Polycomb-associated Non-Coding RNAs

• US 20140142160A1
• Filed: 11/12/2011
• Published: 05/22/2014
• 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: Borowsky, Mark, Lee, Jeannie T., Zhao, Jing, Sarma, Kavitha, Ohsumi, Toshiro Kendrick
• Granted Patent: US 9,328,346 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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

1-71. -71. (canceled)

72. A single stranded oligonucleotide of 5 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 regulates expression of a target gene, wherein at least one nucleotide of the oligonucleotide is a modified nucleotide.

73. The single stranded oligonucleotide of claim 72, wherein the RNA is a long non-coding RNA (lncRNA).

74. The single stranded oligonucleotide of claim 72, wherein the PRC2-binding RNA regulates expression of the target gene in a cis manner.

75. The single stranded oligonucleotide of claim 72, wherein the PRC2-binding RNA is transcribed from the same strand as the target gene in a genomic region containing the target gene.

76. The single stranded oligonucleotide of claim 72, 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, intron-exon junction, a translation initiation region, a translation termination region, a promoter, an intron, a 5′

-UTR, or a 3′

-UTR.

77. The single stranded oligonucleotide of claim 72, 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.

78. The single stranded oligonucleotide of claim 72, 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.

79. The single stranded oligonucleotide of claim 72, wherein the oligonucleotide has complementarity to the PRC2-binding RNA in a region of the PRC2-binding RNA that forms a stem-loop structure.

80. The single stranded oligonucleotide of claim 72, wherein at least one nucleotide of the oligonucleotide is an RNA or DNA nucleotide.

81. The single stranded oligonucleotide of claim 72, 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.

82. The single stranded oligonucleotide of claim 81, wherein the ribonucleic acid analogue comprises a methylene bridge between the 2′

-oxygen and the 4′

-carbon.

83. The single stranded oligonucleotide of claim 72, wherein at least one nucleotide of the oligonucleotide comprises a modified sugar moiety.

84. The single stranded oligonucleotide of claim 83, 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.

85. The single stranded oligonucleotide of claim 72 comprising at least one modified internucleoside linkage.

86. The single stranded oligonucleotide of claim 85, 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.

87. The single stranded oligonucleotide of claim 72 configured such that hybridization of the single stranded oligonucleotide to the PRC2-binding RNA does not activate an RNAse H pathway in a cell.

88. The single stranded oligonucleotide of claim 72 configured such that hybridization of the single stranded oligonucleotide to the PRC2-binding RNA does not induce substantial cleavage or degradation of the PRC2-binding RNA in a cell.

89. The single stranded oligonucleotide of claim 72 configured such that hybridization of the single stranded oligonucleotide to the PRC2-binding RNA interferes with interaction of the RNA with PRC2 in a cell.

90. A compound comprising the single stranded oligonucleotide of claim 72 conjugated to a second single stranded oligonucleotide of 5 to 40 nucleotides in length having a region of complementarity that is complementary with at least 5 contiguous nucleotides of a PRC2-binding RNA.

91. A single stranded oligonucleotide of 5 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 inhibits expression of a target gene in cis, wherein the oligonucleotide has complementarity to the PRC2-binding RNA in a region of the PRC2-binding RNA that is transcribed from the sense strand of the target gene.

92. The single stranded oligonucleotide of claim 91, wherein the RNA is a lncRNA.

93. A method of inducing expression of a target gene in a cell, the method comprising delivering to the cell a single stranded oligonucleotide of 5 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 inhibits expression of the target gene, wherein the oligonucleotide is complementary to and binds specifically to the PRC2-binding RNA.

94. The method of claim 93, wherein the RNA is a lncRNA.

95. The method of claim 93 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.

96. The method of claim 93 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.

97. The method of claim 93 further comprising detecting a change in recruitment of PRC2 to the target gene in the cell following delivery of the single stranded oligonucleotide to the cell, wherein a decrease in recruitment compared with an appropriate control cell indicates effectiveness of the single stranded oligonucleotide.

98. The method of claim 93, wherein the cell is in vitro.

99. The method of claim 93, wherein the cell is in vivo.

100. A method of inducing expression of a target gene in a cell, the method comprising delivering to the cell a single stranded oligonucleotide of 5 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 inhibits expression of the target gene, wherein the oligonucleotide is complementary to and binds specifically to the PRC2-binding lncRNA, and wherein the delivery results in an increase in expression of the target gene of at least 50% compared with an appropriate control cell.

101. The method of claim 100, wherein the delivery results in an increase in expression of the target gene of at least 100% compared with an appropriate control cell.

102. The method of claim 100, wherein the RNA is a lncRNA.