Treatment of colony-stimulating factor 3 (CSF3) related diseases by inhibition of natural antisene transcript to CSF3

• US 9,920,369 B2
• Filed: 06/07/2016
• Issued: 03/20/2018
• Est. Priority Date: 04/02/2010
• 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): Gibbs, Terra C
• Application Number:
  US15/175,607
• Publication Number:
  US 20160273037A1
• Time in Patent Office: 651 Days
• Field of Search: 435 61, 435 911, 435325, 435375
• 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 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 37/02 Immunomodulators
• 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: 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 2310/111 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 2310/315 : Phosphorothioates
• 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
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/178 : miRNA, siRNA or ncRNA
• CPC Class Codes: G01N 25/04 : of melting point; of freezing point; of softening point

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Colony-stimulating factor 3 (CSF3), in particular, by targeting natural antisense polynucleotides of Colony-stimulating factor 3 (CSF3). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of CSF3.

Claims

1. A method of identifying and selecting at least one modified antisense oligonucleotide specific for a target natural antisense polynucleotide of a Colony-stimulating factor 3 (CSF3) gene wherein the function of and/or expression of said CSF3 gene is modulated, comprising:

identifying at least one modified antisense oligonucleotide of 10 to 30 nucleotides in length which are at least 90% complementary to a 10 to 30 nucleotide region of a natural antisense polynucleotide of said CSF3 gene;

measuring the thermal melting point of a hybrid of said modified antisense oligonucleotide and said natural antisense polynucleotide of said CSF3 gene under stringent hybridization conditions to identify and select at least one modified antisense oligonucleotide with said hybrid having a high thermal melting point;

measuring the modulation activity of said modified antisense oligonucleotide identified as having a hybrid with a high thermal melting point; and

identifying and selecting at least one modified antisense oligonucleotide having modulating activity and binding specificity for said target natural antisense polynucleotide 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.

2. The method of claim 1, wherein a function of and/or the expression of the Colony-stimulating factor 3 (CSF3) is increased in vivo or in vitro with respect to a control.

3. The method of claim 1, wherein the at least one modified antisense oligonucleotide targets a natural antisense polynucleotide having coding and/or non-coding nucleic acid sequences of a Colony-stimulating factor 3 (CSF3) polynucleotide.

4. The method of claim 1, wherein the at least one modified antisense oligonucleotide targets a natural antisense polynucleotide having SEQ ID NO:

2.

5. The method of claim 1, wherein the at least one modified antisense oligonucleotide comprises one or more modifications selected from:

at least one modified sugar moiety, at least one modified internucleoside linkage or at least one modified nucleotide.

6. The method of claim 5, 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.

7. The method of claim 5, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from:

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

8. The method of claim 5, 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.

9. The method of claim 1, wherein the at least one modified antisense oligonucleotide comprises at least one oligonucleotide sequence set forth as SEQ ID NOS:

3 to 7.

10. The method according to claim 1 wherein the modified antisense oligonucleotide is a siRNA of 19-30 nucleotides in length.

11. The method of claim 1, wherein the modified antisense 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.

12. The method of claim 1, wherein the modified antisense 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.

13. The method of claim 1, wherein the modified antisense 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.

14. The method of claim 1, wherein the modified antisense 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.