Polycomb-associated non-coding RNAs

• US 10,119,144 B2
• Filed: 02/22/2016
• Issued: 11/06/2018
• 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-GEORGAN, EKATERINA
• Application Number:
  US15/050,273
• Publication Number:
  US 20160355806A1
• Time in Patent Office: 988 Days
• Field of Search: None
• 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 inducing expression of a non-imprinted target gene in a cell, wherein expression of the target gene is inhibited by a PRC2-binding RNA, the method comprising delivering to the cell a composition comprising an isolated single stranded 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 RNA that inhibits expression of the target gene, wherein the PRC2-binding RNA is transcribed from a sequence of the chromosomal locus of the target gene, wherein the target gene is A1BG, ABCB4, ABCC2, ACADM, ACOT12, ACY1, AGXT, AHSG, AIRE, AK4, ALDH1L1, ANG, ANGPTL6, ARF6, ASCL1, ASGR2, ASNS, ASPDH, ASS1, ATF5, AURKA, BDH1, C19ORF80, CBG, CAD, CARD10, CCDC39, CCDC40, CDHR5, CEBPA, CEBPG, CFLAR, CITED2, CLEC4M, COBL, CSNK2A1, CSNK2A2, CSNK2B, CUX2, CX3CR1, CYP1A1, CYP26A1, CYP2A13, CYP2A4, CYP2A5, CYP2A6, CYP3A7, CYP4F13, CYP4F18, CYP4F3, DBP, DDHD2, DNAAF1, DNAJC25, DNMT3L, EGFR, ENTPDS, EPHX1, F2, F5, FABP1, FCN2, FOXA3, FPGS, FUOM, GLDC, GLS2, GLTPD2, GM6484, GNPNAT1, GPBAR1, GPLD1, H2-M3, H2-OA, HES1, HFE2, HLA-C, HLA-DOA, HLA-F, HLA-G, HLX, HMOX1, HNF1B, HNRNPD, IGF2R, IL1B, IL23A, IL6R, IL6RA, ITGA2, ITPA, JARID2, KHK, KNG2, LEAP2, LIPE, LOXL2, LPL, LSR, MAN2A1, MED1, MESP1, MST1, MTTP, MYC, NF1, NFKBIA, NPW, OSGIN1, PAQR9, PCNA, PCSK9, PFKL, PHKA2, PHKG2, PKD1, PKD2, PKM, PLK1, PNPLA1, PNPLA2, PNPLA3, PON3, PPARA, PPP1R3F, PRAP1, PRG4, PRKCE, PRKCSH, PROS1, PRPF31, PTPN1, PTPN3, RAP1A, RB1CC1, RBP4, REEP6, RELA, RIPPLY1, RNASE4, RND1, RPGRIP1L, RPL32, RTP3, RXRA, SAA2-SAA4, SAA4, SDS, SEC14L2, SEC63, SERAC1, SERPINA7, SERPINE1, SLC10A1, SLC13A5, SLC22A1, SLC22A7, SLC25A18, SLC28A1, SLC51A, SOD2, SP3, SRSFS, TAF10, TGFB1, THBS2, THPO, THRSP, TMEM82, TMPRSS6, TRIM55, UGT1A1, UGT1A10, UGT1A2, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A6A, UGT1A6B, UGT1A7, UGT1A8, UGT1A9, UPF2, VDR, WNT1, or ZIC3.

2. The method of claim 1, further comprising detecting expression of the PRC2-binding RNA in the cell, wherein expression of the PRC2-binding RNA in the cell indicates that the single stranded oligonucleotide is suitable for increasing expression of the target gene in the cell.

3. The method of claim 1, further comprising detecting a change in expression of the target gene following delivery of the single stranded oligonucleotide to the cell, wherein an increase in expression of the target gene compared with an appropriate control cell indicates effectiveness of the single stranded oligonucleotide.

4. The method of claim 1, wherein a decrease in recruitment of PRC2 to the target gene in the cell following delivery of the single stranded oligonucleotide to the cell, compared with an appropriate control cell to which the single stranded oligonucleotide has not been delivered, indicates effectiveness of the single stranded oligonucleotide.

5. The method of claim 1, wherein the cell is in vitro.

6. The method of claim 1, wherein the cell is in vivo.

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

8. The method of claim 1, wherein at least one nucleotide of the oligonucleotide is a modified nucleotide.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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 3′

-UTR.

17. 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.

18. 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.

19. 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.

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

21. 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.

22. The method of claim 21, wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′

-oxygen and the 4′

-carbon.

23. The method of claim 1, wherein at least one nucleotide of the oligonucleotide comprises a modified sugar moiety.

24. The method of claim 23, wherein the modified sugar moiety comprises a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, or a bicyclic sugar moiety.

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

26. The method of claim 25, wherein the at least one modified internucleoside linkage is selected from phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

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

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

9. A method of inducing expression of a non-imprinted target gene in a cell, wherein expression of the target gene is inhibited by a PRC2-binding RNA, the method comprising delivering to the cell a composition comprising an isolated single stranded 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 RNA that inhibits expression of the target gene, wherein the PRC2-binding RNA is transcribed from a sequence of the chromosomal locus of the target gene, wherein the oligonucleotide is complementary to and binds specifically within a PRC2-binding region of the PRC2-binding RNA and interferes with binding of PRC2 to the PRC2-binding region without inducing degradation of the PRC2-binding RNA, wherein the PRC2-binding region has a nucleotide sequence protected from nucleases during an RNA immunoprecipitation procedure using an antibody directed against PRC2, and wherein the target gene is A1BG, ABCB4, ABCC2, ACADM, ACOT12, ACY1, AGXT, AHSG, AIRE, AK4, ALDH1L1, ANG, ANGPTL6, ARF6, ASCL1, ASGR2, ASNS, ASPDH, ASS1, ATF5, AURKA, BDH1, C19ORF80, CBG, CAD, CARD10, CCDC39, CCDC40, CDHR5, CEBPA, CEBPG, CFLAR, CITED2, CLEC4M, COBL, CSNK2A1, CSNK2A2, CSNK2B, CUX2, CX3CR1, CYP1A1, CYP26A1, CYP2A13, CYP2A4, CYP2A5, CYP2A6, CYP3A7, CYP4F13, CYP4F18, CYP4F3, DBP, DDHD2, DNAAF1, DNAJC25, DNMT3L, EGFR, ENTPD5, EPHX1, F2, F5, FABP1, FCN2, FOXA3, FPGS, FUOM, GLDC, GLS2, GLTPD2, GM6484, GNPNAT1, GPBAR1, GPLD1, H2-M3, H2-OA, HES1, HFE2, HLA-C, HLA-DOA, HLA-F, HLA-G, HLX, HMOX1, HNF1B, HNRNPD, IGF2R, IL1B, IL23A, IL6R, IL6RA, ITGA2, ITPA, JARID2, KHK, KNG2, LEAP2, LIPE, LOXL2, LPL, LSR, MAN2A1, MED1, MESP1, MST1, MTTP, MYC, NF1, NFKBIA, NPW, OSGIN1, PAQR9, PCNA, PCSK9, PFKL, PHKA2, PHKG2, PKD1, PKD2, PKM, PLK1, PNPLA1, PNPLA2, PNPLA3, PON3, PPARA, PPP1R3F, PRAP1, PRG4, PRKCE, PRKCSH, PROS1, PRPF31, PTPN1, PTPN3, RAP1A, RB1CC1, RBP4, REEP6, RELA, RIPPLY1, RNASE4, RND1, RPGRIP1L, RPL32, RTP3, RXRA, SAA2-SAA4, SAA4, SDS, SEC14L2, SEC63, SERAC1, SERPINA7, SERPINE1, SLC10A1, SLC13A5, SLC22A1, SLC22A7, SLC25A18, SLC28A1, SLC51A, SOD2, SP3, SRSF5, TAF10, TGFB1, THBS2, THPO, THRSP, TMEM82, TMPRSS6, TRIM55, UGT1A1, UGT1A10, UGT1A2, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A6A, UGT1A6B, UGT1A7, UGT1A8, UGT1A9, UPF2, VDR, WNT1, or ZIC3.

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

30. The method of claim 9, 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.

31. The method of claim 9, 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.

32. The method of claim 9, 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.

33. The method of claim 9, 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.

34. The method of claim 9, 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.

35. The method of claim 9, wherein the oligonucleotide has complementarity to a region of the PRC2-binding RNA transcribed from a portion of the target gene corresponding to a 3′

-UTR.

36. The method of claim 9, 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.

37. The method of claim 9, 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.

38. The method of claim 9, wherein the oligonucleotide has complementarity to the PRC2-binding RNA in a region of the PRC2-binding RNA that forms a stem-loop structure.

39. The method of claim 9, wherein at least one nucleotide of the oligonucleotide is an RNA or DNA nucleotide.

40. The method of claim 9, 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.

41. The method of claim 40, wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′

-oxygen and the 4′

-carbon.

42. The method of claim 9, wherein at least one nucleotide of the oligonucleotide comprises a modified sugar moiety.

43. The method of claim 42, wherein the modified sugar moiety comprises a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, or a bicyclic sugar moiety.

44. The method of claim 9, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

45. The method of claim 44, wherein the at least one modified internucleoside linkage is selected from phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.