MODULATION OF TRANSTHYRETIN EXPRESSION

• US 20090082300A1
• Filed: 11/19/2008
• Published: 03/26/2009
• Est. Priority Date: 04/05/2004
• Status: Active Grant

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Jain, Ravi, Brown-Driver, Vickie L.
• Granted Patent: US 8,101,743 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• 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 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• 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
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Compounds, compositions and methods are provided for modulating the expression of transthyretin. The compositions comprise oligonucleotides, targeted to nucleic acid encoding transthyretin. Methods of using these compounds for modulation of transthyretin expression and for diagnosis and treatment of diseases and conditions associated with expression of transthyretin are provided.

Claims

1. A compound comprising a modified oligonucleotide consisting of 8 to 80 nucleobases in length, wherein said oligonucleotide has a nucleobase sequence that is at least 95% complementary to a 3′

untranslated region of a nucleic acid molecule encoding human transthyretin recited in SEQ ID NO;

4, and wherein said compound is double-stranded.

2. The compound of claim 1, wherein said modified oligonucleotide consists of 12 to 50 nucleobases in length.

3. The compound of claim 1, wherein said modified oligonucleotide consists of 15 to 30 nucleobases in length.

4. The compound of claim 1, wherein said modified oligonucleotide is a DNA oligonucleotide.

5. The compound of claim 1, wherein said modified oligonucleotide is an RNA oligonucleotide.

6. The compound of claim 1, wherein said modified oligonucleotide is a chimeric oligonucleotide.

7. The compound of claim 1, wherein at least a portion of said modified oligonucleotide hybridizes with RNA to form an oligonucleotide-RNA duplex.

8. The compound of claim 1 having at least one modified internucleoside linkage, modified sugar, or modified nucleobase.

9. The compound of claim 8, wherein at least one sugar comprises a 2′

-O-methoxyethyl.

10. The compound of claim 8, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage.

11. The compound of claim 8, wherein at least one modified nucleobase is a 5-methylcytosine.

12. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue in vitro with the compound of claim 1 so that expression of human transthyretin is inhibited.

20. The compound of claim 8, wherein the modified sugar is a bicyclic sugar.

13. A method of screening for a modulator of human transthyretin, the method comprising the steps of

contacting a preferred target segment of a nucleic acid molecule encoding human transthyretin with one or more candidate modulators of human transthyretin, and

identifying one or more modulators of human transthyretin expression which modulate the expression of human transthyretin.

14. The method of claim 13 wherein the modulator of transthyretin expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.

15. A diagnostic method for identifying a disease state comprising identifying the presence of human transthyretin in a sample using at least one of the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:

7.

16. A compound comprising a modified oligonucleotide consisting of 12 to 50 nucleobases in length targeted to a nucleic acid molecule encoding human transthyretin, wherein said compound is at least 95% complementary to SEQ ID NO:

4, wherein said compound comprises at least contiguous nucleobases complementary to a nucleotide sequence within nucleotides 572 through 619 of SEQ ID NO;

4, and wherein said compound is double-stranded.

17. The compound of claim 16, wherein said modified oligonucleotide comprises at least 8 contiguous nucleobases of a nucleobase sequence selected from the group consisting of SEQ ID NO:

83, 84, 85, 86, 87- and 88.

18. The compound of claim 16, wherein said modified oligonucleotide comprises the nucleobase sequence of SEQ ID NO:

84.

19. The compound of claim 18, wherein said compound consists of 20 nucleobases in length.

21. The compound of claim 16, wherein the modified oligonucleotide comprises:

a gap segment consisting of linked 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 and wherein each of said nucleosides of each of said wing segments comprises a modified sugar.

22. The compound of claim 21, wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked 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 of said nucleosides of each of said wing segments comprises a 2′

-O-methoxyethyl sugar; and

wherein each internucleoside linkage is a phosphorothioate linkage.

23. The compound of claim 22, wherein the modified oligonucleotide consists of 20 nucleobases in length.

24. The compound of claim 23, wherein the nucleobase sequence of the modified oligonucleotide consists of SEQ ID NO:

84.

25. A composition comprising:

a modified oligonucleotide consisting of 12 to 50 nucleobases in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to a 3′

untranslated region of the nucleobase sequence recited in SEQ ID NO;

4, wherein said nucleobase sequence is at least 90% complementary to said 3′

untranslated region of SEQ ID NO;

4, and wherein said compound is double-stranded, or a salt thereof and,

a pharmaceutically acceptable carrier or diluent.

26. The compound of claim 25, wherein said nucleobase sequence is at least 95% complementary to said 3′

untranslated region of SEQ ID NO;

4.

27. The compound of claim 25, wherein said nucleobase sequence is at least 99% complementary to said 3′

untranslated region of SEQ ID NO;

4.

28. The compound of 8, wherein the modified sugar is a sugar mimetic.