Treatment of fibroblast growth factor 21 (FGF21) related diseases by inhibition of natural antisense transcript to FGF21

• US 10,337,011 B2
• Filed: 08/23/2017
• Issued: 07/02/2019
• Est. Priority Date: 04/09/2010
• 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): Vivlemore, Tracy
• Application Number:
  US15/684,322
• Publication Number:
  US 20170355995A1
• Time in Patent Office: 678 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/12 of the kidneys
• 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 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• 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: A61P 9/12 Antihypertensives
• 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 2310/111 spanning the whole gene, or a large part of it
• 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 2310/17 Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/312 : Phosphonates
• 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 2310/3181 : Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/32 : of the sugar
• 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: C12N 2330/10 : naturally occurring
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Fibroblast growth factor 21 (FGF21), in particular, by targeting natural antisense polynucleotides of Fibroblast growth factor 21 (FGF21). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of FGF21.

Claims

1. A method of identifying at least one modified oligonucleotide of 10 to 30 nucleotides in length having modulatory activity comprising:

selecting a target FGF21 polynucleotide associated with a disease state;

identifying at least one modified oligonucleotide comprising 10 to 30 consecutive nucleotides which are 100% complementary a natural antisense polynucleotide to the selected FGF21 target polynucleotide;

measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target FGF21 natural antisense polynucleotide under stringent hybridization conditions; and

determining the modulatory activity of the at least one modified oligonucleotide by measuring the increase or decrease in the target FGF21 mRNA relative to a mock-transfected control.

3. The method according to claim 1 or 2 wherein the FGF21 natural antisense polynucleotide comprises nucleotides 1 to 4246 of SEQ ID NO:

2.

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

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

3 to 9.

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 FGF21 gene and modulates expression of said FGF21 gene comprising:

sequencing at least one natural antisense polynucleotide of the target FGF21 gene as a putative target;

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

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

measuring the modulatory activity of said at least one oligonucleotide.

5. The method according to claim 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 6 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′

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

8. The method according to claim 6 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 6 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.