# US 10,119,144 B2 · Polycomb-associated non-coding RNAs

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- Number: US 10,119,144 B2
- Document ID: US10119144B2
- Status: Granted
- Kind: B2
- Application number: 15/050,273
- Filed: 2016-02-22
- Published: 2016-12-08
- Granted: 2018-11-06
- Earliest claimed: 2010-11-12
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: Jeannie T. Lee; Jing Zhao; Kavitha Sarma; Mark Borowsky; Toshiro Kendrick Ohsumi
- Web page: https://patentgrep.com/patent/US10119144B2

## CPC

- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/00: Drugs for disorders of the urinary system
- A61P 13/12: of the kidneys
- A61P 19/00: Drugs for skeletal disorders
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- 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)}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Y 201/01043: Histone-lysine N-methyltransferase (2.1.1.43)
- C12N 2310/113: targeting other non-coding nucleic acids, e.g. antagomirs
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/314: Phosphoramidates
- C12N 2310/315: Phosphorothioates
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/3233: Morpholino-type ring
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/30: Special therapeutic applications
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2330/31: Libraries, arrays
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

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.

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 1)

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.

### Claim 17 (depends on 1)

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.

### Claim 18 (depends on 1)

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.

### Claim 19 (depends on 1)

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.

### Claim 20 (depends on 1)

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

### Claim 21 (depends on 1)

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.

### Claim 22 (depends on 21)

The method of claim 21, wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′-oxygen and the 4′-carbon.

### Claim 23 (depends on 1)

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

### Claim 24 (depends on 23)

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.

### Claim 25 (depends on 1)

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

### Claim 26 (depends on 25)

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.

### Claim 27 (depends on 1)

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

### Claim 28 (depends on 1)

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

### Claim 29 (depends on 9)

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.

### Claim 30 (depends on 9)

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.

### Claim 31 (depends on 9)

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.

### Claim 32 (depends on 9)

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.

### Claim 33 (depends on 9)

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.

### Claim 34 (depends on 9)

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.

### Claim 35 (depends on 9)

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.

### Claim 36 (depends on 9)

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.

### Claim 37 (depends on 9)

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.

### Claim 38 (depends on 9)

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.

### Claim 39 (depends on 9)

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

### Claim 40 (depends on 9)

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.

### Claim 41 (depends on 40)

The method of claim 40, wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′-oxygen and the 4′-carbon.

### Claim 42 (depends on 9)

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

### Claim 43 (depends on 42)

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.

### Claim 44 (depends on 9)

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

### Claim 45 (depends on 44)

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.