Single-stranded RNAi agents containing an internal, non-nucleic acid spacer

Patent metadata

Field	Value
Publication number	US-9243246-B2
Application number	US-201113818306-A
Country	US
Kind code	B2
Filing date	Aug 19, 2011
Priority date	Aug 24, 2010
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

Single-stranded RNA molecules comprise one or more internal, non-nucleotide spacers, covalently linked with nucleotide portions of the molecule are provided. The single-stranded RNA molecules function as guide or antisense strands that are capable of inhibiting gene expression via an RNA interference mechanism, and thus represent single-stranded RNAi agents. The single-stranded RNAi molecules can be used in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

First claim

What is claimed:

1. A single-stranded RNA molecule that mediates RNA interference against a target RNA, wherein said single-stranded RNA molecule comprises:
(i) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2) that are not self complementary, wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target site of the target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides; and,
(ii) an internal spacer portion, wherein said spacer portion comprises at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions,
wherein said single-stranded RNA molecule comprises the following structure:
5′ N1-S1-N2 3′

wherein:
(a) N1 contains either one nucleotide or a contiguous stretch of nucleotides;
(b) S1 contains one or more non-nucleotide spacers covalently linking N1 and N2; and,
(c) N2 contains either one nucleotide or a contiguous stretch of nucleotides;
(d) S1 is at the 3′ end of N1 and at the 5′ end of N2; and
(e) wherein said single-stranded RNA molecule has one 3′ end.

2. The molecule of claim 1 , wherein S1 is an aliphatic or aromatic organic group.

3. The molecule of claim 2 , wherein S1 is a C 1 -C 12 alkyl chain that is optionally substituted.

4. The molecule of claim 3 , wherein said alkyl chain is optionally substituted with cholesterol.

5. The molecule of claim 2 , wherein S1 is selected from a group consisting of a C3 alkyl, a C6 alkyl, and polyethylene glycol.

6. The molecule of claim 1 , wherein N1 is 13 to 20 nucleotides long.

7. The molecule of claim 1 , wherein the total number of nucleotides within the nucleic acid portion is about 19 to about 21 nucleotides.

8. The molecule of claim 1 , wherein the target site is within a untranslated region of the target RNA.

9. The molecule of claim 8 , wherein the at least 8 nucleotides that can base pair with the target site is the whole or a part of a seed sequence of a naturally-occurring, endogenous miRNA nucleotide sequence.

10. The molecule of claim 9 , wherein S1 takes the place of from one to 4 internal nucleotides of the naturally-occurring, endogenous miRNA nucleotide sequence.

11. The molecule of claim 10 , wherein the nucleic acid portion of the molecule is at least 50% homologous to the naturally-occurring, endogenous miRNA nucleotide sequence.

12. The molecule of claim 1 , wherein the target site is within a gene coding region of the target RNA.

13. The molecule of claim 1 , wherein the nucleic acid portion of the molecule is at least 90% complementary to the target site.

14. The molecule of claim 1 , wherein the nucleic acid portion comprises at least 20 nucleotides that can base pair with the target site.

15. The molecule of claim 1 , wherein the nucleic acid portion further comprises a third nucleotide portion (N3) and the internal spacer portion further comprises a second non-nucleotide spacer portion (S2), wherein said single-stranded RNA molecule comprises the following structure:
5′ N1-S1-N2-S2-N3 3′

wherein S2 is at the 3′ end of N2 and at the 5′ end of N3.

16. The molecule of claim 1 , wherein at least one nucleotide has a modified sugar.

17. The molecule of claim 1 , wherein at least one nucleotide has a modified internucleoside linkage.

18. The molecule of claim 1 , having a terminal cap at the 5′-end, the 3′-end, or both the 5′- and 3′-ends.

19. A composition comprising the single-stranded RNA molecule of claim 1 and a pharmaceutically acceptable carrier.

20. The composition of claim 19 , further comprising a liposome, a hydrogel, a cyclodextrin, a biodegradable nanocapsule, a bioadhesive microsphere, or a proteinaceous vector.

21. A method of reducing the expression of an endogenous RNA target gene in a cell comprising administering a composition of claim 19 .

22. A single-stranded RNA molecule that mediates RNA interference against a target RNA, wherein said single-stranded RNA molecule comprises:
(i) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2) that are not self complementary, wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target site of the target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides; and,
(ii) an internal spacer portion, wherein said spacer portion comprises at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions,
wherein said single-stranded RNA molecule comprises the following structure:
5′ N1-S1-N2 3′

wherein:
(a) N1 contains 18 contiguous nucleotides, 19 contiguous nucleotides, and 20 contiguous nucleotides;
(b) S1 contains one or more non-nucleotide spacers covalently linking N1 and N2; and
(c) N2 contains either one nucleotide or a contiguous stretch of nucleotides.

23. The molecule of claim 22 , wherein N1 consists of 18 contiguous nucleotides and N2 consists of 2 contiguous nucleotides.

24. The molecule of claim 22 , wherein N1 consists of 19 contiguous nucleotides and N2 consists of 1 nucleotide.

25. A single-stranded RNA molecule that mediates RNA interference against a target RNA, wherein said single-stranded RNA molecule comprises:
(i) a nucleic acid portion comprising a first nucleotide portion (N1) and a second nucleotide portion (N2) that are not self complementary, wherein said nucleic acid portion comprises at least 8 nucleotides that can base pair with a target site of the target RNA, and wherein the total number of nucleotides within the nucleic acid portion is from 8 to 26 nucleotides; and,
(ii) an internal spacer portion, wherein said spacer portion comprises at least a first non-nucleotide spacer portion (S1) that covalently links the first and second nucleotide portions,
wherein said single-stranded RNA molecule comprises the following structure:
5′ N1-S1-N2 3′

wherein:
(a) N1 contains either one nucleotide or a contiguous stretch of nucleotides;
(b) S1 contains one or more non-nucleotide spacers covalently linking N1 and N2; and
(c) N2 contains either 2 contiguous nucleotides or 1 nucleotide.

Assignees

• Lim Lee
• Chorn Guillaume
• Willingham Aarron
• Zhao Lihong
• Sirna Therapeutics Inc

Inventors

• Lim Lee
• Chorn Guillaume
• Willingham Aarron
• Zhao Lihong

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2310/332

  Abasic residue · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2310/318

  where the PO2 is completely replaced, e.g. MMI or formacetal · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title