SNPs of apolipoprotein B and modulation of their expression

• US 8,916,694 B2
• Filed: 01/28/2011
• Issued: 12/23/2014
• Est. Priority Date: 05/05/2004
• Status: Expired due to Fees

• Current Assignee: Kastle Therapeutics, LLC.
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Crooke, Rosanne M., Graham, Mark J., Mah, Steven
• Primary Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US13/016,917
• Publication Number:
  US 20110319469A1
• Time in Patent Office: 1,425 Days
• Field of Search: 536/23.1, 536/25.3, 536/24.5, 514/441, 435/455
• US Class Current: 536/23.1
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• 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 2320/30 Special therapeutic applications
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

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

Claims

1. An antisense compound 15 to 30 nucleobases in length which specifically hybridizes with an allelic variant of a nucleic acid of SEQ ID NO:

1 encoding human apolipoprotein B, wherein said compound inhibits the expression of apolipoprotein B mRNA by at least 10% and wherein said compound is targeted to a region that includes at least one nucleobase selected from the group consisting of;

(a) C at position 27751 of SEQ ID NO;

1;

(b) G at position 27685 of SEQ ID NO;

1;

(c) G at position 27683 of SEQ ID NO;

1;

(d) G at position 27679 of SEQ ID NO;

1;

(e) A at position 27634 of SEQ ID NO;

1;

(f) T/U at position 27627 of SEQ ID NO;

1; and

(g) G at position 27618 of SEQ ID NO;

1,

wherein G is guanine, C is cytosine, T is thymine, U is uracil, and A is adenine; and

wherein said antisense compound comprises a complementary nucleobase at said position.

2. The antisense compound of claim 1 which is 20 nucleobases in length.

3. The antisense compound of claim 1 comprising an oligonucleotide.

4. The antisense compound of claim 3 comprising a DNA oligonucleotide.

5. The antisense compound of claim 3 comprising an RNA oligonucleotide.

6. The antisense compound of claim 3 comprising a chimeric oligonucleotide.

7. The antisense compound of claim 3 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

8. The antisense compound of claim 1 having at least 80%, at least 85%, at least 90% or, at least 95% or at least 99% complementarity with said nucleic acid molecule encoding apolipoprotein B.

9. The antisense compound of claim 1 having one, two or more types of modifications, wherein the modification comprises a modified internucleoside linkage, sugar moiety, or nucleobase.

10. The antisense compound of claim 1 having at least one 2′

-O-methoxyethyl sugar moiety.

11. The antisense compound of claim 1 having at least one phosphorothioate internucleoside linkage.

12. The antisense compound of claim 1 wherein at least one cytosine is a 5-methylcytosine.

13. A method of inhibiting the expression of apolipoprotein B in a cell or tissue comprising contacting said cell or tissue with the antisense compound of claim 1 so that expression of apolipoprotein B is inhibited.

16. A method of treating an animal having a disease or condition treatable by reducing apolipoprotein B, comprising administering to said animal a therapeutically or prophylactically effective amount of the antisense compound of claim 1 so that expression of apolipoprotein B is inhibited.

17. The method of claim 16, wherein the disease or condition is a disorder of lipid metabolism.

14. A method of screening for a modulator of apolipoprotein B, the method comprising the steps of contacting a preferred target segment of a nucleic acid molecule encoding apolipoprotein B with one or more candidate modulators of apolipoprotein B, and identifying one or more modulators of apolipoprotein B expression which modulate the expression of apolipoprotein B, wherein said preferred target segment comprises at least one of:

(a) C at position 27751 of SEQ ID NO;

1;

(b) C at position 27735 of SEQ ID NO;

1;

(c) G at position 27685 of SEQ ID NO;

1;

(d) G at position 27683 of SEQ ID NO;

1;

(e) G at position 27679 of SEQ ID NO;

1;

(f) A at position 27634 of SEQ ID NO;

1;

(g) T/U at position 27627 of SEQ ID NO;

1;

or

(h) G at position 27618 of SEQ ID NO;

1,

wherein G is guanine, C is cytosine, T is thymine, U is uracil, and A is adenine.

15. The method of claim 14 wherein the modulator of apolipoprotein B 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.

18. An antisense compound 15 to 30 nucleobases in length which specifically hybridizes with an allelic variant of a nucleic acid of SEQ ID NO:

1 encoding human apolipoprotein B, wherein said compound inhibits the expression of apolipoprotein B mRNA by at least 10% and wherein said compound is targeted to a region that includes a C at position 27735 of SEQ ID NO;

1, and wherein said antisense compound has one, two or more types of modifications, wherein the modification comprises a modified internucleoside linkage, sugar moiety, or nucleobase; and

wherein the antisense compound is a chimeric antisense compound comprising;

(i) a gap segment consisting of linked deoxynucleotides,

(ii) a 5′

wing segment consisting of linked nucleotides, and

(iii) a 3′

wing segment consisting of linked nucleotides,

wherein the gap segment is positioned between the 5′

wing segment and the 3′

wing segment.

19. The antisense compound of claim 18 which is 20 nucleobases in length.

20. The antisense compound of claim 18 comprising an oligonucleotide.

21. The antisense compound of claim 20 comprising a DNA oligonucleotide.

22. The antisense compound of claim 20 comprising an RNA oligonucleotide.

23. The antisense compound of claim 20 comprising a chimeric oligonucleotide.

24. The antisense compound of claim 20 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.

25. The antisense compound of claim 18 having at least 80%, at least 85%, at least 90% or, at least 95% or at least 99% complementarity with said nucleic acid molecule encoding apolipoprotein B.

26. The antisense compound of claim 18 having at least one 2′

-O-methoxyethyl sugar moiety.

27. The antisense compound of claim 18 having at least one phosphorothioate internucleoside linkage.

28. The antisense compound of claim 18 wherein at least one cytosine is a 5-methylcytosine.

30. The antisense compound of claim 18, wherein the gap segment is flanked on both the 5′

and 3′

sides by wing segments of the same length.

31. The antisense compound of claim 18, wherein the gap segment is flanked on both the 5′

and 3′

sides by wing segments of different lengths.

32. The antisense compound of claim 30, wherein the antisense compound is 20 nucleobases in length, and wherein:

(i) the gap segment is 8 nucleotides in length, and the 5′ and

3′

wing segments are each 6 nucleotides in length;

(ii) the gap segment is 10 nucleotides in length, and the 5′ and

3′

wing segments are each 5 nucleotides in length;

(iii) the gap segment is 12 nucleotides in length, and the 5′ and

3′

wing segments are each 4 nucleotides in length;

(iv) the gap segment is 14 nucleotides in length, and the 5′ and

3′

wing segments are each 3 nucleotides in length;

(v) the gap segment is 16 nucleotides in length, and the 5′ and

3′

wing segments are each 2 nucleotides in length;

or

(vi) the gap segment is 18 nucleotides in length, and the 5′ and

3′

wing segments are each 1 nucleotide in length.

33. The antisense compound of claim 31, wherein the antisense compound is 20 nucleobases in length, and wherein the gap segment is 10 nucleotides in length, flanked on one side by a wing segment 6 nucleotides in length and flanked on the other side by a wing segment 4 nucleotides in length.

29. A method of inhibiting the expression of apolipoprotein B in a cell or tissue comprising contacting said cell or tissue with an antisense compound so that expression of apolipoprotein B is inhibited, wherein said antisense compound is 15 to 30 nucleobases in length and specifically hybridizes with an allelic variant of a nucleic acid of SEQ ID NO:

1 encoding human apolipoprotein B, wherein said antisense compound inhibits the expression of apolipoprotein B mRNA by at least 10%, wherein said antisense compound is targeted to a region that includes a C at position 27735 of SEQ ID NO;

1, and wherein said antisense compound has one, two or more types of modifications, wherein the modification comprises a modified internucleoside linkage, sugar moiety, or nucleobase.