RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality

• US 10,479,992 B2
• Filed: 06/30/2017
• Issued: 11/19/2019
• Est. Priority Date: 02/04/2009
• Status: Active Grant

• Current Assignee: Phio Pharmaceuticals Corp.
• Original Assignee: Phio Pharmaceuticals Corp.
• Inventors: Woolf, Tod M., Kamens, Joanne, Salomon, William, Khvorova, Anastasia
• Primary Examiner(s): Zara, Jane J
• Application Number:
  US15/638,586
• Publication Number:
  US 20180155718A1
• Time in Patent Office: 872 Days
• Field of Search: 435 61, 435 911, 435 9131, 435455, 514 44, 536 231, 536 245
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12N 2310/533 having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The present invention relates to RNAi constructs and their use in gene silencing. RNAi constructs associated with the invention contain a double stranded region connected to a single stranded region of phosphorothioate modified nucleotides.

Claims

1. An isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the passenger strand is 8-16 continuous nucleotides in length, wherein the isolated double stranded nucleic acid molecule comprises a double stranded region that is 8-16 nucleotides in length, wherein the guide strand is 19-25 nucleotides in length, and wherein the double stranded region is connected through a cleavable chemical linker to a single stranded region of at least six phosphorothioate modified nucleotides.

2. The isolated double stranded nucleic acid molecule of claim 1, wherein the double stranded nucleic acid molecule includes at least one of the following properties:

a) at least one 2′

O methyl or 2′

fluoro modification; and

b) a lipophilic group.

4. The isolated double stranded nucleic acid molecule of claim 1 wherein the nucleic acid has one end having a single nucleotide single stranded region, optionally wherein the single stranded region is connected to the guide strand and/or wherein the nucleic acid has one blunt end.

5. The isolated double stranded nucleic acid molecule of claim 1 wherein the single stranded region occurs at the 3′

end of the passenger strand, the 5′

end of the passenger strand, the 3′

end of the guide strand or the 5′

end of the guide strand, and wherein the single stranded region is DNA or RNA.

6. The isolated double stranded nucleic acid molecule of claim 1 wherein the double stranded region of the nucleic acid molecule is a perfect duplex or the double stranded region of the nucleic acid molecule contains at least one bulge region.

7. The isolated double stranded nucleic acid molecule of claim 1 wherein the double stranded region contains at least one nucleotide that is phosphorothioate modified, the single stranded region comprises at least eight phosphorothioate modified nucleotides, and/or wherein the single stranded region comprises 9-17 phosphorothioate modified nucleotides.

8. The isolated double stranded nucleic acid molecule of claim 1 wherein the nucleic acid molecule is chemically modified, optionally wherein the chemical modification is 2′

O methyl and/or 2′

Fluoro and optionally wherein more than one chemical modification is present in the same molecule.

9. The isolated double stranded nucleic acid molecule of claim 8 wherein the chemical modification is on the passenger strand, and/or the guide strand.

10. The isolated double stranded nucleic acid molecule of claim 1 wherein the single stranded region has complementarity to a mammalian gene and/or wherein the single stranded region functions as an antisense molecule.

11. The isolated double stranded nucleic acid molecule of claim 1 wherein the single stranded region is at least twelve nucleotides long.

12. The isolated double stranded nucleic acid molecule of claim 1 wherein the nucleic acid is a bifunctional nucleic acid molecule, wherein the double stranded region functions in RNA interference and the single stranded region functions in antisense.

13. The isolated double stranded nucleic acid molecule of claim 1 wherein the guide strand has complementarity to a target gene, and wherein the guide strand has at least one 2′

O-methyl modification or 2′

-fluoro modification and/or wherein the passenger strand is 8-14 nucleotides long and has complementarity to the guide strand, wherein the passenger strand is linked to a lipophilic group.

14. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule of claim 1.

15. The method of claim 14 wherein the isolated double stranded nucleic acid molecule includes a chemical modification that increases stability, increases evasion of immune regulation and/or prevents off-target gene silencing.

16. A method of inducing RNAi in a subject comprising:

administering to a subject an effective amount for inducing RNAi of an mRNA of a target gene, an isolated double stranded nucleic acid molecule of claim 1, optionally wherein the step of administering is systemic, intravenous, intraperitoneal, intradermal, topical, intranasal, inhalation, oral, intramucosal, local injection, subcutaneous, oral tracheal, or intraocular.

17. The method of claim 16, wherein the subject is a human and optionally wherein the target gene is PPIB, MAP4K4 or SOD1.

18. A composition of an isolated double stranded nucleic acid molecule of claim 1 for inhibiting the expression of a target gene or treating a disease associated with expression of the target gene.

19. The isolated double stranded nucleic acid molecule of claim 1, wherein the guide strand comprises a 5′

phosphate modification.

20. The isolated double stranded nucleic acid molecule of claim 2, wherein the at least one 2′

O methyl or 2′

fluoro modification is located on a C and/or a U nucleotide in the double stranded region of the guide strand.

3. An isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the passenger strand is 8-16 continuous nucleotides in length, wherein the isolated double stranded nucleic acid molecule comprises a double stranded region that is 8-16 nucleotides in length, wherein the guide strand is 19-25 nucleotides in length, and wherein the double stranded region is connected through a cleavable chemical linker to a single stranded region of at least three phosphorothioate modified nucleotides.