Antagonat compositions and methods of use

• US 10,000,752 B2
• Filed: 11/17/2011
• Issued: 06/19/2018
• Est. Priority Date: 11/18/2010
• Status: Active Grant

• Current Assignee: CURNA, Inc. (Opko Health, Inc.)
• Original Assignee: CURNA, Inc. (Opko Health, Inc.)
• Inventors: Collard, Joseph, Khorkova Sherman, Olga, De Leon, Belinda
• Primary Examiner(s): POLIAKOVA-GEORGAN, EKATERINA
• Application Number:
  US13/988,063
• Publication Number:
  US 20130245099A1
• Time in Patent Office: 2,406 Days
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 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 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2330/10 naturally occurring

Abstract

Provided herein are compositions, compounds, and methods of modulating gene expression. In certain embodiments described herein is a composition, wherein the composition comprises an antagoNAT. In some embodiments, the antagoNAT is an oligonucleotide comprising modified and unmodified sugar subunits, wherein the antagoNAT hybridizes with a natural antisense transcript. Certain embodiments of the present invention provide a method for modulating gene expression in a cell comprising contacting the cell with an antagoNAT. In some embodiments, the method includes forming a hybrid comprising the antagoNAT and a natural antisense transcript of the gene, wherein the hybrid sterically blocks the normal function of the natural antisense transcript.

Claims

1. A method of upregulating the expression of a single gene in a cell comprising contacting the cell with an antagoNAT, wherein the antagoNAT is a single stranded oligonucleotide comprising 12-27 nucleoside subunits and which:

is 100% complementary to and specifically hybridizes with a complementary 12-27 nucleotide region of a non-coding natural antisense transcript of the gene; and

comprises at least one sugar modified nucleoside subunit at the 3′

terminus and at least one sugar modified nucleoside subunit at the 5′

terminus; and

further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′

nucleoside subunit and the 3′

nucleoside subunit, and at least one internal nucleoside is modified; and

wherein said antagoNAT upregulates the expression of said gene and wherein the antagoNAT comprises a compound of Formula (I), or a salt thereof;

C-A_u-[B_v-A′

_w]_x-B_y-A″

₂-C 

 

Formula (I)

wherein

each A, A′

, and A″

independently has the structure of;

2. The method according to claim 1 wherein the sugar modified and sugar unmodified nucleoside subunits each comprise a pyrimidine base or purine base and said modified sugar comprises a locked nucleic acid.

3. The method according to claim 1 wherein the internal sugar modified nucleoside subunits each comprises a pyrimidine base or purine base and wherein said internal modified subunits comprise at least one locked nucleic acid.

4. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′

position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, or O-alkylene bound to the C4′

carbon.

5. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′

position with alkoxy, halogen, or O-alkylene bound to the C4′

carbon.

6. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′

position with methoxy.

7. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′

position with O-methoxyethyl.

8. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′

position with O-methylene bound to the C4′

carbon (2′

-OCH₂-4′

) or O-ethylene bound to the C4′

carbon (2′

-OCH₂CH₂-4′

).

9. The method according to claim 1, wherein the unmodified nucleoside subunit comprises a ribose sugar.

10. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphodiester, phosphotriester, phosphorothioate, phosphorodithiate, alkylphosphonate, phosphoramidate, boranophosphate, carbonate, carbamate, acetamidate, thioether, thioformacetal internucleotide linkages, or combinations thereof.

11. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphodiester and phosphorothioate internucleotide linkages.

12. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphorothioate internucleotide linkages.

13. The method according to claim 1, wherein no more than five internal unmodified nucleosides with 2′

-deoxyribose sugar moieties are consecutive, wherein

(a) the 3′

terminus segment comprises a bicyclic 2′

-modified sugar nucleoside and the 5′

terminus segment comprises a non-bicyclic 2′

-modified sugar nucleoside;

or

(b) the 3′

terminus segment comprises a non-bicyclic 2′

-modified sugar nucleoside and the 5′

terminus segment comprises a bicyclic 2′

-modified sugar nucleoside.

14. The method according to claim 1 wherein the antagoNAT comprises a compound of Formula (I), or a salt thereof:

C-A_u-[B_v-A′

_w]_x-B_y-A″

_z-C 

 

Formula (I)

wherein

each A, A′

, and A″

independently has the structure of;

15. The method according to claim 14 wherein each heterocyclic base is independently selected from a purine or pyrimidine base and wherein a internucleoside linkage is selected from a phosphorthioate.

16. The method according to claim 14 wherein each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, cytosine, 2-aminoadenine, 5-methylcytosine, 5-bromouracil, or hypoxanthine and wherein at least one internucleoside linkage is selected from a phosphorothiate.

17. The method of claim 14, wherein each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, or cytosine.

18. The method of claim 14, wherein the heterocyclic base of each A′

is independently selected from uracil, thymine, or cytosine.

19. The method of claim 14, wherein each A, A′

, or A″

independently has the structure of;

20. The method of claim 14, wherein each E is independently methoxy, ethoxy, O-methylethyl, or fluoro.

21. The method of claim 14, wherein each E is methoxy.

22. The method of claim 14, wherein each E is O-methylethyl.

23. The method of claim 14, wherein each G is independently —

OP(O)₂O—

, —

OP(O)(OR)O—

, or —

OP(O)(S)O—

.

24. The method of claim 14, wherein each G is —

OP(O)(S)O—

.

25. The method of claim 14, wherein each C is hydroxy.

26. The method of claim 14, wherein v and y are independently integers of 1, 2, or 3 when K is hydroxy and x is at least one.

27. The method of claim 14, wherein v and y are independently integers of 1, 2, 3, 4, or 5 when K is hydrogen, and

(a) wherein at least one A has the structure of (Id) or (Ie) and at least one A″

has the structure of (Ic);

or

(b) wherein at least one A has the structure of (Ic) and at least one A″

has the structure of (Id) or (Ie).