RNAi modulation of MLL-AF4 and uses thereof

• US 20060166918A1
• Filed: 12/14/2005
• Published: 07/27/2006
• Est. Priority Date: 12/14/2004
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Heidenreich, Olaf, Hadwiger, Philipp, Vornlocher, Hans-Peter
• Granted Patent: US 7,361,752 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/14 interfering N.A.

Abstract

The invention relates to compositions and methods for modulating the expression of the MLL-AF4 fusion gene, and more particularly to the downregulation of MLL-AF4 by chemically modified oligonucleotides.

Claims

1. An iRNA agent comprising a sense strand, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides from the sense strand sequences of agents 1-12 provided in Table 1 (SEQ ID NOs 5, 10, 12, 14, 18, 20, 22, 24, 26, 30, and 32), and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides from the antisense sequences of agents 1-12 provided in Table 1 (SEQ ID NOs 6, 7, 11, 13, 15, 19, 21, 23, 25, 27, 31, and 33).

3. The iRNA agent of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides from the sense strand sequences of agents 1, 2, 5, and 9 provided in Table 1 (SEQ ID NOs 5, 14, and 24), and wherein the antisense strand comprises at least 15 contiguous nucleotides of the antisense sequences of agents 1, 2, 5, and 9 provided in Table 1 (SEQ ID NOs 6, 7, 15, and 25), and wherein the iRNA agent reduces the amount of MLL-AF4 mRNA present in cultured human SEM cells after incubation with these agents by more than 60% compared to cells which have not been incubated with the agent.

4. The iRNA agent of claim 1, wherein the antisense RNA strand is 30 or fewer nucleotides in length, and the duplex region of the iRNA agent is 15-30 nucleotide pairs in length.

5. The iRNA agent of claim 1, comprising at least one nucleotide overhang having 1 to 4 unpaired nucleotides.

6. The iRNA agent of claim 5, wherein the nucleotide overhang has 2 or 3 unpaired nucleotides.

7. The iRNA agent of claim 5, wherein the nucleotide overhang is at the 3′

-end of the antisense strand of the iRNA agent.

8. The iRNA agent of claim 1, wherein the iRNA agent consists of

(a) a double stranded structure formed by a sense strand consisting of a nucleotide sequence chosen from the group of:

the sequence of nucleotides of from position (5′

to 3′

) 1 to 19, or 1 to 21, of the sense strand sequences SEQ ID NOs 5, 10, 12, 14, 18, 20, 22, 24, 26, 30, and 32, and an antisense strand consisting of a nucleotide sequence chosen from the group of;

the sequence of nucleotides of from positions (5′

to 3′

) 1 to 21, or 3 to 21, of the antisense strand sequences SEQ ID NOs 6, 11, 13, 15, 19, 21, 23, 25, 27, 31, and 33, if the sense strand is chosen to consist of a sequence of nucleotides of from positions 1 to 21 of said sense strand sequences, and the sequence of nucleotides of from positions 3 to 21 of the antisense strand sequences SEQ ID NOs 6, 11, 13, 15, 19, 21, 23, 25, 27, 31, and 33, if the sense strand is chosen to consist of a sequence of nucleotides of from positions 1 to 19 of said sense strand sequences,

(b) at least one single stranded overhang of 1 to 4 unpaired nucleotides at the 3′

-end of the sense and/or antisense strand, wherein said single stranded overhang, if at the 3′

-end of the sense strand, optionally comprises the nucleotides in positions 20 and 21 of the said sense strand sequences in their respective positions, and/or, if at the 3′

-end of the antisense strand, optionally comprises the nucleotides in positions 22 and 23 of the said antisense strand sequences in their respective positions,

wherein the iRNA agent does not comprise a single stranded overhang of 1 to 4 unpaired oligonucleotides on the 3′

-end of the sense strand, if the double stranded structure is formed by a sense strand consisting of a sequence of nucleotides of from position 1 to 21 of the said sense strand sequences and an antisense strand consisting of a sequence of nucleotides of from position 1 to 21 of said antisense strand sequences.

9. The iRNA agent of claim 8, wherein the nucleotide sequence of the sense strand consists of the nucleotide sequence of SEQ ID NO:

14, and the nucleotide sequence of the antisense strand consists of the nucleotide sequence of SEQ ID NO;

15.

10. The iRNA agent of claim 1, wherein the iRNA agent reduces the amount of MLL-AF4 mRNA present in cultured human SEM cells after incubation with the iRNA agent by more than 40% compared to cells which have not been incubated with the agent.

11. The iRNA agent of claim 1, further comprising a modification that causes the iRNA agent to have increased stability in a biological sample.

12. The iRNA agent of claim 1, comprising a phosphorothioate or a 2′

-modified nucleotide.

13. The iRNA agent of claim 1, comprising at least one 5′

-uridine-adenine-3′

(5′

-UA-3′

) dinucleotide wherein the uridine is a 2′

-modified nucleotide;

at least one 5′

-uridine-guanine-3′

(5′

-UG-3′

) dinucleotide, wherein the 5′

-uridine is a 2′

-modified nucleotide;

at least one 5′

-cytidine-adenine-3′

(5′

-CA-3′

) dinucleotide, wherein the 5′

-cytidine is a 2′

-modified nucleotide;

or at least one 5′

-uridine-uridine-3′

(5′

-UU-3′

) dinucleotide, wherein the 5′

-uridine is a 2′

-modified nucleotide.

14. The iRNA agent of claim 12, wherein the 2′

-modification is chosen from the group of;

2′

-deoxy, 2′

-deoxy-2′

-fluoro, 2′

-O-methyl, 2′

-O-methoxyethyl (2′

-O-MOE), 2′

-O-aminopropyl (2′

-O-AP), 2′

-O-dimethylaminoethyl (2′

-O-DMAOE), 2′

-O-dimethylaminopropyl (2′

-O-DMAP), 2′

-O-dimethylaminoethyloxyethyl (2′

-O-DMAEOE), and 2′

-O—

N-methylacetamido (2′

-O-NMA).

15. The iRNA agent of claim 1, comprising a cholesterol moiety.

16. The iRNA agent of claim 15, wherein the cholesterol moiety is conjugated to the 3′

-end of the sense strand of the iRNA agent.

17. The iRNA agent of claim 1, wherein the iRNA agent is targeted for uptake by cells of the liver.

18. The iRNA agent of claim 1, wherein the iRNA agent is targeted for uptake by cells of the gut.

19. A pharmaceutical composition, comprising:

(a) an iRNA agent of claim 1;

and

(b) a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, further comprising a formulating agent which prolongs the half-life of the iRNA agent in human and/or mouse serum, or which facilitates uptake of the iRNA agent into cells.

21. The pharmaceutical composition of claim 20, wherein the formulating agent comprises a polycation.

22. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is useful in the treatment of a proliferative disorder.

23. The pharmaceutical composition of claim 22, wherein the proliferative disorder is cancer, preferably leukemia, more preferably acute lymphoblastic leukemia.

24. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is capable of reducing MLL-AF4 fusion gene expression in a cell or tissue of a subject and/or of inhibiting the rate of proliferation of t(4;

11)-positive cells upon administration of the pharmaceutical composition to the subject, e.g. a human.

25. A method of treating a human subject having or at risk for developing a proliferative disorder comprising the step of:

administering an iRNA agent of claim 1 to the human subject.

26. The method of claim 25, wherein the proliferative disorder is cancer, preferably acute lymphoblastic leukemia.

27. The method of claim 25, wherein the iRNA agent is administered in an amount sufficient to reduce the expression of MLL-AF4 in a cell or tissue of the subject and/or to inhibit the rate of proliferation of t(4;

11)-positive cells.

28. A method of making a pharmaceutical composition, comprising the step of:

formulating the iRNA agent of claim 1 with a pharmaceutical carrier.

29. The method of claim 28, further comprising the step of:

formulating the iRNA agent with a formulating agent which prolongs the half-life of the iRNA agent in human and/or mouse serum, or which facilitates uptake of the iRNA agent into cells.

30. The method of claim 29, wherein the formulating agent comprises a polycation.

31. The method of claim 28, wherein the pharmaceutical composition is useful in the treatment of a proliferative disorder.

32. The method of claim 31, wherein the proliferative disorder is cancer, and preferably a leukemia, and more preferably acute lymphoblastic leukemia.

33. The method of claim 28, wherein the pharmaceutical composition is capable of reducing MLL-AF4 fusion gene expression in a cell or tissue of a subject and/or of inhibiting the rate of proliferation of t(4;

11)-positive cells upon administration of the pharmaceutical composition to the subject, e.g. a human.

34. A method of reducing the amount of MLL-AF4 RNA in a cell and/or of inhibiting the rate of proliferation of t(4;

11)-positive cells, comprising the step of;

contacting the cell or cells with the iRNA agent of claim 1.

35. The method of claim 34, wherein the method is performed in vitro.

36. The method of claim 34, wherein the cell is a cell of a subject, and the subject is diagnosed as having a proliferative disorder, and preferably cancer, and more preferably an acute lymphoblastic leukemia.

37. A method of making an iRNA agent of claim 1, the method comprising the step of:

synthesizing the sense and the antisense strand of the iRNA agent, wherein the sense and/or antisense strands comprise at least one nucleotide modification

38. The method of claim 37, wherein said nucleotide modification is a modification that causes the iRNA agent to have increased stability in a biological sample.

39. The method of claim 37, further comprising administering the iRNA agent to a subject.

40. The method of claim 39, wherein the subject is diagnosed as having a proliferative disorder, preferably cancer.

41. The method of claim 39, wherein the subject is diagnosed as having an acute lymphoblastic leukemia.

42. The method of claim 39, wherein the subject is a human.

44. The method of claim 43, wherein the iRNA agent is an iRNA agent of claim 1.

2. An iRNA agent comprising a nucleotide sequence in the sense strand and a nucleotide sequence in the antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical to one of the sequences of agents 1-12 of Table 1 (sense strand:

SEQ ID NOs 5, 10, 12, 14, 18, 20, 22, 24, 26, 30, and 32;

antisense strand;

SEQ ID NOs 6, 7, 11, 13, 15, 19, 21, 23, 25, 27, 31, and

     33), except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit MLL-AF4 expression in cultured human SEM cells.

43. A method of evaluating an iRNA agent thought to inhibit the expression of an MLL-AF4 gene, the method comprising:

(a) providing an iRNA agent, wherein a first strand is sufficiently complementary to a nucleotide sequence of an MLL-AF4 mRNA, and a second strand is sufficiently complementary to the first strand to hybridize to the first strand;

(b) contacting the iRNA agent to a cell comprising an MLL-AF4 gene;

(c) comparing MLL-AF4 gene expression before contacting the iRNA agent to the cell, or of uncontacted control cells, to the MLL-AF4 gene expression after contacting the iRNA agent to the cell; and

(d) determining whether the iRNA agent is useful for inhibiting MLL-AF4 gene expression, wherein the iRNA is useful if the amount of MLL-AF4 RNA present in the cell, or protein secreted by the cell, is less than the amount present or secreted prior to contacting the iRNA agent to the cell, or less than the amount present or secreted by cells not so contacted.