Polycomb-associated non-coding RNAS

Patent metadata

Field	Value
Publication number	US-10053694-B2
Application number	US-201615171883-A
Country	US
Kind code	B2
Filing date	Jun 2, 2016
Priority date	Nov 12, 2010
Publication date	Aug 21, 2018
Grant date	Aug 21, 2018

Abstract

This invention relates to long non-coding RNAs (lncRNAs), libraries of those ncRNAs that bind chromatin modifiers, such as Polycomb Repressive Complex 2, inhibitory nucleic acids and methods and compositions for targeting lncRNAs.

First claim

What is claimed is:

1. A method of producing an oligonucleotide for inducing expression of a non-imprinted target gene, the method comprising:
i) performing an immunoprecipitation assay using an antibody directed against PRC2 to immunoprecipitate a PRC2-binding RNA from a cell, wherein the PRC2-binding RNA is not ANRIL lncRNA, is transcribed from a sequence of the chromosomal locus of the non-imprinted target gene and inhibits expression of the target gene in the cell, wherein the immunoprecipitation assay comprises:
a) providing a sample comprising RNA bound nuclear proteins from the cell;
b) contacting the sample with an antibody directed against PRC2 under conditions sufficient to form complexes between the antibody and PRC2; and
c) isolating the complexes, wherein the isolated complexes comprise the PRC2-binding RNA bound to PRC2;

ii) detecting a PRC2-binding region of the PRC2-binding RNA by subjecting the PRC2-binding RNA of the isolated complexes to a sequencing assay, wherein the PRC2 binding region is detected as a nucleotide sequence of the PRC2-binding RNA that is protected from nucleases during the immunoprecipitation assay; and
iii) producing an isolated oligonucleotide of 8 to 40 nucleotides in length having a region of complementarity that is complementary with at least 5 contiguous nucleotides of the PRC2-binding region of the PRC2-binding RNA, wherein the oligonucleotide induces expression of the non-imprinted target gene.

2. The method of claim 1 , wherein the oligonucleotide interferes with binding of PRC2 to the PRC2-binding region without inducing degradation of the PRC2-binding RNA.

3. The method of claim 1 , further comprising:
iv) delivering the oligonucleotide to a test cell; and
v) detecting a change in expression of the target gene following delivery of the oligonucleotide to the test cell, wherein an increase in expression of the target gene compared with an appropriate control cell indicates effectiveness of the oligonucleotide.

4. The method of claim 3 , further comprising detecting a decrease in recruitment of PRC2 to the target gene in the cell following delivery of the oligonucleotide to the test cell, wherein the decrease in recruitment of PRC2 to the target gene in the test cell, compared with an appropriate control cell to which the oligonucleotide has not been delivered, indicates effectiveness of the oligonucleotide for inducing expression of the target gene.

5. The method of claim 1 , wherein the RNA is a lncRNA.

6. The method of claim 3 , further comprising, prior to step iv), detecting expression of the PRC2-binding RNA in the test cell, wherein expression of the PRC2-binding RNA in the test cell indicates that the oligonucleotide is suitable for inducing expression of the target gene in the test cell.

7. The method of claim 3 , wherein the test cell is in vitro.

8. The method of claim 3 , wherein the test cell is in vivo.

9. The method of claim 1 , wherein the PRC2-binding RNA is transcribed from the same strand as the target gene in a genomic region containing the target gene.

10. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to an exon.

11. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from the same strand as the target gene within a chromosomal region within −2.0 kb to +0.001 kb of the transcription start site of the target gene.

12. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from the opposite strand of the target gene within a chromosomal region within −0.5 to +0.1 kb of the transcription start site of the target gene.

13. The method of claim 1 , wherein the oligonucleotide has complementarity to the PRC2-binding RNA in a region of the PRC2-binding RNA that forms a stem-loop structure.

14. The method of claim 1 , wherein at least one nucleotide of the oligonucleotide is an RNA or DNA nucleotide.

15. The method of claim 1 , wherein the target gene is a protein-coding gene.

16. The method of claim 1 , wherein the target gene is a gene of an autosomal chromosome.

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

18. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to an intron-exon junction or an intron.

19. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to a translation initiation region or a translation termination region.

20. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to a promoter.

21. The method of claim 1 , wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to a 5′-UTR or a 3′-UTR.

22. The method of claim 1 , wherein the oligonucleotide comprises at least one nucleotide having a 2′-O-methoxyethyl modified sugar moiety and locked nucleic acid (LNA) nucleotides.

23. The method of claim 1 , wherein at least one nucleotide of the oligonucleotide is a ribonucleic acid analogue comprising a ribose ring having a bridge between its 2′-oxygen and 4′-carbon.

24. The method of claim 23 , wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′-oxygen and the 4′-carbon.

25. The method of claim 1 , wherein at least one nucleotide has a 2′-O-methoxyethyl modified sugar moiety.

26. The method of claim 1 , wherein the oligonucleotide comprises at least one modified internucleoside linkage.

27. A method of producing an oligonucleotide for inducing expression of a non-imprinted target gene in a cell, the method comprising:
producing an isolated oligonucleotide of 8 to 40 nucleotides in length having a region of complementarity that is complementary with at least 5 contiguous nucleotides of a PRC2-binding RNA that is not ANRIL lncRNA, is transcribed from a sequence of the chromosomal locus of the non-imprinted target gene and that inhibits expression of the target gene, wherein the oligonucleotide is complementary to and bind specifically within a PRC2-binding region of the PRC2-binding RNA, wherein the PRC2-binding region is determined to have a nucleotide sequence that is protected from nucleases during an RNA immunoprecipitation procedure using an antibody directed against PRC2, wherein the oligonucleotide interferes with binding of PRC2 to the PRC2-binding region without inducing degradation of the PRC2-binding RNA, and wherein the oligonucleotide induces expression of the non-imprinted target gene.

Assignees

• Massachusetts Gen Hospital

Inventors

• Lee Jeannie T
• Zhao Jing
• Sarma Kavitha
• Borowsky Mark
• Ohsumi Toshiro Kendrick

Classifications

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title