TREATMENT OF HEPATOCYTE GROWTH FACTOR (HGF) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO HGF

• US 20120329855A1
• Filed: 12/23/2010
• Published: 12/27/2012
• Est. Priority Date: 12/23/2009
• Status: Active Grant

• Current Assignee: CURNA, Inc. (Opko Health, Inc.)
• Original Assignee: CURNA, Inc. (Opko Health, Inc.)
• Inventors: Khorkova Sherman, Olga, Collar, Joseph
• Granted Patent: US 8,940,708 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• 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 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• 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 17/04 Antipruritics
• CPC Class Codes: A61P 17/08 Antiseborrheics
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• 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 25/08 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 25/28 : 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 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/04 : Antibacterial agents
• CPC Class Codes: A61P 31/18 : for HIV
• 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 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 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/08 : Vasodilators for multiple indications
• 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 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 2310/113 : targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/31 : of the backbone
• CPC Class Codes: C12N 2310/311 : Phosphotriesters
• CPC Class Codes: C12N 2310/3125 : Methylphosphonates
• CPC Class Codes: C12N 2310/313 : Phosphorodithioates
• CPC Class Codes: C12N 2310/314 : Phosphoramidates
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/316 : Phosphonothioates
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/3231 : having an additional ring, e.g. LNA, ENA
• 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 2600/136 : Screening for pharmacological compounds

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Hepatocyte Growth Factor (HGF), in particular, by targeting natural antisense polynucleotides of Hepatocyte Growth Factor (HGF). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of HGF.

Claims

1. A method of modulating a function of and/or the expression of a Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within nucleotides 1 to 514 of SEQ ID NO;

2, nucleotides 1 to 936 of SEQ ID NO;

3 or 1 to 1075 nucleotides of SEQ ID NO;

4;

thereby modulating a function of and/or the expression of the Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro.

13. The method of claim 1, wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS:

5 to 12.

2. A method of modulating a function of and/or the expression of a Hepatocytc Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a Hepatocytc Growth Factor (HGF) polynucleotide;

thereby modulating a function of and/or the expression of the Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro.

3. A method of modulating a function of and/or the expression of a Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the Hepatocyte Growth Factor (HGF) polynucleotide;

thereby modulating a function of and/or the expression of the Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro.

4. A method of modulating a function of and/or the expression of a Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Hepatocyte Growth Factor (HGF) polynucleotide;

thereby modulating a function of and/or the expression of the Hepatocyte Growth Factor (HGF) polynucleotide in patient cells or tissues in vivo or in vitro.

5. The method of claim 4, wherein a function of and/or the expression of the Hepatocyte Growth Factor (HGF) is increased in vivo or in vitro with respect to a control.

6. The method of claim 4, wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a Hepatocyte Growth Factor (HGF) polynucleotide.

7. The method of claim 4, wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a Hepatocytc Growth Factor (HGF) polynucleotide.

8. The method of claim 4, wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Hepatocyte Growth Factor (HGF) polynucleotide.

9. The method of claim 4, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from:

at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

10. The method of claim 9, wherein the one or more modifications comprise at least one modified sugar moiety selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

11. The method of claim 9, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from:

a phosphorothioate, 2′

-Omethoxyethyl (MOE), 2′

-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

12. The method of claim 9, wherein the one or more modifications comprise at least one modified nucleotide selected from:

a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof.

14. A method of modulating a function of and/or the expression of a Hepatocyte Growth Factor (HGF) gene in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 5 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for an antisense polynucleotide of a Hepatocyte Growth Factor (HGF) polynucleotide, wherein said at least one siRNA oligonucleotide has at least 50% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense nucleic acid molecule of the Hepatocyte Growth Factor (HGF) polynucleotide; and

, modulating a function of and/or the expression of Hepatocyte Growth Factor (HGF) in mammalian cells or tissues in vivo or in vitro.

15. The method of claim 14, wherein said oligonucleotide has at least 80% sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the Hepatocyte Growth Factor (HGF) polynucleotide.

16. A method of modulating a function of and/or the expression of Hepatocyte Growth Factor (HGF) in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide of about 5 to 30 nucleotides in length specific for noncoding and/or coding sequences of a sense and/or natural antisense strand of a Hepatocyte Growth Factor (HGF) polynucleotide wherein said at least one antisense oligonucleotide has at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS;

1 to 4; and

, modulating the function and/or expression of the Hepatocyte Growth Factor (HGF) in mammalian cells or tissues in vivo or in vitro.

17. A synthetic, modified oligonucleotide comprising at least one modification wherein the at least one modification is selected from:

at least one modified sugar moiety;

at least one modified internucleotide linkage;

at least one modified nucleotide, and combinations thereof;

wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a Hepatocyte Growth Factor (HGF) gene in vivo or in vitro as compared to a normal control.

18. The oligonucleotide of claim 17, wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate. carboxymethyl ester, and combinations thereof.

19. The oligonucleotide of claim 17, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

20. The oligonucleotide of claim 17, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.

21. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from:

a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.

22. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:

phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

23. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from:

peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.

24. The oligonucleotide of claim 17, wherein the oligonucleotide comprises at least one modified sugar moiety selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

25. The oligonucleotide of claim 17, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

26. The oligonucleotide of claim 17, wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a Hepatocyte Growth Factor (HGF) polynucleotide wherein said oligonucleotide has at least about 20% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the Hepatocyte Growth Factor (HGF) polynucleotide.

27. The oligonucleotide of claim 17, wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the Hepatocyte Growth Factor (HGF) polynucleotide.

28. The oligonucleotide of claim 17, wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Hepatocyte Growth Factor (HGF) polynucleotide in vivo or in vitro, as compared to a normal control.

29. The oligonucleotide of claim 17, wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS:

5 to 12.

30. A composition comprising one or more oligonucleotides specific for one or more Hepatocyte Growth Factor (HGF) polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof.

31. The composition of claim 30, wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS:

5 to 12.

32. The composition of claim 30, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS:

5 to 12.

33. The composition of claim 32, wherein the oligonucleotides set forth as SEQ ID NOS:

5 to 12 comprise one or more modifications or substitutions.

34. The composition of claim 33, wherein the one or more modifications are selected from:

phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.

35. A method of preventing or treating a disease associated with at least one Hepatocyte Growth Factor (HGF) polynucleotide and/or at least one encoded product thereof, comprising:

administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Hepatocyte Growth Factor (HGF) polynucleotide and modulates expression of said at least one Hepatocyte Growth Factor (HGF) polynucleotide;

thereby preventing or treating the disease associated with the at least one Hepatocyte Growth Factor (HGF) polynucleotide and/or at least one encoded product thereof.

36. The method of claim 35, wherein a disease associated with the at least one Hepatocyte Growth Factor (HGF) polynucleotide is selected from:

a disease or disorder associated with abnormal function and/or expression of HGF, an ischemic disease (e.g., ischemic heart disease and peripheral vascular disease), an arterial disease or disorder, a disease or disorder caused by disturbance which mainly involves abnormal proliferation of vascular smooth muscle cells (e.g., restenosis after percutaneous transluminal coronary angioplasty (PTCA), arterioselerosis, insufficiency of peripheral circulation, etc.), a cardiovascular disease or disorder (e.g., myocardial infarction, myocardia, peripheral angiostenosis, cardiac insufficiency, etc.), a neurological disease or disorder (e.g., nerve degeneration, peripheral neuropathy, diabetic neuropathy, neurotoxin induced lesions, injury of nerve cell by surgery, lesions of nerve cell by infection, epilepsy, head trauma, dementia, senile dementia of Alzheimer type, cerebral stroke, cerebral infarction, amyotrophic lateral sclerosis, Parkinson'"'"'s disease, Alzheimer'"'"'s disease Huntington'"'"'s disease and tumors of nerve cell and the like,), cancer, tumor, a cell proliferative disorder, an immune (such as autoimmune) disorder, an angiogenesis-related disorder, liver cirrhosis, Nonalcoholic fatty liver disease, a renal disease or disorder, renal fibrosis, rhabdomyolysis, a disease or disorder related to deficient epithelial cell growth, a pulmonary disease or disorder, gastrointestinal damage, cranial nerve disorder, a cartilage disorder, an arterial disease, pulmonary fibrosis, a cartilage injury, a hepatic disease or disorder, blood coagulopathy, plasma hypoproteinosis or wound, adenosine deaminase deficiency, Inflammatory bowel disease (e.g., Chronic Ulcerative Colitis, Crohn'"'"'s Disease, necrotizing enterocolitis, severe acute gastroenteritis, chronic gastroenteritis, cholera, chronic infections of the bowel), an immunologic disease or disorder affecting the intestine, immunodeficiency syndrome affecting the intestine, AIDS, pustulous fibrosis, fibrosis, a diseases or disorder related to reduced collagenase activity (for example, osteopetrosis or the like), a fibrosis disorder (e.g., Arterial sclerosis, chronic glomerulonephritis, cutis keloid formation, progressive systemic sclerosis (PSS), liver fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft versus host disease, scleroderma (local and systemic), Peyronie'"'"'s disease, penis fibrosis, urethrostenosis after the test using a cystoscope, inner accretion after surgery, myclofibrosis, idiopathic retroperitoneal fibrosis) characterized by excessive production of fibroblast-derived connective tissue matrix, hemophilia, a skin disease or disorder (e.g., wound, alopecia (baldness), skin ulcer, decubitus ulcer (bedsore), scar (keloid), atopic dermatitis, or skin damage following a skin graft including autotransplantation and crosstransplantation).

37. A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising:

selecting a target polynucleotide associated with a disease state;

identifying at least one oligonucleotide comprising at (cast five consecutive nucleotides which are complementary to the selected target polynucleotide or to a polynucleotide that is antisense to the selected target polynucleotide;

measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and

selecting at least one oligonucleotide for in vivo administration based on the information obtained.

38. A method of preventing or treating a skin condition associated with at least one Hepatocytc Growth Factor (HGF) polynucleotide and/or at least one encoded product thereof, comprising:

administering to a patient having a skin condition or at risk of developing a skin condition a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Hepatocyte Growth Factor (HGF) polynucleotide and modulates expression of said at least one Hepatocyte Growth Factor (HGF) polynucleotide;

thereby preventing or treating the disease skin condition associated with the at least one Hepatocyte Growth Factor (HGF) polynucleotide and/or at least one encoded product thereof.

39. The method of claim 38, wherein the skin condition is caused by caused by inflammation, light damage or aging.

40. The method of claim 39, wherein the skin condition is the development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullosa disease, exfoliative dermatitis, seborrheic dermatitis, an erythema, discoid lupus erythematosus, dermatomyositis, skin cancer, or an effect of natural aging.