Treatment of erythropoietin (EPO) related diseases by inhibition of natural antisense transcript to EPO

• US 10,358,645 B2
• Filed: 08/23/2017
• Issued: 07/23/2019
• Est. Priority Date: 12/04/2008
• Status: Active Grant

• Current Assignee: CURNA, Inc. (Opko Health, Inc.)
• Original Assignee: CURNA, Inc. (Opko Health, Inc.)
• Inventors: Collard, Joseph, Khorkova Sherman, Olga
• Primary Examiner(s): Whiteman, Brian
• Application Number:
  US15/684,587
• Publication Number:
  US 20180010131A1
• Time in Patent Office: 699 Days
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• 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 17/02 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 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• 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 25/22 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 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• 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 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/02 : Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• 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: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07K 14/505 : Erythropoietin [EPO]
• 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 2310/11 : Antisense
• CPC Class Codes: C12N 2310/113 : targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/178 : miRNA, siRNA or ncRNA
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

Oligonucleotide compounds modulate expression and/or function of Erythropoietin (EPO) polynucleotides and encoded products thereof. Methods for treating diseases associated with Erythropoietin (EPO) comprise administering one or more oligonucleotide compounds designed to inhibit the EPO natural antisense transcript to patients.

Claims

1. A method of identifying at least one modified oligonucleotide of 10 to 30 nucleotides in length having upregulatory activity for a target EPO polynucleotide having SEQ ID NO:

1;

determining oligonucleotides 10 to 30 consecutive nucleotides in length which are at least 95 percent complementary to an antisense polynucleotide of the EPO polynucleotide;

measuring the thermal melting point of a hybrid of said modified antisense oligonucleotide and the antisense polynucleotide of said EPO polynucleotide under stringent hybridization conditions; and

, measuring the upregulatory activity of said modified oligonucleotides by measuring EPO mRNA expression relative to a mock-transfected control.

3. The method according to claim 1 or 2 wherein the EPO natural antisense polynucleotide comprises SEQ ID NO:

2.

4. The method according to claim 1 or 2 wherein the at least one antisense oligonucleotide is a siRNA.

5. The method according to claim 1 or 2 wherein the at least one antisense oligonucleotide is a single stranded, modified antisense oligonucleotide.

6. The method according to claim 5 wherein the modifications are selected from the group consisting of at least one modified sugar moiety;

at least one modified internucleotide linkage;

at least one modified nucleotide, and combinations thereof.

7. The method according to claim 5 wherein the at least one modified sugar moiety is selected from the group consisting of a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-0-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.

8. The method according to claim 5 wherein the at least one modified nucleotide is selected from the group consisting of peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.

9. The method according to claim 5 wherein the least one modified internucleotide linkage is selected from the group consisting of phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.

10. The method according to claim 1 or 2 wherein the antisense oligonucleotides are selected from the group consisting of SEQ ID NOS:

4-6.

2. A method of identifying at least one active modified antisense oligonucleotide of 10 to 30 nucleotides in length which specifically hybridizes to a natural antisense polynucleotide of a EPO polynucleotide having SEQ ID NO:

1 and upregulates expression of said EPO polynucleotide comprising;

sequencing at least one antisense polynucleotide of the target EPO gene as a putative target;

identifying at least one modified antisense oligonucleotide which is 100 percent complementary to the antisense polynucleotide target;

measuring the thermal melting point of a hybrid of said at least one modified antisense oligonucleotide and the target antisense polynucleotide under stringent hybridization conditions; and

measuring the upregulatory activity of said at least one modified oligonucleotide.