RNAi modulation of MLL-AF4 and uses thereof

• US 7,674,779 B2
• Filed: 02/27/2008
• Issued: 03/09/2010
• 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
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US12/038,808
• Publication Number:
  US 20080233584A1
• Time in Patent Office: 741 Days
• Field of Search: 514/44
• US Class Current: 514/44.00R
• 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. A method of reducing the amount of MLL-AF4 RNA in a cell or of inhibiting the rate of proliferation of t(4;

11)-positive cells, comprising the step of;

contacting the cell or cells with an iRNA agent, wherein said iRNA agent comprises a sense strand and an antisense strand and the antisense strand consists of SEQ ID NO;

15.

2. The method of claim 1, wherein the method is performed in vitro.

3. The method of claim 1, wherein the iRNA agent comprises at least one nucleotide modification.

4. The method of claim 3, wherein the nucleotide modification is a modification that causes the iRNA agent to have increased stability in a biological sample.

5. The method of claim 1, wherein the cell is a cell of a subject.

6. The method of claim 5, wherein the subject is diagnosed as having a proliferative disorder.

7. The method of claim 5, wherein the subject is diagnosed as having an acute lymphoblastic leukemia.

9. The method of claim 3, wherein the nucleotide modification is a phosphorothioate or a 2′

-modified nucleotide.

10. The method of claim 3, wherein the nucleotide modification is a 5′

-uridine-adenine-3′

(5′

-UA-3′

) dinucleotide wherein the uridine is a 2′

-modified nucleotide;

a 5′

-uridine-guanine-3′

(5′

-UG -3′

) dinucleotide, wherein the 5′

-uridine is a 2′

-modified nucleotide;

a 5′

-cytidine-adenine-3′

(5′

-CA-3′

) dinucleotide, wherein the 5′

-cytidine is a 2′

-modified nucleotide;

or a 5′

-uridine-uridine-3′

(5′

-UU-3′

) dinucleotide, wherein the 5′

-uridine is a 2′

-modified nucleotide.

11. The method of claim 9, wherein the 2′

-modification is chosen from the group consisting 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).

12. The method of claim 1, wherein the iRNA agent comprises a cholesterol moiety.

13. The method of claim 12, wherein the cholesterol moiety is conjugated to the 3′

-end of the sense strand of the iRNA agent.

14. The method of claim 1, wherein the sense strand consists of SEQ ID NO:

14.

16. The method of claim 1, wherein the cell or cells are cultured human SEM cells and the iRNA agent reduces the amount of MLL-AF4 mRNA present in the cells by more than 40% compared to cells which have not been contacted with the agent.

18. The method of claim 1, wherein the iRNA agent comprises at least one nucleotide overhang having 1 to 4 unpaired nucleotides.

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

(a) providing an iRNA agent, wherein a first strand of the iRNA agent is sufficiently complementary to a nucleotide sequence of an MLL-AF4 mRNA, and a second strand of the iRNA agent is sufficiently complementary to the first strand to hybridize to the first strand, wherein the second strand consists of SEQ ID NO;

15;

(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 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.

15. The method of claim 8, wherein the first strand consists of SEQ ID NO:

14.

17. The method of claim 8, wherein the amount of MLL-AF4 RNA present in the cell, or protein secreted by the cell, is at least 40% 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.

19. The method of claim 8, wherein the iRNA agent comprises at least one nucleotide overhang having 1 to 4 unpaired nucleotides.