DOUBLE-STRANDED AND SINGLE-STRANDED RNA MOLECULES WITH 5' TRIPHOSPHATES AND THEIR USE FOR INDUCING INTERFERON

• US 20160369280A1
• Filed: 09/02/2016
• Published: 12/22/2016
• Est. Priority Date: 02/04/2005
• Status: Active Grant

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: ROSSI, John J., Kim, Dongho, Lundberg, Patric, Cantin, Edouard
• Granted Patent: US 9,938,529 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/1131 against viruses
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/117 Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/17 Immunomodulatory nucleic acids
• CPC Class Codes: C12N 2310/18 acting by a non-sequence specific mechanism
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell

Abstract

Double-stranded and single-stranded RNA molecules, and their use in methods for inducing interferon are provided. The interferon induction provides anti-viral and other medically useful effects, such as anti-cancer effects. Also provided are methods for reducing or inhibiting interferon induction exhibited by such molecules, particularly siRNA and shRNA molecules produced in vitro.

Claims

1. A method for inducing an anti-viral response in a cell, comprising introducing into a cell an effective amount of an RNAi molecule having a 5′

-triphosphate, wherein said RNAi molecule induces interferon and elicits an anti-viral response mediated by said interferon induction.

2. The method of claim 1, wherein said RNAi molecule is produced in vitro by a phage polymerase.

3. The method of claim 2, wherein said polymerase is T7 RNA polymerase.

4. The method of claim 1, wherein said RNAi molecule is an siRNA or an shRNA molecule.

5. An in vitro method for producing an RNAi molecule which reduces interferon induction while maintaining the efficacy of said RNAi molecule, comprising:

removing one or more 5′

triphosphates from an RNAi molecule produced in vitro, wherein said removal reduces interferon induction while maintaining the efficacy of said RNAi molecule.

6. The method of claim 5, wherein said removal occurs by cleaving said one or more 5′

-triphosphates from said RNAi molecule with one or both of a ribonuclease and a phosphatase.

7. The method of claim 6, wherein said ribonuclease is a T1 ribonuclease.

8. The method of claim 6, wherein said phosphatase is a calf intestinal phosphatase (CIP).

9. The method of claim 5, wherein said RNAi molecule contains at least two bases at the 3′

terminus which prevent base pairing with a 5′

guanine of said RNAi molecule prior to said removal of one or more 5′

-triphosphates.

10. The method of claim 9, wherein the two bases are adenosines.