about 12 to about 20 residues in length, or about 20 to about 25 residues in length.

16. The method of claim 12, wherein said nucleic acid molecule:

comprises a modified backbone;

is a chimera;

is chemically linked to one or more moieties or conjugates;

is composed of linked regions;

comprises at least one modified or substituted nucleobase; and

/or combinations thereof.

17. The method of claim 16, wherein said at least one modified or substituted nucleobase comprises a modified sugar moiety or a modified base moiety.

18. The method of claim 16, wherein said at least one modified or substituted nucleobase comprises a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, or a 2′

-O-alkyl modified sugar moiety.

19. The method of claim 16, wherein said at least one modified or substituted nucleobase comprises:

an LNA;

hypoxanthine;

6-methyladenine;

5-Me pyrimidine;

5-methylcytosine;

5-hydroxymethylcytosine (HMC);

glycosyl HMC;

gentobiosyl HMC;

2-aminoadenine;

2-(methylamino)adenine;

2-(imidazolylalkyl)adenine;

2-(aminoalklyamino)adenine;

a heterosubstituted alkyladenine;

2-thiouracil;

2-thiothymine;

5-bromouracil;

5-hydroxymethyluracil;

8-azaguanine;

7-deazaguanine;

N₆ (6-aminohexyl)adenine;

or 2,6-diaminopurine.

20. The method of claim 16, wherein said nucleic acid comprising a modified backbone comprises:

a PNA backbone linkage;

a phosphorothioate backbone linkage;

a morpholino backbone linkage;

a phosphotriester backbone linkage;

a methyl phosphonate backbone linkage;

a short chain alkyl or cycloalkyl intersugar linkage;

a short chain heteroatomic or heterocyclic intersugar linkage; and

/or combinations thereof.

21. The method of claim 16, wherein said one or more moiety or conjugate comprises:

a lipid;

a cholesteryl;

a cholic acid;

a thioether;

a thiocholesterol;

an aliphatic chain;

a phospholipid;

a polyamine;

a polyethylene glycol chain;

or adamantane acetic acid.

22. The method of claim 12, wherein the nucleic acid molecule is a ribozyme, small interfering RNA (sRNAi), double stranded RNA (dsRNA), inverted repeat, short hairpin RNA (shRNA), small temporally regulated RNA, clustered inhibitory RNA (cRNAi), radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, or a complex or compound clustered inhibitory RNA.

23. The method of claim 12, wherein the nucleic acid molecule is targeted wholly or in part to noncoding ribonucleotides.

24. The method of claim 12, wherein expression of the gene encoding the drug target is up-regulated by at least about 33%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100%.

28. The method of claim 12, wherein modulating expression of the targeted gene is down-regulating expression of the targeted gene, the method further comprising targeting a nucleic acid molecule to a sense transcript of the target gene, wherein the sense transcript is complementary to the sense strand of the targeted gene, whereby expression of the sense strand in the target cell is down-regulated.

25. A method of modulating expression of a targeted gene encoding a drug target, comprising:

identifying a nucleic acid molecule that binds to a naturally-occurring anti-sense transcript of a sense strand of the targeted gene encoding the drug target;

targeting the nucleic acid molecule to the anti-sense transcript of the sense strand, wherein the nucleic acid molecule targeting the anti-sense transcript is complementary to the anti-sense transcript and the nucleic acid binds to the anti-sense transcript;

wherein the anti-sense transcript is a coding RNA transcript or a noncoding RNA transcript lacking any extensive open reading frame, wherein the targeted gene is selected from the group consisting of PINK1, HIF1α

, Gnbp3g, Adrenomedullin AM1 receptor, 3-oxoacid CoA transferase, Cathepsin W and BACE1, whereby expression of the sense strand is modulated and expression of the gene encoding the drug target is up-regulated or down-regulated.

29. The method of claim 25, wherein modulating expression of the targeted gene is down-regulating expression of the targeted gene, the method further comprising targeting a nucleic acid molecule to a sense transcript of the target gene, wherein the sense transcript is complementary to the sense strand of the targeted gene, whereby expression of the sense strand in the target cell is down-regulated.

26. A method of modulating expression of a protein, comprising:

identifying a nucleic acid molecule that binds to a naturally-occurring anti-sense transcript of a sense strand of a targeted gene encoding the protein;

targeting the nucleic acid molecule to the anti-sense transcript of the sense strand, wherein the nucleic acid molecule targeting the anti-sense transcript is complementary to the anti-sense transcript and the nucleic acid binds to the anti-sense transcript;

wherein the targeted gene is selected from the group consisting of CD97, TS-α

, C/EBP delta, CDC23, PINK1, HIF1α

, Gnbp3g, Adrenomedullin AM1 receptor, 3-oxoacid CoA transferase, Cathepsin W and BACE1.

30. The method of claim 26, wherein modulating expression of the targeted gene is down-regulating expression of the targeted gene, the method further comprising targeting a nucleic acid molecule to a sense transcript of the target gene, wherein the sense transcript is complementary to the sense strand of the targeted gene, whereby expression of the sense strand in the target cell is down-regulated.