Treatment of PAR4 related diseases by inhibition of natural antisense transcript to PAR4

• US 8,980,857 B2
• Filed: 05/14/2011
• Issued: 03/17/2015
• Est. Priority Date: 05/14/2010
• 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 ANN
• Application Number:
  US13/697,875
• Publication Number:
  US 20130065947A1
• Time in Patent Office: 1,403 Days
• Field of Search: None
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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 35/00 Antineoplastic agents
• 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 15/1138 against receptors or cell surface proteins
• 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 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/323 modified ring structure
• 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 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of PAR4, in particular, by targeting natural antisense polynucleotides of PAR4. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of PAR4.

Claims

1. A method of upregulating a function of and/or the expression of a PAR4 polynucleotide in a biological system comprising:

contacting said system with at least one antisense oligonucleotide 12 to 26 nucleotides in length wherein said at least one oligonucleotide is specific for and specifically hybridizes to a natural antisense polynucleotide of the PAR4 gene and has at least 80% sequence identity to a reverse complement of a natural antisense of a PAR4 polynucleotide;

thereby upregulating a function of and/or the expression of the PAR4 polynucleotide wherein said natural antisense polynucleotide consists essentially of SEQ ID NO;

2.

2. A method of upregulating a function of and/or the expression of a PAR4 polynucleotide in a biological system according to claim 1 comprising:

contacting said biological system with at least one antisense oligonucleotide 14 to 22 nucleotides in length wherein said at least one oligonucleotide is specific for and specifically hybridizes to a natural antisense polynucleotide of the PAR4 gene and has at least 80% sequence identity to a reverse complement of as polynucleotide comprising 14 to 22 nucleotides within the natural antisense polynucleotide having SEQ ID NO;

2;

thereby upregulating a function of and/or the expression of the PAR4 polynucleotide.

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

3 to 9.

3. A method of upregulating a function of and/or the expression of a PAR4 polynucleotide in a biological system comprising:

contacting said system with at least one antisense oligonucleotide of 14 to 24 nucleotides in length that specifically targets and specifically hybridizes to a region of a natural antisense polynucleotide of the PAR4 polynucleotide wherein said natural antisense polynucleotide comprises SEQ ID NO;

2;

thereby upregulating a function of and/or the expression of the PAR4 polynucleotide.

4. The method of claim 3, wherein a function of and/or the expression of the PAR4 is increased in vivo or in vitro with respect to a control.

5. The method of claim 3, wherein the at least one antisense oligonucleotide targets a natural antisense polynucleotide antisense to coding nucleic acid sequences of a PAR4 RNA polynucleotide.

6. The method of claim 3, wherein the at least one antisense oligonucleotide targets a natural antisense polynucleotide having overlapping sequences with a PAR4 polynucleotide.

7. The method of claim 3, 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.

8. The method of claim 7, 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.

9. The method of claim 7, 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.

10. The method of claim 7, 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.

12. A method of upregulating a function of and/or the expression of a PAR4 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 19 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for and specifically hybridizes to a natural antisense polynucleotide of a PAR4 polynucleotide, wherein said at least one siRNA oligonucleotide has at least 80% sequence identity to at least about 19 consecutive nucleic acids of the non-overlapping RNA adjacent to the PAR4 polynucleotide; and

, upregulating a function of author the expression of PAR4 in mammalian cells or tissues in vivo or in vitro.

13. The method of claim 12, wherein said oligonucleotide has at least 90% sequence identity.

14. A method of upregulating a function of and/or the expression of PAR4 in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one antisense oligonucleotide of about 12 to 24 nucleotides in length specific for and specifically hybridizing to a natural antisense strand of a PAR4 polynucleotide wherein said at least one antisense oligonucleotide has at least 80% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS;

1 or an RNA transcribed from the PAR4 polynucleotide; and

, upregulating the function and/or expression of the PAR4 in mammalian cells or tissues in vivo or in vitro.

15. A method of preventing or treating a disease associated with at least one PAR4 polynucleotide and/or at least one encoded product thereof, comprising:

administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide of 12 to 24 nucleotides in length that specifically hinds to a natural antisense sequence of said at least one PAR4 polynucleotide and upregulates expression of said at least one PAR4 polynucleotide;

thereby preventing or treating the disease associated with the at least one PAR4 polynucleotide and/or at least one encoded product thereof.

16. The method of claim 15, wherein a disease associated with the at least one PAR4 polynucleotide is selected from:

a disease or disorder associated with abnormal function and/or expression of PAR4, cancer, aberrant apoptosis, a proliferative disease or disorder, a cardiovascular disease or disorder, an inflammatory disease or disorder, a neurological disease or disorder and aging.

17. A method of inducing apoptosis in a biological system comprising administration of an oligonucleotide of 14 to 24 nucleotides in length and which is at least 90% identical to 14 to 24 consecutive nucleotides within a reverse complement of a natural antisense transcript to a PAR4 polynucleotide to said system and wherein the natural antisense transcript consists essentially of SEQ ID NO:

2.

18. The method according to claim 17 wherein the biological system is a patient cell or tissue.

19. A method of inducing apoptosis in a biological system comprising administration of an oligonucleotide of about 14 to 22 nucleotides in length to said system wherein said oligonucleotide upregulates a target gene by targeting to a natural antisense transcript of the gene and wherein said oligonucleotide is at least 80% identical to a reverse complement of the natural antisense transcript of the upregulated gene and wherein the target gene is a PAR4 gene and the natural antisense transcript comprises SEQ ID NO:

2.