Inhibition of MAP4K4 through RNAI

• US 9,074,211 B2
• Filed: 11/19/2009
• Issued: 07/07/2015
• Est. Priority Date: 11/19/2008
• Status: Active Grant

• Current Assignee: Phio Pharmaceuticals Corp.
• Original Assignee: Rxi Pharmaceuticals Corporation
• Inventors: Woolf, Tod M., Kamens, Joanne, Khvorova, Anastasia, Salomon, William
• Primary Examiner(s): MCDONALD, JENNIFER SUE PITRAK
• Application Number:
  US13/130,194
• Publication Number:
  US 20120059046A1
• Time in Patent Office: 2,056 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12Y 207/01037 Protein kinase (2.7.1.37) C12Y207/11001, C12Y207/11008 - C12Y207/11013, C12Y207/11021, C12Y207/11022, C12Y207/11024, C12Y207/11025, C12Y207/1103, C12Y207/12001 take precedence

Abstract

RNAi constructs directed to MAP4K4 that demonstrate unexpectedly high gene silencing activities, and uses thereof are disclosed. The blunt-ended constructs have a double-stranded region of 19-49 nucleotides. The constructs have selective minimal modifications to confer an optimal balance of biological activity, toxicity, stability, and target gene specificity. For example, the strands may be modified (e.g., one or both ends of the sense strand is modified by 2′-O-methyl groups), such that the construct is not cleaved by Dicer or other RNAse III, the antisense strand may also be modified by a 2′-O-methyl group at the penultimate 5′-end nucleotide to greatly reduce off-target silencing.

Claims

1. A blunt-ended double-stranded RNA (dsRNA) for inhibiting expression of a MAP4K4 gene, comprising:

(1) a sense strand of 25, 26, 27, 28, 29 or 30 nucleotides in length, having a 5′

-end and a 3′

-end, wherein the sense strand is highly modified with 2′

-modified ribose sugars, comprising a continuous stretch of 2′

-modified ribose sugars on the 5′

-end, a continuous stretch of 2′

-modified ribose sugars on the 3′

-end and a central region that is unmodified, and,

(2) an antisense strand of 25, 26, 27, 28, 29 or 30 nucleotides in length, having a 5′

-end and a 3′

-end, which hybridizes to the sense strand,

wherein the sense strand comprises a sequence selected from the group consisting of;

SEQ ID NO;

1, 3, 5, 7, 11, 12, 13, 15, 17, 19, 21, 25, 27, 31, 33, 35, 37, 45 and 50, and

wherein the dsRNA does not form a hairpin.

2. The dsRNA of claim 1, wherein the dsRNA comprises a modification of a base, and/or a phosphodiester linkage.

3. The dsRNA of claim 1, wherein the dsRNA comprises a hydrophobic modification of one or more bases.

4. The dsRNA of claim 1, wherein the dsRNA is 25-27 base pairs in length, and wherein the sense strand of the dsRNA comprises 12-14 and 10-12 consecutive 2′

-modified ribose sugars at the 5′

-end of the sense strand and the 3′

-end of the sense strand, respectively.

5. A composition comprising the dsRNA of claim 1, and a pharmaceutically acceptable carrier or diluent.

6. A method for inhibiting the expression of a MAP4K4 gene in a mammalian cell, comprising contacting the mammalian cell with the dsRNA of claim 1.

7. A method of treating a patient for a disease characterized by overexpression of a MAP4K4 gene, comprising administering to the patient the dsRNA of claim 1, wherein the dsRNA mediates reduction in MAP4K4 expression.

8. The dsRNA of claim 1, wherein the sense strand comprises SEQ ID NO:

11.

9. The dsRNA of claim 1, wherein the sense strand comprises SEQ ID NO:

19.

10. The dsRNA of claim 1, wherein the sense strand comprises SEQ ID NO:

50.

11. The dsRNA of claim 1, wherein the antisense strand comprises a sequence selected from the group consisting of SEQ ID NO. 2, 4, 6, 8, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 52.