Polycomb-associated non-coding RNAS

• US 10,053,694 B2
• Filed: 06/02/2016
• Issued: 08/21/2018
• Est. Priority Date: 11/12/2010
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lee, Jeannie T., Zhao, Jing, Sarma, Kavitha, Borowsky, Mark, Ohsumi, Toshiro Kendrick
• Primary Examiner(s): POLIAKOVA-GEORGA, EKATERINA
• Application Number:
  US15/171,883
• Publication Number:
  US 20160376598A1
• Time in Patent Office: 810 Days
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 9/1271 Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers liposome as conjugate A61K47/6911
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/113 targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 : Phosphorothioates
• CPC Class Codes: C12N 2310/3181 : Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 : having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3233 : Morpholino-type ring
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/32 : Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/31 : Libraries, arrays
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6881 : for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6886 : for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/178 : miRNA, siRNA or ncRNA
• CPC Class Codes: C12Y 201/01043 : Histone-lysine N-methyltransferase (2.1.1.43)

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.

Claims

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.