Identification of novel pathways, genes and promoter motifs regulating adipogenesis

• US 7,759,318 B1
• Filed: 05/27/2005
• Issued: 07/20/2010
• Est. Priority Date: 05/28/2004
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Koo, Seongjoon, Marcusson, Eric G., Dean, Nicholas M., Perera, Ranjan
• Primary Examiner(s): Chong; Kimberly
• Application Number:
  US11/140,437
• Time in Patent Office: 1,880 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: C07K 14/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• 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 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy

Abstract

The present invention provides compounds, compositions, and methods for identification of motifs within regulatory regions of genes involved in adipogenesis and adipocyte differentiation. Compounds and compositions are provided for modulating expression of nuclear receptor/transcription factor-regulated genes. Methods for modulating the function of PPAR-γ as well as PPAR-γ-regulated gene promoters, and methods of using oligomeric compounds for diagnosis and treatment of diseases such as obesity and diabetes are also provided.

Claims

1. A method of decreasing triglyceride accumulation in a preadipocyte or adipocyte cell comprising contacting a preadipocyte or adipocyte cell with an effective amount of a compound comprising an oligonucleotide consisting of 12 to 50 linked nucleosides, wherein said oligonucleotide has a nucleobase sequence that is at least 90% complementary to SEQ ID NO:

29 as measured over the entirety of said oligonucleotide, wherein the oligonucleotide comprises at least one modified nucleobase, modified sugar, or modified internucleoside linkage, wherein said triglyceride accumulation is decreased, and wherein the compounds is single-stranded.

2. The method of claim 1 wherein the modified sugar is a 2′

-O-methoxyethyl sugar moiety, wherein the modified oligonucleoside linkage is a phosphorothioate internucleoside linkage, and wherein the modified nucleobase is a 5-methylcytosine.

3. The method of claim 1 wherein the preadipocyte or adipocyte cell is in an animal that has a metabolic disease.

4. The method of claim 3 wherein the metabolic disease is obesity or diabetes.

5. The method of claim 1, wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate internucleoside linkage.

6. The method of claim 1, wherein the modified sugar is a bicyclic sugar.

7. The method of claim 1, wherein the modified sugar is a 2′

-O-methoxyethyl sugar or a 4′

-(CH₂)_n-O-2′

bridge, wherein n is 1 or 2.

8. The method of claim 1, wherein the modified nucleobase is a 5-methylcytosine.

9. The method of claim 1, wherein the oligonucleotide comprises:

a gap segment consisting of linked 2′

-deoxynucleosides;

a 5′

wing segment consisting of linked nucleosides;

a 3′

wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′

wing segment and the 3′

wing segment, each nucleoside of each wing segment comprises a modified sugar, and each internucleoside linkage is a phosphorothioate internucleoside linkage.

10. The method of claim 1, wherein the oligonucleotide comprises:

a gap segment consisting of ten linked 2′

-deoxynucleosides;

a 5′

wing segment consisting of five linked nucleosides;

a 3′

wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′

wing segment and the 3′

wing segment, wherein each nucleoside of each wing segment comprises a 2′

-O-methoxyethyl sugar; and

wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate linkage.

11. The method of claim 10, wherein the oligonucleotide comprises at least one modified cytosine, wherein the modified cytosine is a 5-methylcytosine.

12. The method of claim 11, wherein each cytosine is a 5-methylcytosine.

13. The method of claim 12, wherein the oligonucleotide has a nucleobase sequence comprising at least 15 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85.

14. The method of claim 13, wherein the oligonucleotide has a nucleobase sequence that is 100% complementary to SEQ ID NO:

29.

15. The method of claim 12, wherein the oligonucleotide has a nucleobase sequence consisting of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85.

16. The method of claim 1, wherein the oligonucleotide has a nucleobase sequence that is 100% complementary to SEQ ID NO:

29.

17. The method of claim 1, wherein the oligonucleotide consists of 20 linked nucleosides.

18. The method of claim 1, wherein the oligonucleotide has a nucleobase sequence comprising at least 15 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85.

19. The method of claim 1, wherein the oligonucleotide has a nucleobase sequence consisting of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85.

20. The method of claim 1 further comprising inhibiting adipocyte differentiation in said preadipocyte cell.

21. The method of claim 1, wherein said oligonucleotide consists of 15 to 30 linked nucleosides.

22. The method of claim 1, wherein said nucleobase sequence is at least 95% complementary to SEQ ID NO:

29 as measured over the entirety of said oligonucleotide.

23. The method of claim 10, wherein said oligonucleotide consists of 20 linked nucleosides.